Download Regulation of tubulin heterodimer partitioning during interphase and

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New series No. 1220
ISSN: 0346-6612
ISBN: 978-91-7264-670-4
Editor: The Dean of the Faculty of Medicine
Regulation of tubulin heterodimer partitioning
during interphase and mitosis
Per Holmfeldt
Department of Molecular Biology
Umeå University, Sweden 2008
Copyright © Per Holmfeldt
Printed by Print & Media
Umeå 2008
TABLE OF CONTENTS
1.
2.
3.
LIST OF PUBLICATIONS
LIST OF ABBREVIATIONS
ABSTRACT
INTRODUCTION
4.
INTRODUCTION TO THE CYTOSKELETON
4.1
The cytoskeleton
4.2
Evolution of cytoskeletal systems that organize the cell
4.3
The cytoskeleton reorganizes during the cell cycle
4.4
Lateral bonds between adjacent protofilaments are required
for the stability of cytoskeletal filaments
4.5
Nucleation is the rate limiting step for a polymerization reaction
5.
BASIC STRUCTURE AND PROPERTIES OF MICROTUBULES
Microtubules are polar polymers of α/β-tubulin heterodimers
Microtubules are nucleated from microtubule-organizing centers
The spatial organization of the microtubule system may differ
between specialized cell types
5.4
The energy of GTP hydrolysis drives the intrinsic dynamic
instability of microtubules
PROTEINS RELEVANT TO THE STRUCTURE AND FUNCTION
OF THE MICROTUBULE SYSTEM
6.1
Regulation of microtubule dynamics in intact cells
6.2
Microtubule regulatory proteins
6.3
Unidirectional movement of molecular motors along microtubules
MICROTUBULE FUNCTION DURING THE INTERPHASE AND MITOSIS
OF THE CELL CYCLE
7.1
A brief introduction to the mammalian cell cycle and its check points
7.2
Microtubule dependent molecular motors organize the intracellular space
7.3
Microtubules segregate the duplicated chromosomes during mitosis
7.4
The role of kinetochore microtubule treadmilling to ensure bipolar
kinetochore attachment
THE IMPORTANCE OF CHROMOSOMAL INSTABILITY FOR TUMOR
PROGRESSION
8.1
Tumor progression requires on genomic instability
8.2
Genomic instabilities of tumors depends on two distinct mechanisms
MAJOR REGULATORS OF MICROTUBULE POLYMERIZATION
9.1
MCAK is a conserved catastrophe-promoting protein
9.2
The function of MCAK during the cell cycle
9.3
The evolutionary conserved XMAP215/TOGp protein family
9.4
Function of XMAP215/TOGp family proteins during mitosis
9.5
Counteractive activities between MCAK and XMAP215 family members
9.6
Op18 - a developmentally regulated microtubule-destabilizing protein
9.7
Phosphorylation-inactivation of Op18 during interphase and mitosis
9.8
Dysregulation of Op18 in human malignancies
5.1
5.2
5.3
6.
7.
8.
9.
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9.9
9.10
9.11
Relations between Op18 and the other Op18/Stathmin family members
Microtubule stabilization by the ubiquitous expressed MAP4
Regulation of MAP4 during interphase and mitosis
RESULTS AND DISCUSSION
10.
AIMS
11.
PROLOGUE
12.
THE MCAK AND XMAP215/TOGp PROTEINS ARE NOT IMPORTANT
FOR THE CONTROL OF TUBULIN SUBUNIT PARTITIONING BUT
HAVE ESSENTIAL INTERCONNECTED FUNCTIONS DURING
SPINDLE ASSEMBLY
12.1
Background
12.2
XMAP215/TOGp is not essential to enhance the polymerization rate
in human somatic cells
12.3
Differential phenotypes of XMAP215/TOGp and MCAK
depletion in human cells and Xenopus egg extracts
12.4
The overexpression phenotypes of MCAK and TOGp reveal
an unexpected interplay
12.5
Confirmation that MCAK function is important for spindle fidelity
12.6
TOGp provides protection from centrosomal MCAK activity, which is
essential for bipolar spindle formation
18
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12.7
13.
14.
15.
16.
17.
18.
The functions of both CaMKIIγ and TOGp are essential for protection
of spindle microtubules from centrosome-directed MCAK activity
PHOSPHORYLATION-REGULATION OF Op18 AND MAP4 CONTROLS THE
PARTITIONING OF TUBULIN SUBUNITS IN THE INTERPHASE
MICROTUBULE SYSTEM
13.1
Background
13.2
Evidence for the importance of MAP4 and Op18 as interphase-specific
counteractive regulators of tubulin subunit partitioning
13.3
The significance of MAP4 and Op18 as phosphorylation-responsive
regulators of tubulin subunit partitioning
IDENTIFICATION OF Op18 AS A MAJOR SIGNAL TRANSDUCTION
RESPONSIVE REGULATOR OF TUBULIN SUBUNIT PARTITIONING
14.1
Background
14.2
Identification of an Op18 dependent signal transduction chain for
microtubule polymerization in response to T cell antigen receptor
triggering
EXCESSIVE Op18 ACTIVITY DURING MITOSIS RESULTS IN SPINDLEDESTABILIZATION AND CHROMOSOMAL INSTABILITY
15.1
Background
15.2
The relationship between the oncogenic Q18ÆE substitution of Op18
and leukemia-associated up-regulation of the wild-type protein
15.3
The significance of life-span of animals for the demand for increased
chromosomal instability during tumor-progression
FINAL CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
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1. LIST OF PUBLICATIONS
This thesis is based on the following papers, which will be referred to in the text by their roman numerals:
I.
Holmfeldt, P., Stenmark S., and Gullberg M., (2004)
Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis.
EMBO Journal 23:627-637.
II.
Holmfeldt P., Zhang X., Stenmark S., Walczak C., and Gullberg M., (2005)
CaMKIIgamma-mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation.
EMBO Journal 24:1256–1266.
III.
Holmfeldt P., Brännström K., Stenmark S., and Gullberg M., (2006)
Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18->E mutation leukemic cells.
Molecular Biology of the Cell 17:2921-30
IV.
Holmfeldt, P., Stenmark S., and Gullberg M., (2007)
Interphase-specific phosphorylation-mediated regulation of tubulin dimer partitioning in human cells.
Molecular Biology of the Cell 18:1909-17
2. LIST OF ABBREVIATIONS
MARK, Microtubule-Affinity Regulating Kinase
MCAK, Mitotic Centromere-Assoociated Kinesin
Mor1, Microtubule ORganisation gene-product 1
Msps, Mini spindles
MT, MicroTubule
NuMA, Nuclear Mitotic Apparatus protein
Op18, Oncoprotein of 18 kDa
PAR1, PARtitioning-defective mutant 1
PI-3 kinase, PhosphatidylIonositol-3 kinase
PKA, Protein Kinase A (also known as cAMP dependent protein
kinase)
SCG10, Superior Cervical Ganglion 10
SCLIP, SCG10-LIke Protein
shRNA, short hair-pin RiboNucleic Acid
Stu2, Suppressor of a TUbulin mutation 2
Ras, Rat sarcoma virus oncogene
Ran, Ras-related nuclear protein
Rb, Retinoblastoma susceptibility protein
RB3, Rat homolog of the Xenopus gene XB3
Rho, Ras homology
RNA, RiboNucleic Acid
TACC, Transforming Acidic Coiled-Coil
TBCE, TuBulin-folding Cofactor E
+TIP, plus-TIP(/end) binding protein
TOGp, Tumour Overexpressing Gene protein
TPX2, Targeting Protein for Xklp2
XMAP215, Xenopus Microtubule Associated Protein of 215 kDa
ZAP-70, Zeta-chain-Associated Protein kinase of 70 kDa
Zyg-9, ZYGote defective 9
APC, Adenomatous Polyposis Coli
APC/C, Anaphase Promoting Complex/Cyclosome
ADP, Adenosine DiPhosphate
Alp14, ALtered Polarity gene-product 14
ATP, Adenosine TriPhosphate
CaMKII, Ca2+/CalModulin-dependent protein Kinase II
CaMKIV, Ca2+/CalModulin-dependent protein Kinase IV
cAMP, cyclic Adenosine MonoPhosphate
CDC6, Cell Division Control mutant 6
CDC42, Cell Division Control mutant 42
CDK, Cyclin-Dependent Kinase
CLASP, CLIP-ASsociated Protein
CLIP, Cytoplasmic LInker Protein
Dis1, Defect In Sister chromatid disjoining 1
DdCP224, Dictyostelium discoideum Centrosomal Protein of 224
kDa
DLG1, Drosophila discs LarGe 1
DNA, DeoxyriboNucleic Acid
EB1, End-Binding protein 1
EBV, Epstein-Barr Virus
ELKS, protein rich in E (glutamic acid), L (leucine), K (lysine)
and S (serine)
FtsZ, Filamenting temperature-sensitive mutant Z
GDP, Guanosine DiPhosphate
GTP, Guanosine TriPhosphate
KIF17, KInesin Family member 17
KIN I, KINesin Internal motor domain
MAP2, Microtubule-Associated Protein 2
MAP4, Microtubule-Associated Protein 4
MAPK, Mitogen-Activated Protein Kinase
1
evolutionary context, the cooperation of these filaments was
first required for segregation of duplicated chromosomes, and
has subsequently evolved to facilitate complex cell shapes
and evolution of multi-cellular organisms. The cytoskeleton
encompasses microtubules, intermediate filaments and actin
filaments (also known as microfilaments), which are
polymers of evolutionary conserved protein subunits. Given
that polymerization of these subunits in general is rapid and
reversible, the cytoskeleton can rapidly reorganize during the
cell cycle and in response to changes of the environment. A
large set of accessory proteins has evolved to facilitate
specific nucleation, assembly and the dynamic behavior of
the cytoskeleton. In addition, proteins that serve as molecular
motors are central for the function of the microtubule and
actin cytoskeletons. These motor proteins generate forces by
directional movement along polymers and by this mean
moves cargo to selected cellular locations. Thus, the
cytoskeletal systems involve many more proteins than their
structural subunits. While the cytoskeleton systems have been
described in eukaryotic cells, recent reports on structural
similarities with prokaryotic proteins important for cell
division have lead to the insight that the microtubule and
actin cytoskeletons have an ancient origin (reviewed in
Erickson, 2007).
3. ABSTRACT
The microtubule cytoskeleton, which consists of dynamic
polymers of α/β tubulin heterodimers, organizes the
cytoplasm and is essential for chromosome segregation
during mitosis. My thesis addresses the significance and
potential interplay between four distinct microtubuleregulatory proteins. The experimental approach included the
development of a replicating vector system directing either
constitutive expression of short hairpin RNAs or inducible
ectopic expression, which allows stable depletion and/or
conditional exchange of gene-products.
Based on the originally observed activities in frog
egg extracts, MCAK and TOGp have been viewed as major
antagonistic proteins that regulate microtubule-dynamics
throughout the cell cycle. Surprisingly, while my thesis work
confirmed an essential role of these proteins to ensure mitotic
fidelity, tubulin subunits partitioning is not controlled by the
endogenous levels of MCAK and TOGp in human somatic
cells. Our major discovery in these studies is that the
activities of both CaMKIIγ and TOGp are essential for
spindle bipolarity through a mechanism involving protection
of spindle microtubules against MCAK activity at the
centrosome.
In our search for the major antagonistic activities
that regulates microtubule-dynamics in interphase cells, we
found that the microtubule-destabilizing activity of Op18 is
counteracted by MAP4. These studies also established Op18
and MAP4 as the predominant regulators of tubulin subunit
partitioning in all three human cell model systems studied.
Moreover, consistent with phosphorylation-inactivation of
these two proteins during mitosis, we found that the
microtubule-regulatory activities of both MAP4 and Op18
were only evident in interphase cells. Importantly, by
employing a system for inducible gene product replacement,
we found that site-specific phosphorylation-inactivation of
Op18 is the direct cause of the demonstrated hyperpolymerization in response to T-cell antigen receptor
triggering. This provides the first formally proven example of
a signal transduction pathway for regulation of interphase
microtubules.
Op18 is frequently upregulated in various types of
human malignancies. In addition, a somatic mutation of Op18
has recently been identified in an adenocarcinoma. This
thesis work revealed that the mutant Op18 protein exerts
increased microtubule-destabilizing activity. The mutant
Op18 protein was also shown to be partially resistant to
phosphorylation-inactivation during mitosis, which was
associated with increased chromosome segregation
aberrancies. Interestingly, we also observed the same
phenotype by overexpressing the wild type Op18 protein.
Thus, either excessive levels of wild type Op18 or normal
levels of mutated hyper-active Op18 seems likely to
contribute to tumor progression by exacerbating
chromosomal instability.
4.2 Evolution of cytoskeletal systems that organize the
cell
During the early evolution of the eukaryotic cell the genome
increased both in size and complexity. This was associated
with evolution of strategies to ensure correct transmission of
the genetic material to the two daughter cells during cell
division, such as splitting up the genome into chromosomes
that are segregated by microtubules of the mitotic spindle
during cell division (Fig. 1A (I)). This process has been
coined mitosis based on the thread-like appearance of the
condensed chromatin (from Greek mitos, thread)(reviewed in
Paweletz, 2001). In addition to division of the genetic
material, the contractile ring built from actin filaments has
evolved to execute the cellular division, a process known as
cytokinesis (Fig. 1A (II)).
A)
B)
I
II
II
III
I
IV
V
INTRODUCTION
Fig. 1. Cellular functions that depend on the cytoskeleton
4. INTRODUCTION TO THE CYTOSKELETON
4.1 The cytoskeleton
All eukaryotic cells contain three distinct types of filament
forming proteins, collectively termed the cytoskeleton. In an
During interphase, the period in between two
division events, cytoskeletal filaments serve a primordial role
to protect against mechanical stress, which made the cell wall
redundant in the ancient eukaryotic cell. This was probably a
2
facilitate the contraction of the whole cell. During cell
division, actin filaments together with myosin form the
contractile ring that is positioned just beneath the plasma
membrane at the cellular equator (Fig. 2).
prerequisite for the development of actin-filament based
phagocytosis (Fig. 1B (I)) and for subsequent acquisition of
endosymbiotic organelles, such as mitochondria. Thus, the
loss of the cell wall paved the way for the current
organization of the eukaryotic cells into distinct
compartments. To allow a cytoskeleton dependent spatial
organization of the cytoplasm, the molecular motor proteins
evolved. Through the utilization of energy released during
ATP hydrolysis, a molecular motor can move directionally
along either microtubules or actin filaments. By attachment
to motor proteins, various cargos such as organelles, vesicles
and macromolecules is moved to specific locations in the cell
(Fig. 1B (II)).
Alterations of cell shape depends on the protruding
action of polymerizing actin-filaments exerted on the plasma
membrane (Fig. 1B (III)). The resulting protrusions are
thereafter in general stabilized by intermediate filaments. If
the protrusive activity of actin-filaments at one side of a cell
is coupled to simultaneous actin-filament dependent
contractions at the opposite side, this will result in directional
movement of the cell (Fig. 1B (IV)).
A central aspect during evolution of multicellular
organisms is cell surface molecules that facilitate specific
adhesion between neighboring cells. These connections
depends on anchorage of the adhesion molecules to the
intermediate filaments, which provides resistance towards
mechanical stress (Fig. 1B (V)).
Microtubules
Actin filaments
Intermediate
filaments
Interphase
Mitosis
Fig. 2. Reorganization of the three distinct cytoskeleton systems
during the cell cycle
4.4 Lateral bonds between adjacent protofilaments are
required for the stability of cytoskeletal filaments
Many of the functions of the cytoskeleton require filaments
to assemble over long distances and still have the potential to
disassemble rapidly to allow recycling of subunits at different
cellular locations, for example during reorganization of cells
in response to signaling events. The small size of the protein
subunits, in the range of a few nanometers, enables rapid
diffusion over long distances. Soluble subunits are thereby
available for polymerization at any given location within the
cell. To generate polymers with the potential of rapid
disassembly, the subunits are held together by weak noncovalent bonds (Fig. 3A). However, a polymer containing
just a single row of subunits, held together by weak
interactions, has a high probability to break at any given
point of the polymer, which would prevent formation of
polymers of the required length (Fig. 3B). Thus, to combine
the requirements of rapid disassembly with stability, all
cytoskeletal filaments are stabilized by lateral interactions
between adjacent rows of subunits, termed protofilaments.
This arrangement provides a stable core with dynamic ends
(Fig. 3C).
4.3 The cytoskeleton reorganizes during the cell cycle
As illustrated in Fig. 2, all parts of the cytoskeleton undergo
major reorganizations between interphase and mitosis. The
microtubule system, which during interphase is organized
into a single array that originates from the centre of the cell,
transforms into the bipolar mitotic spindle. The interphase
microtubule system constitutes the track system on which
molecular motors move to localize cellular components over
long distances. In certain cell types specialized microtubules
are essential for the motile behavior of cellular appendages,
called cilia and flagella. During mitosis, the microtubules of
the mitotic spindle organize the duplicated chromosomes
onto the cellular equator and thereafter segregate them such
that each daughter cell receives a complete set of
chromosomes (Fig. 2).
Nuclear intermediate filaments termed lamins are
localized just beneath the inner nuclear membrane and are
important for the integrity of the nuclear envelope during
interphase in all eukaryotic cells. Cytosolic intermediate
filaments have only been identified with certainty in
metazoan cells, in which they form a web-like network
throughout the cell. These cytosolic intermediate filaments
provide mechanical resistance and stabilize connections with
neighboring cells or the extracellular matrix (Fig. 2).
Actin filaments are generally found directly beneath
the plasma membrane. These filaments provide structural
integrity of the plasma membrane and are important for
formation of cellular protrusions, thus serving a central role
in establishment of cell shape. Actin filaments also have the
ability to form contractile structures together with bipolar
filaments of the motor protein myosin-II. These contractile
structures can either be positioned just beneath the plasma
membrane and mediate contractions of specific parts of the
cell, or, as in muscle cells, fill the interior of the cell and
A)
Monomers
+
B)
C)
+
Polymer
+
+
Breakage of longitudinal monomer binding
due to ambient thermal energy
Stabilizing lateral
bindings between
adjacent protofilaments
Fig. 3. Cytoskeletal filaments stabilized by laterally interacting
protofilaments
In the case of microtubules, lateral interactions are
provided by the arrangements of 13 protofilaments that
interact to form a hollow tube. This tubular structure is stiff
3
1967), α- and β-tubulin were subsequently identified as the
subunits of microtubules (Stephens, 1970). The α- and βtubulin subunits are ~40% identical in sequence and are
highly conserved among eukaryotes (Little & Seehaus,
1988). The atomic structure of tubulin heterodimers, solved
by electron crystallography of zinc induced tubulin sheets,
revealed a very similar globular structure of the α- and βtubulin subunits (Fig. 6B; Nogales et al., 1998).
but brittle, which provides the ability to generate pushing
forces. Intermediate filaments are composed of eight
protofilaments, which wrap around each other to form a
ropelike bundle. This arrangement is strong and flexible and
obviously well suited to withstand stress without rupturing.
In actin filament two protofilaments twist around each other
to form a stable but yet dynamic structure (Fig. 4).
Microtubule
Intermediate
filament
Actin
filament
A)
Protofilament
B)
β-tubulin
α-tubulin
Subunits:
25 nm
10 nm
7 nm
Tubulin
heterodimer
Variable
Actin
Fig. 6. Microtubule and tubulin structure
Fig. 4. Structure of the three cytoskeletal filaments of eukaryotic
cells
The α- and β-tubulin subunits do not exist as monomers and
their folding and subsequent assembly into the tubulin
heterodimer is dependent on several specific chaperonin
cofactors (reviewed in Lopez-Fanarraga et al., 2001). The
significance of this folding pathway is illustrated by the
finding that a mutation in one of its components, TBCE, is
the genetic defect in the human inherited disease KennyCaffey syndrome (Parvari et al., 2002).
Both α- and β-tubulin subunits are encoded by
multiple genes, with a pronounced cell-type specific
expression in animals. The tubulin heterodimer isotypes
appear largely functionally redundant and co-polymerize into
mixed microtubules in vitro. It is still unclear how the
different isotypes affect the microtubule system in intact
cells; however, it seems clear that the neural specific isotypes
are important for the properties of neural cells in which the
microtubule system has highly specialized roles during e.g.
axon outgrowth (reviewed in Katsetos et al., 2003).
Moreover, the presence of isotypes is also of clinical
significance, since changes in isotype composition can
mediate resistance to tubulin directed drugs in tumor cells
(reviewed in Verrills & Kavallaris, 2005).
Tubulin heterodimers form polar protofilaments
through head-to-tail assembly (Fig. 6A). Parallel
protofilaments form hollow tubes by lateral interactions.
Microtubules assembled under in vitro conditions may
contain between 8 and 19 protofilaments (Chretien & Wade,
1991), suggesting flexibility in the interactions between
adjacent protofilaments. Cellular microtubules, however,
always contain 13 protofilaments (Evans et al., 1985), a
number probably explained by the mechanism of nucleation,
which is based on a template (see Fig. 7).
Microtubules are polar polymers with α-tubulin
exposed at one end and β-tubulin at the opposite and the
polymerization rate differs ~5-fold between opposite ends.
The faster growing end is termed the plus end while the
slower growing end the minus end. By docking the crystal
structure of the tubulin heterodimer into a microtubule model
it has been established that α-tubulin is exposed at the minus
end and β-tubulin consequently is exposed at the plus end of
the microtubule (Nogales et al., 1999).
4.5 Nucleation is the rate limiting step for a
polymerization reaction
During in vitro assembly of filaments (Fig. 5A), formation of
an initial aggregate of subunits, the nucleus, is the first step
of the polymerization reaction. This process is called
nucleation and depends on the formation of short oligomers.
Since the subunits will only be able to interact with few other
subunits, the short oligomers are unstable and are likely to
dissociate. This creates a kinetic barrier for spontaneous
polymerization, which provides a mean for a cell to control
the localization of cytoskeletal assembly. To facilitate
nucleation, cells are equipped with dedicated proteins, termed
nucleation factors, which serve as templates on to which
subunits can efficiently form polymers (Fig. 5B). The
subcellular localization and activation of nucleation factors
will therefore be a major determinant for the location of
cytoskeletal assembly.
A)
Polymerization
Spontaneous nucleation
B) Nucleation factor
Template facilitated
nucleation
Polymerization
Fig. 5. Initiation of polymerization is regulated by nucleation factors
that decrease the kinetic barrier of nucleation
5. BASIC STRUCTURE AND PROPERTIES OF
MICROTUBULES
5.1 Microtubules are polar polymers of α/β-tubulin
heterodimers
The α/β-tubulin heterodimer is the subunit of microtubules. It
consists of two homologous proteins, α- and β- tubulin, each
of a molecular weight of 55 kDa (Fig. 6A). Tubulin
heterodimers were initially identified because of their affinity
to the spindle poisoning drug colchicine (Borisy & Taylor,
4
Tubulin heterodimers are subject to complex posttranslational modifications (reviewed in Westermann &
Weber, 2003), but the physiological significance is unclear
and the extent of modifications seems largely to reflect the
age of individual microtubules. Most of these modifications
affect the carboxy-terminal region, which in both α- and βtubulin is located on the outside of the microtubule and
therefore positioned to influence interactions between the
microtubule and other proteins.
The prokaryotic protein FtsZ has a limited sequence
homology with α- and β-tubulin (reviewed in Erickson,
1995) and is virtually identical in structure (Lowe & Amos,
1998). FtsZ can assemble into protofilaments (Mukherjee &
Lutkenhaus, 1994), which form a contractile ring at the
division site of the bacterial cell (Ma et al., 1996). Inhibitors
of FtsZ function is currently developed as novel therapeutic
agents against pathogenic bacteria (reviewed in Vollmer,
2006). In the bacteria Prosthecobacter dejongeii two genes
with sequence homology to α- and β-tubulin have been
identified (Jenkins et al., 2002), which have probably been
acquired through horizontal gene transfer (Schlieper et al.,
2005). These bacterial versions of tubulin assemble into
protofilaments, which form bundles rather than tubular
microtubule structures (Sontag et al., 2005). The function of
the Prosthecobacter tubulin-like proteins is still unclear.
these centrioles, termed mother, is characterized by two
distinct sets of nine appendages at distal and subdistal
locations (Paintrand et al., 1992).
In the currently favored model for γ-tubulin
mediated microtubule nucleation, the γ-tubulin ring complex
forms a lock-washer shaped template, on which tubulin
heterodimers can readily polymerize (Fig. 7) (reviewed in
Raynaud-Messina & Merdes, 2007). Following their
nucleation by γ-tubulin ring complexes, the minus end of
microtubules are believed to be anchored to the appendages
of the mother centriole inside the centrosome (Fig. 8A; Piel
et al., 2000), through a ninein dependent mechanism
(Mogensen et al., 2000), leaving the minus ends inert. Thus,
the γ-tubulin ring complex has potential to be recycled for
many round of nucleation.
5.2 Microtubules are nucleated from microtubuleorganizing centers
Provided that nucleation is facilitated by e.g., volumeexcluding glycerol or the microtubule-stabilizing drug taxol,
tubulin heterodimers have potential to self-assemble in vitro.
In intact cells, however, a template structure is required to
initiate polymerization. A key structural element for
microtubule nucleation in all eukaryotes is provided by γtubulin, a paralogue of α- and β-tubulin (reviewed in Oakley,
2000). Multiple γ-tubulin molecules associates laterally to
form a ring –shaped structure, first detected by electron
microscopy studies of purified complexes from Xenopus eggs
(Fig. 7) (Zheng et al., 1995). Many proteins are important for
assembly, localization and the structure of the γ-tubulin ring
complex (reviewed in Raynaud-Messina & Merdes, 2007).
Cytosolic γ-tubulin, which constitutes the major part
of cellular γ-tubulin, does not exert significant microtubule
nucleation activity (Moudjou et al., 1996). Active γ-tubulin is
primarily located at structures termed microtubule organizing
centers, exemplified by the animal centrosome and the
spindle pole body of Saccharomyces cerevisiae. Based on
morphological studies, the centrosome has been characterized
as a large, ~1 μm in diameter, non-membranous organelle,
which is attached to the cell nucleus in what appears to be a
microtubule dependent mechanism (reviewed in Gonczy,
2004). Centered on a pair of perpendicular arranged
centrioles and surrounded by a cloud of pericentriolar
material, the centrosome is believed to contain at least 200
different proteins. Centrioles, which are required for
centrosomal integrity (Bobinnec et al., 1998), are barrel
shaped structures with walls made of nine parallel triplet
microtubules, with each triplet tilted inward like the blades of
a turbine. The centrioles are duplicated once during the cell
cycle (reviewed in Azimzadeh & Bornens, 2007) and the
individual centrioles consequently differ in age. The older of
During mitosis, centrosomal microtubules are
excised by the microtubule severing protein katanin (Fig. 8B;
McNally & Thomas, 1998) and released from the
centrosome. The minus ends of the microtubules are
thereafter focused by the molecular motor dynein (Gaglio et
al., 1997) and re-attached to the centrosome, by a mechanism
that leaves the minus end exposed (Fig. 8A). This
arrangement allows for a flux of tubulin heterodimers
through the polymer, with addition of subunits at the plus end
coupled to simultaneous disassociation at the minus end, a
process termed treadmilling.
γ-tubulin
ring complex
Fig. 7. Cellular microtubule formation is facilitated by templated
nucleation mediated by γ-tubulin ring complexes
A)
Interphase
Mitosis
Centrosome
B) Microtubule severing by katanin
Fig. 8. Microtubule nucleation, release and anchorage at the
centrosome
Generation of mitotic microtubules is also
dependent on extra-centrosomal nucleation (reviewed in
Walczak & Heald, 2008), believed to occur primarily in the
vicinity of chromosomes through either a Ran regulated
TPX2 dependent mechanism (Carazo-Salas et al., 2001;
Gruss et al., 2002) or a γ-tubulin mediated mechanism
5
extracellular fluids or the cells themselves (Fig. 9D). These
appendages, termed cilia and flagella, respectively, are
formed around a central core, the axoneme, consisting of nine
doublet microtubules arranged in a circle surrounding two
singlet microtubules (reviewed in Downing & Sui, 2007).
Through sliding of the axonemal microtubules, mediated by
specific molecular motors belonging to the dynein family, the
appendage will acquire an oscillating motion. The axonemal
microtubules are nucleated from a structure termed the basal
body, which is situated right beneath each individual
appendage during their assembly. The basal body contains a
single centriole, which is analogous to the mother centriole of
the centrosome, and may mediate the actual nucleation event
(reviewed in Dawe et al., 2007).
(Mahoney et al., 2006). The randomly nucleated
microtubules are sorted into bipolar arrays by the action of
microtubule dependent molecular motors (reviewed in Gadde
& Heald, 2004). This mechanism allows spindle formation to
occur even in cells with ablated centrosomes (Khodjakov et
al., 2000).
In vitro studies have revealed that many proteins,
such as dynein (Malikov et al., 2004), EB1 (Vitre et al.,
2008) and XMAP215 (Popov et al., 2001), posses significant
microtubule nucleation activity. Whether this is of
physiological significance remains unclear.
5.3 The spatial organization of the microtubule system
may differ between specialized cell types
Most mammalian cells exhibit a radial array of microtubules
with minus ends focused at the centrosome (Fig. 9A).
However, in certain specialized cells, a non-radial array of
microtubules lacking centrosomal attachment may dominate
(reviewed in Bartolini & Gundersen, 2006).
+
A)
B)
+
+
+
+
C)
- - - - --
5.4 The energy of GTP hydrolysis drives the intrinsic
dynamic instability of microtubules
Tracking of individual microtubules in vitro, through dark
field video microscopy, revealed that microtubules exhibit
non-equilibrium dynamics, characterized by periods of
polymerization and depolymerization, with stochastic
transitions between them (Fig. 10; Horio & Hotani, 1986).
The transition between polymerization and depolymerization
is termed a catastrophe and the opposite event a rescue.
Similar dynamic behavior was recorded in intact cells, but
with a general increase in dynamicity (Fig. 10; Cassimeris et
al., 1988). The observed dynamic behavior had indeed
previously been postulated, based on analysis of the length
distribution of fixed microtubules, and termed dynamic
instability (Mitchison & Kirschner, 1984a; Mitchison &
Kirschner, 1984b).
The energy released during GTP hydrolysis provides
the driving force for the dynamic instability of microtubules
(Hyman et al., 1992). Both α- and β-tubulin have a GTPbinding site termed the N-site (Non-exchangeable) and E-site
(Exchangeable), respectively (Fig. 6B). While α-tubulin
binds GTP prior to assembly and the GTP molecule is buried
at the monomer-monomer interface within the tubulin
heterodimer, the β-tubulin GTP is exposed on the
longitudinal end of the heterodimer. This leaves the E-site
bound GTP of soluble tubulin heterodimers freely
exchangeable and in equilibrium with the free GTP pool.
During polymerization of tubulin, the E-site GTP will be
buried at the newly formed heterodimer interface, making it
non-exchangeable and at the same time exposed to a catalytic
loop on α-tubulin that promotes its hydrolysis (Nogales et al.,
1999).
+
- - --
+-
++
+
+
+
D)
+
+
+ + + + ++
Fig. 9. Cell type specific organization of microtubules
Length of MT (μM)
Neurons have a combination of centrosomal
attached microtubules, that form a radial array in the cell
body, and non-centrosomal microtubules that form bundles in
both axons and dendrites (Fig. 9B). The non-centrosomal
bundles are thought to provide both structural support and the
transport tracks needed for the delivery of various cargos by
molecular motors. All neuronal microtubules are believed to
be nucleated at the centrosome and that the severing activity
of katanin (Ahmad et al., 1999) and spastin (Evans et al.,
2005) serves to release microtubules from the centrosome.
The released microtubules are subsequently arranged and
localized by molecular motors and accessory proteins.
In polarized epithelial cells, microtubules are
nucleated at the centrosome and are thereafter released to
organize into an array of parallel microtubules along the
apical-basal axis of the cell (Keating et al., 1997), with minus
ends at the apical side and the plus ends at the basal side (Fig.
9C). The minus ends are potentially stabilized by ninein
(Mogensen et al., 1997) through a mechanism that requires
functional adherence junctions (Chausovsky et al., 2000).
This microtubule organization is important for the
establishment and maintenance of the highly polarized
structure of the epithelial cell, through polarized transport of
cellular components along the microtubules by molecular
motors.
Certain specialized epithelial cells and sperms are
equipped with motile appendages that are used to either move
8
Catastophe
6
Rescue
In
In vivo
intact cells
4
2
In vitro
vitro
In
0
0
20
40 60 80 100 120
Time (seconds)
Fig. 10. Dynamic behavior of microtubules in vitro and in intact
cells
GTP hydrolysis at the E-site induces a straight-tocurved conformational change in the tubulin heterodimer
(reviewed in Nogales & Wang, 2006) and the classical GTP-
6
cap model postulates that this generates an energetically
unstable microtubule lattice, stabilized by a layer of straight
GTP loaded heterodimers at the plus-end (Fig. 11). The
stochastic losses of the GTP-cap, due to either hydrolysis or
heterodimer disassociation, allows the GDP-containing
protofilaments to relax into a more energetically favorable
curved conformation, which disrupts the stabilizing lateral
interactions between adjacent protofilaments. The individual
protofilaments will consequently rapidly depolymerize due to
that longitudinal interactions alone are not sufficient to
maintain the polymer (see Fig. 3). During this
depolymerization-phase, the ends of microtubules have a
distinct appearance revealed by electron microscopy, i.e.
frayed ends in which the curling protofilaments have a ram
horn like appearance (Fig. 11; Mandelkow et al., 1991).
The difference in microtubule dynamics in intact
cells and during assembly of purified tubulin in vitro is
primarily due to an array of microtubule regulatory proteins.
A recent proteomic search in Drosophila embryos identified
270 microtubule interacting proteins (Hughes et al., 2008),
out of which about 100 was not previously known to bind
microtubules. The task of determining which ones of these
proteins that are important for regulation of microtubule
function and dynamics is challenging.
6.2 Microtubule regulatory proteins
Several evolutionary conserved regulatory mechanisms
important for microtubule dynamics have been identified,
primarily through work on Xenopus egg extracts. These
mechanisms can generally be divided into microtubule
stabilizing and destabilizing activities. Microtubule
stabilizing proteins are subdivided in two main categories,
namely proteins that interact alongside microtubules or
proteins that specifically interact with microtubule ends.
Among the proteins interacting along the outside of
microtubules the best characterized are the so called classical
Microtubule-Associated-Proteins (MAPs), which include the
neuronal members MAP2 and tau and the ubiquitously
expressed MAP4. These proteins are thought to stabilize
microtubules by crosslinking adjacent protofilaments and
thereby prevent depolymerization (Gustke et al., 1994),
which explains the rescue promoting activity of these
proteins (Ookata et al., 1995; Pryer et al., 1992).
Proteins that specifically interact with the plus end
of microtubules are termed +TIP-proteins (reviewed in
Akhmanova & Steinmetz, 2008). Members of the +TIP
family include XMAP215/TOGp, APC and CLASP. The
latter two proteins have been show to be involved in the
selective stabilization of microtubules at specialized cellular
locations, such as the kinetochores of the mitotic
chromosomes and polarized sites in the plasma membrane
(Fig. 13). It appears therefore that APC and CLASP allow
dynamic microtubules to probe the cellular space only to be
stabilized at special sites (see section 7.2-3; reviewed in
Akhmanova & Steinmetz, 2008). Given that MAP4 and
XMAP215/TOGp have been in focus during my thesis work,
a more detailed account of their structure and function will be
given in Chapter 9.
GTP
GTP-tubulin
GDP
+
Depolymerization
Polymerization
GDP-tubulin
Catastrophe
Rescue
-
Fig. 11. Dynamic instability implies stochastic switches between
polymerization and depolymerization that depend on hydrolysis of a
stabilizing GTP-cap
Rescues, i.e., the transition from depolymerization
to polymerization, are also frequent in a system consisting
only of pure tubulin heterodimers and GTP. It can not be
excluded that rescues are the result of contaminating
microtubule stabilizing proteins. Nevertheless, rescues in the
presumed absence of microtubule-binding proteins are
thought to involve breakage of protofilaments close to the
microtubule wall and the simultaneous association of GTP
loaded heterodimers to generate a new GTP-cap.
6. PROTEINS RELEVANT TO THE STRUCTURE
AND FUNCTION OF THE MICROTUBULE SYSTEM
6.1 Regulation of microtubule dynamics in intact cells
The dynamicity of cellular microtubules differs distinctively
compared to microtubules assembled from purified tubulin
(Fig. 10). Importantly, during entry into mitosis microtubule
dynamic is altered such that very short microtubules become
predominant during prometaphase (Fig. 12A). This alteration
of microtubule length distribution can be fully explained by
an ~7-fold increase of catastrophe-promotion in mitotic frog
egg extracts (Fig. 12B).
A)
Interphase
Long, less
dynamic
microtubules
Mitosis
Short, more
dynamic
microtubules
1
Xenopus egg extractsa
B)
Interphase
Mitosis
Catastrophe frequency (sec-1)
0.018
0.12
Rescue frequency (sec-1)
0.011
0.02
Growth rate (μm/min)
9.3
12.3
Shrinkge rate (μm/min)
12.8
15.3
2
3
Fig. 13. Selective stabilization of microtubules through interaction
with +TIP proteins
Microtubule destabilizing proteins can be
subdivided into three categories, i) severing proteins, which
facilitate breakage of the microtubule (reviewed in Quarmby,
2000), exemplified by katanin (see Fig. 8B), ii) catastrophe
promoters, which interact with microtubule ends and
destabilize the GTP-cap and thereby promoting catastrophes,
a
(Belmont et al., 1990)
Fig. 12. Dynamic parameters of microtubules during interphase and
mitosis
7
apparatus (Corthesy-Theulaz et al., 1992) and mitochondria
(Varadi et al., 2004), ii) vesicles (Gilbert & Sloboda, 1989)
and iii) specific proteins (reviewed in Kamal & Goldstein,
2002) and iv) specific mRNAs (Schnorrer et al., 2000).
Motor proteins translocate along microtubules
through conformational changes driven by cycles of ATP
binding, hydrolysis and product release (Fig. 14). Since at
least one of the two motor domains of both kinesins and
dyneins is attached to the microtubule at any given time
point, the stability of attachment and processivity of these
motors is high, i.e. translocation can occur over great lengths
without de-attachment (reviewed in Amos, 2008).
6.3 Unidirectional movement of molecular motors along
microtubules
Microtubule dependent molecular motors are proteins that
through cycles of conformational changes have the ability to
move directionally along microtubules. The two classes of
structurally distinct microtubule dependent motor proteins
translocate to either the plus-end, which are prototype
members of the large kinesin superfamily, or the minus-end,
which are members of the dynein family. Kinesins and
dyneins share the same general features, even though they are
structurally unrelated. Each motor unit is centered on two
heavy chains, each with a globular head motor domain and an
elongated tail, which is responsible for the dimerization of
the heavy chains. Accessory light and intermediate chains are
primarily used to facilitate the association of different cargos
to the motor unit.
The kinesin superfamily constitutes a diverse array
of related proteins, with about 45 members in human,
grouped into 14 distinct subfamilies (Lawrence et al., 2004).
Most kinesins have the motor domain of the heavy chain
localized to the carboxy-terminal part of the protein, which
will facilitate plus-end translocation. However, members of
the kinesin-14 subfamily have the motor domain localized to
the amino terminal of the heavy chain, which alters the
direction of translocation to the minus-end. Members of the
kinesin-13 subfamily have the motor domain localized to the
central part of the heavy chain, which results in loss of the
original motor activity and instead facilitate the binding and
destabilization of microtubule ends. The best characterized
kinesin-13 protein is MCAK, which is described in detail in
Chapter 9.
Through the direct interaction via their light chains
or a connection mediated by various adaptor proteins kinesins
can bind to, i) organelles, such as the endoplasmic reticulum
(Dabora & Sheetz, 1988), lysosomes (Hollenbeck &
Swanson, 1990) and mitochondria (Nangaku et al., 1994), ii)
vesicles (Sheetz et al., 1986), iii) specific proteins (reviewed
in Wozniak et al., 2004) and iv) specific mRNAs (reviewed
in Tekotte & Davis, 2002). The critical question regarding
the regulation of motor-cargo binding has only recently
started to be unfolded when it was shown that the kinesin
KIF17 ability to interact with cargo was inhibited through
CaMKII phosphorylation (Guillaud et al., 2008).
Dynein comprises a small homogeneous family
compared to the kinesins and are either located in the
cytoplasm or in cilia/flagellas as a part of the axoneme.
Cytoplasmic dyneins are homodimers of heavy-chains and
are expressed in all eukaryotes, while the axonemal dyneins
are heterodimers or heterotrimers and are only expressed in
specialized cells containing cilia or flagella. Cytoplasmic
dyneins have been shown to interact directly or through
adaptor proteins with, i) organelles, such as the Golgi
1.
2.
ATP
3.
ADP
A
A B
-
4.
ATP ADP
ADP ATP
A
B
+ -
B
B
+ -
5.
+ -
A
i.e. the transition from polymerizing to depolymerizing
(reviewed in Howard & Hyman, 2007) and iii) tubulin
heterodimer binding proteins, which function by sequestering
tubulin from the polymerization process (reviewed in
Howard & Hyman, 2007). In this thesis I have focused on
functional analysis of two prominent microtubule
destabilizing proteins, MCAK and Op18, and a detailed
description on their structure and function will be given in
Chapter 9.
ATP
B A
+ -
+
Fig. 14. ATP-dependent unidirectional translocation of kinesin
along a microtubule
7. MICROTUBULE FUNCTION DURING THE
INTERPHASE AND MITOSIS OF THE CELL CYCLE
7.1 A brief introduction to the mammalian cell cycle and
its check points
The term cell cycle is used to describe the series of events
that culminates in cell division and the term interphase refers
to all stages of the cell cycle except mitosis (Fig. 15). During
the G1-phase the cell commits itself to enter the cell cycle
and increase in size. DNA replication together with centriole
duplication occurs during S-phase, followed by quality
control of the replicated DNA during the G2-phase (Fig. 15).
Cellular division compromises two main events, the
segregation of the duplicated genome, termed mitosis, and
the subsequent cytoplasmic division of the cell, termed
cytokinesis (Fig. 15).
G0
3.
M
G1
1.
2.
G2
Interphase
S
1. G1/S checkpoint
Sense: Surroundings and DNA status
Block: DNA replication
2. G2/M checkpoint
Sense: DNA status and cell size
Block: Mitotic entry
3. Spindle assembly checkpoint
Sense: Chromosome attachment
to the mitotic spindle
Block: Chromosome separation and
cytoplasmic division
Mitosis
Fig. 15. The cell cycle and its major checkpoints
Various surveillance systems, collectively termed
checkpoints, ensure the correct sequential order of cell cycle
phases. Check points also monitor that each cell cycle phase
is accurately completed before progression into the next
phase is allowed. The mammalian cell cycle contains three
major checkpoints (Fig. 15) that are in principle conserved
among all eukaryotes. Progression through the cell cycle is
regulated by the family of cyclin dependent kinases (CDKs).
Both the activity and substrate specificity of the
constitutively expressed CDK-proteins depend on specific
cyclin partners, which are proteins that oscillate during the
8
interior for membrane proximal stabilization cues, i.e. +TIP
proteins (Fig. 18).
cell cycle. The temporal pattern of cyclin expression and
degradation allows for the correct sequential order of the cell
cycle. It is notable from the outline of key molecular events
in Fig. 16 that the CDKs important for G1 and S-phases are
primarily involved in transcriptional regulation, while the
mitotic CDK1/cyclin B kinase is an executor of mitosis and
many structural proteins are direct or indirect targets.
1.
External
stimuli
Golgi
2.
Selective stabilization of microtubules that
randomly encounter membrane localized tip
stabilizing proteins, which have been
activated at the signaling site.
Expression induced
by mitogen signaling
S
Production of cyclin E
3.
Cyclin
E
CDK 2
Golgi
P Rb P
Cyclin
D
Cyclin
A
CDK 1
Cyclin
A
Production of S phase
components and cyclin A
Dynein dependent re-orientation of the
centrosome.
Firing of replication origins
4.
Golgi
CDK 2
P Rb P
P
P
Golgi
G1
CDK 4/6
G2
P
Prevention of re-replication
CDC6
P
Cyclin
CDK 1
B
M
P
Nuclear lamins
CDC42 is a member of the Rho-GTPase family and
has been shown to be essential for polarization of astrocytes
(Etienne-Manneville & Hall, 2001). After local activation at
the leading edge, CDC42 activates a signaling pathway that
de-repress the activity of the +TIP protein APC in the
proximity of the leading edge (Etienne-Manneville & Hall,
2003), which mediates local stabilization of microtubules
(general principle outlined in Fig. 18). These microtubules
are anchored to the plasma membrane through the interaction
between APC and DLG1 (Etienne-Manneville et al., 2005).
Stabilized microtubules at the leading edge are used for the
re-orientation of the centrosome towards the leading edge by
the dynein dependent pulling force (Etienne-Manneville &
Hall, 2001; Palazzo et al., 2001).
Migration of fibroblasts involves different
regulatory proteins but polarization is achieved according to
the same search and capture mechanism as in astrocytes. In
fibroblasts microtubules are stabilized at the leading edge by
members of the CLASP family of +TIP proteins, which are
locally activated by a phosphatidylionositol-3 kinase (PI-3
kinase) dependent pathway (Akhmanova et al., 2001). The
stabilized microtubules are thereafter anchored to the plasma
membrane through the interaction between CLASPs and
LL5beta or ELKS (Lansbergen et al., 2006).
T-lymphocyte recognition of antigens on virally
infected cells or specialized antigen presenting cells is
associated with centrosome re-orientation (Geiger et al.,
1982). In the case of a cytotoxic T-lymphocyte, the secretory
apparatus is thereby localized in proximity to the target cell.
Centrosomal reorientation in T-lymphocytes depends on Tcell antigen receptor mediated activation of the downstream
effectors CDC42 (Lowin-Kropf et al., 1998; Stowers et al.,
1995), ZAP-70 (Blanchard et al., 2002) and dynein (Combs
et al., 2006).
Fig. 16. The sequential activation of a family of cyclin-dependent
kinases drive cell cycle progression
7.2 Microtubule dependent molecular motors organize
the intracellular space
Interphase microtubules are organized by the centrosome in
most cells (see section 5.3 for exceptions), with the minus
ends anchored within the centrosomal matrix and the plus
ends extending out towards the cell periphery, resulting in a
microtubule array focused at the center of the cell. The major
function of the interphase microtubule system is to facilitate
intracellular organization, which to a large extent involves
kinesin and dynein mediated transport of different cargos
along microtubules (see section 6.3). By this mean,
microtubules organize cellular organelles and facilitate
vesicle transport, e.g. in the secretory- and endocytotic
pathways (Fig. 17).
+
1. Nucleus
2. Endoplamic reticulum
3. Golgi apparatus
4. Mitochondrion
5. Vesicle in the secretory pathway
6. Vesicle in the endocytotic pathway
1.
+
2.
+
3.
6.
Kinesin
+
5.
+
4.
Stable microtubules serve as rail tracks,
transporting membrane vesicles and actin
regulatory proteins to support directional cell
migration.
Fig. 18. Selective stabilization of interphase microtubules results in
cellular polarization
Chromatin condensation
and nuclear envelope
disassembly, i.e.
mitotic entry
Condensin
Chomosome separation and mitotic exit triggered by activation of APC/C
Dynein
In a non-polarized cell, microtubules
exhibit dynamic instability, i.e. they
constantly grow and shrink in a
random manor.
+
Fig. 17. Microtubule mediated organization of organelles during
interphase
Polarized intracellular transport of various cargos,
e.g. membrane vesicles or actin regulatory proteins, is
important during cellular migration. Cell polarization during
migration involves re-orientation of the centrosome, which is
accompanied by the Golgi apparatus, towards the leading
edge of the cell. The search-and-capture mechanism of
microtubules provides the cell with a system to probe its
7.3 Microtubules segregate the duplicated chromosomes
during mitosis
During mitosis, chromosomes duplicated during the S-phase
are segregated into two daughter cells. This is accomplished
by assembly of an elaborate microtubule structure, termed the
9
and attach to all kinetochores, which is the hallmark of the
prometaphase (Fig. 19 & 22; reviewed in Walczak & Heald,
2008).
mitotic spindle, that is responsible for organizing the sister
chromatid pairs onto the cellular equator and subsequently
segregate them to the opposite cell poles (Fig. 19; reviewed
in Walczak & Heald, 2008).
At the entry of mitosis, the interphase microtubule
system depolymerizes and the centrosomes duplicated during
the S-phase separate to initialize the formation of two
separate microtubule arrays. During the initial phase of
mitosis, termed prophase (Fig. 19), the two arrays are
separated by sliding of anti-parallel microtubules by
members of the kinesin-5 family (Fig. 20A&C; Kapitein et
al., 2005). The positioning of the division plane is thereafter
determined by interactions between specifically located
dyneins and the astral microtubules as outlined in Fig. 21
(reviewed in Dhonukshe et al., 2007).
Interphase
1. Astral microtubules are stabilized by tip
binding proteins at cell poles
2. Membrane anchored
dynein translocates
along astral microtubules
-
1. +
2.
3. Dynein mediated
pulling forces relocate
spindle poles to generate
the correct division plane
3.
Prophase
Telophase/
cytokinesis
Fig. 21. Establishment of correct division plane by astral
microtubules and dynein
Prometaphase
1 Microtubules restlessly search the cellular space...
Anaphase
2 ...and are stabilized at the
Metaphase
kinetochores of the chromosomes
AstralOverlapKinetochore MT
Fig. 19. Microtubule dependent processes during different stages of
mitosis
Prophase
A)
*
*
*
*
3 Finally both kinetochores capture
microtubules from opposite centrosomes
and the sister chromatid pair is
positioned at the cellular equator by the
polar ejection forces generated by
microtubules marked with an asterisk
Anaphase B
B)
Fig. 22. Search and capture of chromosomes by microtubules during
the assembly of the mitotic spindle
C)
Plus end
During prometaphase, kinetochores of sister
chromatid pairs will progressively become attached to
microtubules from the opposite spindle poles. Sister
chromatid pairs are thereafter positioned near the cellular
equator to form the metaphase plate (Fig. 19). This process is
primarily thought to be mediated by pushing forces on the
chromatids
by
non–kinetochore
attached
spindle
microtubules; a phenomenon termed polar ejection forces
(Fig. 22; Rieder et al., 1986). Since these forces are inversely
proportional to the distance to the spindle poles, sister
chromatid pairs will line up close to the cell equator, i.e. at an
equal distance to each spindle pole.
Prometaphase proceeds into metaphase at a stage in
which all sister chromatid pairs are aligned at the cellular
equator (Fig. 19). This is followed by anaphase, at which
sister chromatids segregate to opposite poles (Fig. 19).
Segregation requires dissociation of sister chromatids, which
is executed by proteases that are activated by the Anaphase
Promoting Complex (also known as the cyclosome).
Following dissociation, individual sister chromatids are
transported to respectively cell pole by two distinct
mechanisms, namely i) depolymerizing kinetochoremicrotubules pull sister chromatids toward the pole (Fig 19),
Bipolar kinesins
Plus end
Fig. 20. Bipolar kinesins mediate sliding of anti-parallel
microtubules during mitosis
A protein complex, termed the kinetochore,
assemble during prophase on distinct structures on the
chromosomes, termed centromeres. Each sister chromatid
contains only one centromere and consequently only have
one kinetochore. Several +TIP proteins localize to the
kinetochore and are thought to selectively stabilize
microtubules that randomly encounter the kinetochore during
prometaphase (Fig. 22; reviewed in Maiato et al., 2004).
At the entry to mitosis the microtubules become
more dynamic (see section 6.1), which facilitates probing of
the cytosol for sister chromatid pairs by the search-andcapture mechanism. By this stochastic trial and error
mechanism, microtubules have potential to rapidly localize
10
exposed, which is indeed a characteristic of microtubules
during mitosis (see Fig. 8A).
Defects in the spindle checkpoint increase the
probability for aneuploidy, i.e. abnormal chromosome
content, which is a characteristic of >90% of all human
tumors. It is therefore not surprising that several of the
components in the spindle checkpoint have been found to be
mutated in cancer (reviewed in Kops et al., 2005).
ii) sliding of anti-parallel overlap-microtubules pushes
spindle poles further apart (Fig. 20B&C).
The genetic cell division is completed in telophase
(Fig. 19), when most of the microtubules return to their
interphase configuration and the remnants of the overlapmicrotubules determine the position of the actin containing
contractile ring, which will mediate the final stage of cell
division, cytokinesis (reviewed in Barr & Gruneberg, 2007).
Labeled tubulin
7.4 The role of kinetochore microtubule treadmilling to
ensure bipolar kinetochore attachment
The spindle checkpoint is a control mechanism that ensures
that all sister chromatid pairs are aligned on the cellular
equator before initiation of anaphase. The high degree of
fidelity of this check point is explained by the fact that a
single non-attached kinetochore is sufficient to prevent
activation of the Anaphase Promoting Complex (reviewed in
Kops, 2008).
The spindle checkpoint is also responsive to errors
in the attachment of microtubules at kinetochores, e.g. if
microtubules from the same spindle pole are attached to both
kinetochores of the sister chromatid pair, i.e. syntelic
attachment (Fig. 23). Kinetochores distinguish between
correct and incorrect attachments by a sensing mechanism
that register the amount of tension applied over the
kinetochores. Thus, given that bipolar attachment is required
for tension, syntelic attachment will be sensed and the repair
involves induced depolymerization at the kinetochore
followed by a new round of search-and-capture (reviewed in
Tan et al., 2005).
Kinetochores with incorrect microtubule attachment
recruit and activate proteins that cause depolymerization of
the incorrectly attached microtubules. One of these
depolymerizing proteins is the kinesin-13 member MCAK
(Kline-Smith et al., 2004), which has been in focus during
my thesis work, and detailed account of the structure and
function of MCAK will be given in Chapter 9.
Kinetochores
under tension
Metaphase
-
+
Fig. 24. Treadmilling of microtubules visualized by labeled tubulin
8. THE IMPORTANCE OF CHROMOSOMAL
INSTABILITY FOR TUMOR PROGRESSION
8.1 Tumor progression requires genomic instability
Formation of human tumors is a multi-step process (Fig. 25),
termed tumor progression, which requires alteration at
several distinct levels of cell function.
1.- Uncontrolled cellular growth and avoidance of cellular death
X X
X
X X
X
X
X
X
X
X X
Anaphase
2.- Formation of new
blood vessels
3.- Metastasis
STOP
Aneuploidy
Kinetochores
without tension
Fig. 25. Key events during the development of a metastasizing
tumor
Genomic instability
In solid tumors, alterations at six levels of cell
function appear essential for malignant growth (Fig. 26), and
these functional alterations have been termed the “Hallmarks
of cancer” by Hanahan and Weinberg (Hanahan & Weinberg,
2000). These alterations are the result of accumulation and
selection of random mutations in genes operationally termed
oncogenes and tumor suppressor genes. Mutations in
unrelated proteins may result in the same distinct change of
cellular physiology. While certain mutations are frequent
among various diagnostic groups of tumors, it has still been
established during recent years that each tumor is unique with
respect to the whole set of accumulated mutations (Fig. 27;
Wood et al., 2007).
Fig. 23. Tension dependent detection of misaligned chromosomes
during prometaphase
The source of tension is the treadmilling behavior of
kinetochore microtubules. Treadmilling implies simultaneous
association of subunits at the plus end and dissociation of
subunits at the minus end, which result in an intrinsic flux of
subunits without altering the length of the microtubule (Fig.
24; reviewed in Rogers et al., 2005). A prerequisite for
treadmilling is that the minus ends at the centrosome are
11
also microtubule-regulatory proteins that are normally not
involved in spindle-formation may acquire alterations in
expression levels or function that serve to promote
chromosomal instability in tumors (See section 9.8).
Mutation
1 Self-sufficiency in proliferative signals
1.
- Defective DNA repair
2 Insensitivity to anti-growth signals
2.
Normal cell
3.
3 Evading cell death
Tumor cell
4.
4 Limitless replicative potential
5 Sustained blood vessel formation
5.
- Errors during chromosome segregation
6 Metastasis capability
6.
Fig. 26. Essential traits that are acquired during tumor progression
of solid tumors
Fig. 28. Two distinct mechanisms for genomic instability in tumor
cells
2
X
X
5
3
6
X
4
5
X
1
4
X
3
X
2
X
1
9. MAJOR REGULATORS OF MICROTUBULE
POLYMERIZATION
9.1 MCAK is a conserved catastrophe-promoting protein
The conserved MCAK protein belongs to a unique group of
kinesins, the kinesin-13 family (previously known as KIN I
for their internal motor domain), which depolymerize
microtubules rather than translocate along them (reviewed in
Kinoshita et al., 2006). Human MCAK is preferentially
expressed in proliferative cells (Kim et al., 1997) and has also
been shown to be overexpressed in various solid tumors
(Nakamura et al., 2007; Shimo et al., 2008). The pioneering
study on the microtubule regulatory activity of MCAK was
done using the Xenopus ortholog, originally termed XKCM1
(Walczak et al., 1996).
The inactivation of MCAK in mitotic Xenopus egg
extracts was shown to be accompanied by longer, less
dynamic microtubules, and quantification of dynamic
parameters revealed a 4-fold decrease in the catastrophe
frequency (Walczak et al., 1996). Studies on purified MCAK
revealed an interaction with both minus and plus ends of
microtubules (Desai et al., 1999) and it has been proposed
that ATP-independent diffusion along the sides of
microtubules facilitates rapid targeting of MCAK to the
microtubule ends (Helenius et al., 2006). The binding of
MCAK to the microtubule ends promotes the curling of
protofilaments, thereby structurally destabilizing the GTPcap, thus promoting a catastrophe, i.e. the transition from
polymerization to depolymerization (Fig. 29; Desai et al.,
1999). For recycling of MCAK, the energy from ATP
hydrolysis is utilized to release the protein from the curling
protofilament (Desai et al., 1999).
Cancer #1
6
Cancer #2
Fig. 27. Mutations in many types of proteins are required for tumor
progression
8.2 Genomic instabilities of tumors depend on two
distinct mechanisms
Given random acquisition of oncogenic mutations and that
the mutation frequency in a normal cell is very low, cancer
development is statistically improbable. However, mutations
in genes encoding proteins involved in different types of
surveillance and repair systems, which normally protect
against genomic instability, are known to cause an increase in
mutational frequency in tumors (reviewed in Cahill et al.,
1999). Thus, inactivating mutations in genes encoding for
such gene-products can be considered as a prerequisite for
tumor progression.
The most common causes for increased mutational
frequency in tumor cells are mutations that either alter the
function of proteins involved in DNA repair or proteins
involved at some levels of chromosome segregation during
mitosis (Fig. 28). The later mechanism behind genomic
instability, termed chromosomal instability, is thought to be
the major factor that causes genomic instability in human
tumors (reviewed in Kops et al., 2005).
Chromosomal instability seems primarily to be a
consequence of defective microtubule attachments to
kinetochores that evade detection by the spindle checkpoint.
Several +TIP proteins, e.g. APC, which has been implicated
in stabilizing microtubule attachments to kinetochores have
been found mutated in tumors, which seems likely to promote
chromosomal instability (Fodde et al., 2001; Kaplan et al.,
2001). In this thesis, by analyzing the phenotype of a tumor
specific point mutation in the Op18 gene, I will argue that
9.2 The function of MCAK during the cell cycle
Studies in Xenopus egg extracts suggest that the activity of
MCAK is low during interphase, which has been proposed to
be an effect of the dominant counteractive activity of the
microtubule-associated protein XMAP215. Moreover, the
predominant microtubule-destabilizing activity of MCAK
during mitosis has been explained by phosphorylation–
inactivation of XMAP215 by mitotic kinases (Tournebize et
al., 2000). The predominant and counteractive activities of
12
Beside its association to microtubules, TOGp, the human
homologue of XMAP215, has been shown to bind tubulin
heterodimers in vitro (Spittle et al., 2000) and XMAP215 coimmunoprecipitates with tubulin heterodimers in Xenopus
egg extracts (Niethammer et al., 2007).
XMAP215 and MCAK in frog egg extracts have formed the
current paradigm on how the microtubule-system is regulated
during interphase and mitosis. This paradigm is also
presented in the most influential of current text books (e.g.,
Alberts et al., Molecular biology of the cell 5th edition, page
1080 and Lewin et al., Cells, page 341). However, our own
studies in human cell-lines depleted for MCAK and the
human homologue of XMAP215, TOGp, fail to reproduce
the importance of these counteractive activities during both
interphase and mitosis (Paper I). As will be extended on in
the “Result and Discussion” section, I believe that our results
from human cells refutes that MCAK represents a major
microtubule-destabilizing activity.
TOG-domain
TOGp
1972 a.a.
XMAP215
2065
Msps
2050
2015
DdCP224
Mor1
1979
Zyg-9
MCAK
1415
Dis1
882
Alp14
809
Stu2p
Fig. 29. MCAK destabilizes both microtubule ends
During mitosis, MCAK concentrates to kinetochores
and spindle poles (Wordeman & Mitchison, 1995).
Inactivation of MCAK, which was achieved by either
microinjection of a dominant negative mutant (Kline-Smith
et al., 2004; Maney et al., 1998) or RNA interference
mediated protein knockdown (Paper I; Cassimeris &
Morabito, 2004; Ganem et al., 2005) has only small effects
on mitotic spindle assembly. Significantly, however, an
increase of lagging chromosomes during both prometaphase
and anaphase was observed (Kline-Smith et al., 2004; Maney
et al., 1998; Andrews et al., 2004; Lan et al., 2004; Walczak
et al., 2002). The current well established model implies that
MCAK is specifically recruited to kinetochores with
incorrect microtubule attachments, and subsequently
destabilize these faulty attachments, see model in Paper II
(Fig. 7), (reviewed in Walczak & Heald, 2008). The
regulation of recruitment and activation of MCAK during this
process is complex and is shown to involve the Aurora B
kinase at multiple levels (reviewed in Walczak & Heald,
2008).
888
Fig. 30. The XMAP215/TOGP family of microtubule regulators.
In vitro studies using purified tubulin heterodimers
have revealed that XMAP215 causes a 7-fold increase in the
polymerization rate specifically at the plus end (Gard &
Kirschner, 1987). Two distinct mechanisms and mutually
exclusive models have been proposed to explain the
accelerated polymerization rate, namely i) XMAP215 serves
as a template for tubulin oligomers and thereby facilitates
delivery of preassembled protofilaments to the plus-end of
microtubules (Fig. 31A; Kerssemakers et al., 2006; Slep &
Vale, 2007) ii) XMAP215 binds processively to the plus end
of the microtubule and facilitates repeated rounds of addition
of tubulin heterodimers into polymerizing protofilaments
(Brouhard et al., 2008), possibly through the recruitment of
tubulin heterodimers by high affinity binding (Fig. 31B).
9.3 The evolutionary conserved XMAP215/TOGp protein
family
Members of the XMAP215/TOGp family are +Tip-proteins
(see section 6.2) and are found in all eukaryotes (Fig. 30). In
most species XMAP215/TOGp proteins are represented as a
single essential gene. The most conserved feature of the
XMAP215/TOGp family proteins is amino-terminal TOG
domains, which mediate binding to tubulin (Al-Bassam et al.,
2007). XMAP215/TOGp has been implicated in many levels
of microtubule regulation, potentially by multiple distinct
mechanisms (reviewed in Howard & Hyman, 2007). Electron
microscopy studies have revealed an elongated shape for both
Xenopus XMAP215 (Cassimeris et al., 2001) and the S.
cerevisiae family member Stu2p (Al-Bassam et al., 2006).
The crystal structure of a TOG-domain of the C. elegans
family member Zyg-9 reveals a flat and paddle-shaped
structure (Al-Bassam et al., 2007). The number of TOG
domains in the protein varies between two and five,
depending on species (reviewed in Ohkura et al., 2001).
A)
B)
Pre-assembled
protofilament
High affinity
surface
Fig. 31. Possible mechanisms for the XMAP215 dependent increase
in polymerization rate at microtubule plus ends
Decreased amounts of XMAP215 in Xenopus egg
extracts (Wilde & Zheng, 1999; Tournebize et al., 2000) has
been shown to result in shorter microtubules, which was also
observed when the protein was inhibited in intact Xenopus
oocytes (Becker et al., 2003). Given the high degree of
conservation among the XMAP215/TOGp family members
(reviewed in Kinoshita et al., 2002), it has been assumed that
the prominent microtubule stabilizing activity of XMAP215
observed in Xenopus model systems is general among
eukaryotes. This concept was supported by the pronounced
decrease of microtubule stability caused by mutations of the
Arabidopsis homologue Mor1 (Whittington et al., 2001), the
C. elegans homoluge Zyg-9 (Srayko et al., 2005), and the
Drosophila homologue Msps (Moon & Hazelrigg, 2004).
13
However, depletion of the human homologue of XMAP215,
TOGp, does not reveal detectable effects on the levels or
stability of microtubules (Gergely et al., 2003; Paper I;
Cassimeris & Morabito, 2004).
By studies using purified components, it have also
been shown that XMAP215 (Shirasu-Hiza et al., 2003) and
Stu2p (van Breugel et al., 2003) have the potential to
destabilize microtubules. This paradoxical finding might be
explained by the documented ability of XMAP215/TOGp
family members to bind to soluble tubulin heterodimers and
possibly sequester them from microtubule polymerization
under certain conditions. In intact cells, the microtubule
destabilization activity has also been documented for
overexpressed TOGp (Paper I). Interestingly, this activity
was shown to be dependent on the microtubule destabilizer
MCAK, but the mechanism behind this phenomenon remains
unclear. Thus, current literature on the function of
XMAP215/TOGp family members in various species is
clearly difficult to boil down into a simple coherent picture.
This is in part due to that these family members found in
evolutionary divergent species are quite different with respect
to size and numbers of the microtubule-binding TOG
domains, which makes it unclear whether all of these proteins
are true orthologs or just paralogs.
9.5 Counteractive activities between MCAK and
XMAP215 family members
A series of classical experiments in Xenopus egg extracts
have had major influence over the current perception on the
regulation of microtubule-polymer mass and dynamics
through the cell cycle (reviewed in Andersen & Wittmann,
2002; Heald, 2000). In these experiments, XMAP215 and
MCAK were shown to be the major counteractive
microtubule regulatory activities. Thus, based on antibody
mediated depletion of egg extracts, their relative activities
appeared to largely determine the dynamic properties and
polymer-content of the microtubule system (Figure 32;
Tournebize et al., 1997).
Interphase extract
Mitotic extract
Control
1
9
XMAP215 inhibited
7
21
2
3
(only 60% depletion)
XMAP215 and
MCAK inhibited
9.4 Function of XMAP215/TOGp family proteins during
mitosis
XMAP215/TOGp family proteins concentrate to spindle
poles and spindle microtubules during mitosis (reviewed in
Gard et al., 2004). This localization of XMAP215 family
members has been shown to be dependent on TACC proteins
(reviewed in Gergely, 2002) and possibly regulated by the
Aurora A kinase (Barros et al., 2005; Giet et al., 2002).
Studies on cells from various species lacking the
XMAP215/TOG protein have revealed a prominent role in
the organization of the mitotic spindle. Disorganized spindle
poles and aberrant spindle morphology have been recorded in
human cell lines (Gergely et al., 2003), Drosophila (Cullen et
al., 1999) and C. elegans (Matthews et al., 1998). In human
cells, depletion of TOGp results in multipolar spindles
(Gergely et al., 2003), which is a phenotype that depends on
the microtubule destabilizing activity of MCAK (Paper I;
Cassimeris & Morabito, 2004). To explain the TOGp
depletion phenotype in human cells, we have proposed a
model in which TOGp is essential for inhibiting MCAK at
the spindle poles, thereby ensuring the bipolarity of the
spindle. We have subsequently extended our model to
include the CaMKIIγ, which we have shown has an equal
essential role in suppressing the activity of MCAK at the
spindle poles (Paper II).
Studies on XMAP215/TOGp family members in the
two diverse yeast species S. pombe (Garcia et al., 2001) and
S. cerevisiae (Kosco et al., 2001) have revealed a prominent
kinetochore localization, which has not been observed in
animal cells. Loss-of-function mutational studies in these two
yeast model systems showed impaired microtubule
attachment to kinetochores and failure to form bipolar
spindles. These studies thereby reveal an essential role at the
opposite end of microtubules compared to the human
XMAP215/TOGp family member TOGp, which indicates
major differences in the function of these proteins in diverse
species.
Relative catastrophe frequency
Fig. 32. Counteractive activities of MCAK and XMAP215 in
Xenopus egg extracts (adopted from Heald, 2000)
The proposed model implies that the dominant microtubule
stabilizing activity of XMAP215 generates the long and
modestly dynamic microtubules seen during interphase. At
the entry into mitosis, inactivation of XMAP215 is thought to
relieve inhibition of MCAK, which would explain how the
catastrophe promoting activity of MCAK mediates formation
of short and highly dynamic microtubules during mitosis, but
not during interphase (Fig. 32 and 33). Decreased activity of
XMAP215 during mitosis is thought to be mediated by
activation of Cdk1/Cyclin B, since XMAP215 is an in vitro
substrate for this kinase (Vasquez et al., 1999).
The polymerization rate of purified tubulin is slower
than observed in intact cells and the frequency of
catastrophes is low as depicted in Fig. 12. However, if
purified XMAP215 and MCAK are added to purified tubulin,
both the microtubule growth rate and catastrophe frequency
will increase and under certain conditions be virtually
identical to what is observed in intact cells (see Fig. 12)
(Kinoshita et al., 2001). Taken together, these data were
conceived as compelling evidence for a model in which
microtubule dynamics in cells are primarily determined by
the combined actions of XMAP215 and MCAK (Kinoshita et
al., 2002). According to this model on collaborating activities
of XMAP215 and MCAK, the counteractive activities earlier
described in Xenopus frog egg extracts can be perceived as
primarily important to determine the average length of
microtubules. Subsequent studies in human cells (Paper I;
Cassimeris & Morabito, 2004) have provided a very different
picture of both the functions and interplay of
XMAP215/TOGp and MCAK. The implications of these
findings will be discussed in the “Results and Discussion”
section (see section 12).
14
(Sellin et al., 2008) to regulate the level of tubulin
heterodimers, which in the Drosophila study was shown to be
an essential role.
CDK1/Cyclin B
MCAK
TOGp
A)
B)
N
II
I
α
β
C
Fig. 34. Microtubule destabilization by Op18
Fig. 33. Proposed model of counteractive microtubule-regulating
activities of MCAK and XMAP215/TOGp during the cell cycle
9.7 Phosphorylation-inactivation of Op18 during
interphase and mitosis
Op18 is phosphorylated at four Ser residues (Ser-16, -25, -38,
and -63) by multiple signal transducing kinases and mitotic
kinases, which results in a partial or complete inactivation
(Figure 35A; reviewed in Cassimeris, 2002). Expression of
kinase-site deficient mutants of Op18, combined with
detailed analysis of the phosphorylation state of endogenous
Op18,
indicate
that
spindle
formation
requires
phosphorylation-inactivation of Op18 at the onset of mitosis
(Figure 35B; Larsson et al., 1997; Larsson et al., 1995;
Marklund et al., 1996)
9.6 Op18 - a developmentally regulated microtubuledestabilizing protein
The ubiquitously expressed cytosolic protein Op18, also
known as Stathmin, was initially isolated due to its
phosphorylation in the response to external signals and
upregulation in various malignancies (reviewed in Sobel,
1991). Op18 is developmentally regulated with expression
levels up to 50 fold higher in neonatal tissue compared with
the corresponding adult tissue (Koppel et al., 1990). Op18 is
highly conserved among vertebrates and has also been
identified in Drosophila (Ozon et al., 2002).
Op18 was identified as a microtubule destabilizer in
Xenopus egg extracts (Belmont & Mitchison, 1996) and in
intact human cells (Marklund et al., 1996). Studies with
purified components demonstrated that Op18 can specifically
stimulate microtubule catastrophes (Fig. 34A (I); Belmont &
Mitchison, 1996) or sequester tubulin heterodimers (Fig. 34A
(II); Curmi et al., 1997). These discrepancies were resolved
when it was shown that Op18, indeed, excesses both these
activities, which in vitro are dependent on buffer conditions
and can furthermore be attributed to distinct regions of the
protein (Howell et al., 1999b). The Op18–tubulin complex
consists of two tubulin heterodimers arranged head-to-tail
(Steinmetz et al., 2000) with each of the two tandem helical
repeats of Op18 binding alongside one heterodimer (Figure
34B; Gigant et al., 2000).
Consistent with its microtubule regulatory activity,
Op18 has been implicated in cell migration during
Drosophila development (Borghese et al., 2006; Ozon et al.,
2002) and invasive behavior and metastasis of solid tumor
cells (Baldassarre et al., 2005; Belletti et al., 2008).
Surprisingly, however, mice with the Op18 gene inactivated
by gene targeting develop normally (Schubart et al., 1996),
which contrasts to the essential role of Op18 during
Drosophila development (Borghese, et al., 2006).
Nevertheless, later studies revealed subtle neuronal
phenotypes such as impaired regulation of innate and learned
fear (Shumyatsky et al., 2005) and age-dependent
axonopathy of both the central and peripheral nervous
systems (Liedtke et al., 2002).
The physiological relevance of Op18 was initially
established by the finding that antibody mediated inactivation
of Op18 in newt lung cells reduced catastrophe promotion
and increased microtubule polymer content (Howell et al.,
1999a). In human cells, depletion of Op18 by RNA
interference is associated with a substantial shift of the
tubulin pool towards the polymeric state (Paper III and IV).
Furthermore, the Op18 protein has been shown both in
Drosophila (Fletcher & Rorth, 2007) and human cell lines
A)
CaMKIV MAPK
PKA
Ser-16 Ser-25
Ser-38
PKX
Signal transduction
Ser-63
CDK
PKX
Op18 (149 a.a.)
Cell cycle regulation
B)
Interphase
(active)
Op18
P P P P
Mitosis
Op18 (inactive!?)
Fig. 35. Phosphorylation dependent regulation of the
microtubule-destabilizing activity of Op18
Despite a consensus that Op18 is phosphorylationinactivated in human cell lines during mitosis, it appears that
Op18 is sub-stoichiometrically phosphorylated in mitotic
Xenopus egg extracts. Based on this model system, a model
on
phosphorylation
gradients
around
condensed
chromosomes was proposed, which implied that local
phosphorylation-inactivation of Op18 occurs in the vicinity
of condensed chromosomes (Andersen et al., 1997;
Tournebize et al., 1997). However, the Op18 mouse
knockouts are viable and fertile and there is no indication of
chromosomal instability, as evidenced by unaltered incidence
of tumors (Schubart et al., 1996). Finally, my own studies in
human cell lines extensively depleted of Op18 by stable
expression of interfering RNA do not reveal any alteration in
cell growth or distinguishable spindle defects (Paper III).
The basal level of Op18 phosphorylation is low in
interphase cells and increases in response to stimulation of
various signal-transducing receptor systems. Initial
experiments in our research group demonstrated that
phosphorylation of ectopically expressed Op18 on specific
sites results in inactivation (Melander Gradin et al., 1997;
Gradin et al., 1998). By co-expression of constitutively active
protein kinases involved in signal transduction events, it was
shown that site-specific Op18 phosphorylation may provide a
15
responsible for generating local accumulation of tubulin
heterodimers as previously proposed by us (Holmfeldt et al.,
2003a).
direct mechanism to stabilize the microtubule system in
response to external signals but the physiological significance
was unclear. In the present thesis, this was addressed by a
gene-replacement strategy combined with a physiologically
relevant signaling event, as represented by T cell antigen
receptor stimulation. This study revealed that cell surface
receptor stimulation and activation of cognate kinase systems
result in a dramatic microtubule stabilization that is strictly
dependent on site-specific phosphorylation-inactivation of
Op18 (Paper IV).
Op18
P P P
SCG10
P P P
SCLIP
P P P
RB3
P
Ubiquitous
Neuronal
P
Palmitylated membrane
localization domain
9.8 Dysregulation of Op18 in human malignancies
Op18 has frequently been reported as overexpressed in
various malignancies (reviewed in Mistry & Atweh, 2002). In
prostatic adenocarcinomas it has been reported that low Op18
expression correlates with higher levels of differentiation of
the tumor cells and increased patient survival (Friedrich et
al., 1995). Recently, a somatic missense mutation in the
Op18 gene was isolated in a human tumor (Misek et al.,
2002). The mutation results in substitution of glutamine (Q)
to glutamic acid (E) at amino acid position 18 and cells
expressing the Q18ÆE mutant form of Op18 were also
shown to form tumors when injected in immunodeficient
mice (Misek et al., 2002).
From previous studies on leukemia and lymphomas
it is clear that Op18 expression levels are uncoupled from
cellular proliferation (Brattsand et al., 1993; Nylander et al.,
1995) and two recent studies have implicated Op18 mediated
regulation of microtubules as important for tumor invasion
and metastasis (Baldassarre et al., 2005; Belletti et al., 2008).
Finally, we have reported that excessive Op18 activity during
mitosis may serve to promote chromosomal instability (Paper
III), a hallmark of most tumors, and the significance of our
findings will be discussed in the “Results and Discussion”
(see section 15).
Fig. 36. The Op18/stathmin family
9.10 Microtubule stabilization by the ubiquitous
expressed MAP4
Many diverse proteins have been found to associate with
microtubules and in most cases the significance of the
observed association is unclear. However, the classical
microtubule-associated proteins (MAPs), MAP2, MAP4 and
tau, have common features in their organization in distinct
functional domains and are evolutionary related. MAPs bind
via a conserved region to the outer wall of microtubules and
stabilize them (reviewed in Mandelkow & Mandelkow,
1995). Only MAP4 is ubiquitously expressed, while the
others are abundant proteins in neuronal tissues. MAP4
appears conserved among mammals and Xenopus expresses a
protein with similar overall structure but with weak sequence
homology (~30% identity) (reviewed in Andersen, 2000).
The MAP4 primary-transcript can be spliced into
three different isoforms, which differ by encoding tree, four
or five 18 amino-acids imperfect repeats in the microtubule
binding domain of the protein (Chapin et al., 1995). All three
isoforms are large proteins with a molecular mass exceeding
200 kDa, which all share the potential to bind to
microtubules.
MAP4 can promote polymerization of purified
tubulin (Bulinski & Borisy, 1980) and overexpressed MAP4
stabilizes microtubules in intact cells (Holmfeldt et al., 2002;
Nguyen et al., 1997). Studies on the dynamic behavior of
microtubules assembled from purified tubulin documented
that MAP4 have the potential to increase the rescue, i.e. the
transition from depolymerization to polymerization,
frequency without significantly altering the other dynamic
parameters (Fig. 37; Ookata et al., 1995). The microtubule
stabilizing activity of MAP4 has been shown to counteract
the destabilizing activity of katanin in Xenopus egg extracts
(McNally et al., 2002), MCAK and the catastrophepromoting activity of Op18 in intact human cells (Holmfeldt
et al., 2002). The physiological significance of MAP4 for the
stability of microtubules is unclear, however, since antibody
depletion of MAP4 in human fibroblasts and monkey
epithelial cells failed to generate any observable effect on the
microtubule system through the cell cycle (Wang et al.,
1996). Nevertheless, depletion studies in human suspension
growing cell model systems suggest that MAP4 and Op18 are
the major counteractive microtubule regulating activities in
human cells (Paper IV), and the significance of these findings
will be discussed in the “Results and Discussion” (see section
13).
9.9 Relations between Op18 and the other Op18/
Stathmin family members
Op18 is the founding member of a small family of conserved
proteins, in which Op18 is ubiquitously expressed, while the
other members, RB3, SCG10 and SCLIP, are expressed
almost exclusively in the nervous system (Fig. 36; reviewed
in Curmi et al., 1999). The neural-specific family members
are expressed about 100-fold less compared to Op18 in brain
cells (Bieche et al., 2003). In contrast to the cytosolic Op18
protein, all other members of the Op18/Stathmin family have
a membrane targeting region, which mediates binding to
intracellular membranes and concentrates preferably at the
Golgi apparatus and to vesicle-like structures along neurites
and at growth cones (Gavet et al., 2002). The tubulin binding
affinity is much higher for the neural-specific family
members compared to the Op18 protein, shown both in vitro
(Charbaut et al., 2001) and in intact cells (Holmfeldt et al.,
2003a). Tantalizing as it might seem, it is unlikely that the
neuronal-specific family members influence the microtubule
system by the same principle mechanism as the abundant and
cytosolic Op18 protein. Given their low abundance, the
neuronal-specific family members can only be expected to
bind such small amounts of tubulin heterodimers that the
effect on the tubulin monomer-polymer partitioning of the
cell would be negligible. Considering their subcellular
localization, it is instead possible to envision them as proteins
16
MAP4
MAP4
MAP4
MAP4
MAP4
MAP4
Fig. 37. Potential mechanism for MAP4 mediated rescue promotion
9.11 Regulation of MAP4 during interphase and mitosis
Members of the PAR1/MARK family of kinases (reviewed in
Hurov & Piwnica-Worms, 2007) phosphorylate conserved
KXGS motifs that are present in all three classical
microtubule associated proteins, which results in their
detachment from microtubules (Fig. 38; reviewed in Drewes
et al., 1998). MARK kinases phosphorylate MAP4 on Thr802, Ser-914 and Ser-1046 (Fig. 38; Illenberger et al., 1996).
Studies on the cellular effects of MARK mediated
phosphorylation have been centered on neurons, primarily
explained
by
the
Alzheimer´s
disease
specific
phosphorylation on tau at Ser-262, and its significance in
non-neuronal cells have only recently been addressed. In
human cells we have demonstrated that MARK2
phosphorylation-inactivation of MAP4 has a profound effect
on the tubulin partitioning status in cells (Paper IV).
Negative regulation of MAP4 has also been shown
to be mediated by members of the septin family of proteins
(Fig. 38; Kremer et al., 2005). Septins are conserved proteins
found among all eukaryotes except plants and have been
described to have functions during cell division, cytoskeletal
organization and membrane-remodeling events (reviewed in
Weirich et al., 2008). In mammals, at least 12 distinct septins
with multiple splice variants have been identified. A
heterotrimer of Septins 2, 6 and 7 was shown to bind MAP4
and thereby inhibit MAP4 association to microtubules
(Kremer et al., 2005).
Interphase
Mitosis
PAR1/MARK family kinases
CDK1
MAP4
Septins
Septins
Fig. 38. Proposed post-translational regulation of MAP4 during the
cell cycle
Phosphorylation of MAP4 by CDK1/cyclin B on
Ser-696 and Ser-787, decreases its affinity for microtubules
(Fig. 38; Ookata et al., 1997). The significance of this mitotic
phosphorylation has been investigated in Xenopus cells by
over-expressing a kinase-site deficient mutant of MAP4,
which was shown to interfere with chromosome movement
during anaphase (Shiina & Tsukita, 1999). Depletion of
septins in human cells generates a similar phenotype, which
is suppressed by the by co-depletion of MAP4 (Kremer et al.,
2005). Together these data suggest the existence of two
different pathways that are import for inhibition of MAP4
during mitosis, which based on the study of MAP4-septin
interactions appear to be required for proper spindle function.
Consistent with these recent reports, it was earlier shown that
inhibition of MAP4 by injection of inactivating antibodies is
not associated with any detectable effects on the mitotic
spindle (Wang et al., 1996).
17
permitted gene-product replacement with finely tuned
expression levels, which required alterations of codons to
prevent targeting by the co-expressed shRNA.
We have dedicated much time to optimize design of
shRNAs in a shuttle vector context, and during initial
experiments I scored with excitement many interesting
phenotypes suggesting a cell cycle block during G1.
However, it turned out in all cases that the observed growth
arrest did not correlate with gene-product depletion, but
rather that all shRNA sequences caused more or less a G1arrest and that some sequences for obscure reasons were
much more potent in this respect than others. I therefore
dedicated a lot of efforts to screen shRNA sequences and to
adjust shRNA expression levels to achieve optimal depletion
with minimal “non-sequence” specific G1-arrest. It seems
likely that the observed G1-arrest is a consequence of
inhibition of protein synthesis by, e.g., activation of double
stranded RNA activated protein kinase (Sledz et al., 2003). It
is notable that these artificial effects of both small interfering
RNA and shRNA appear to be generally ignored in the
community. Thus, there are several published examples of a
claimed specific G1-arrest in response to RNA interference
that I believe are artifacts.
During optimization of this dual system for
inducible gene-product replacement, I included many
microtubule-regulatory proteins with “known” loss-offunction phenotypes. With the ambition to identify the major
regulators of the microtubule-system during interphase and
mitosis, it also became obvious to include the most
intensively studied of all regulators, namely MCAK and
TOGp in the initial optimization of the system.
RESULTS AND DISCUSSION
10. AIMS:
-
To elucidate which microtubule-regulatory
proteins are the major determinants of tubulin
subunit partitioning
-
To explore the implications of site-specific Op18
phosphorylation-inactivation as a mechanism to
stabilize the interphase microtubule-system in
response to external signals
-
To search for mechanisms behind selection of
Op18 overexpressing clones during tumor
progression
11. PROLOGUE
During my early period as a Ph.D. student, I was involved in
mechanistic studies concerning Op18 mediated microtubule
destabilization. At that time, two different activities of Op18,
tubulin sequestering and microtubule end-specific
catastrophe promotion, were suggested to either individually
or in combination contribute to microtubule destabilization.
However, it was notable that the physiological function of
Op18 was largely unknown at that time and there were
disparate ideas concerning an important role during either
interphase or mitosis (see section 9.6), or alternatively, that
Op18 may not be important at all since the Op18 deficient
mouse strain developed normally and only subtle
neurological defects had been reported.
Based on these disparate views on the relevance of
Op18 and which proteins were the major regulators of the
microtubule system during interphase and mitosis, I
refocused to establish a system that allowed gene-product
depletion and exchange. My aim was a system that allowed
counter-selection to provide stable and homogeneous cell
populations that could be monitored for phenotypes for
extended periods. In addition, it seemed also important to
combine gene-product depletion with inducible gene-product
exchange, to allow temporal studies of reversal of a loss-offunction phenotype. Inducible gene-product exchange was
also essential since kinase-target site deficient mutants of
Op18 were known to block cells during mitosis.
This was at the early time of RNA-interference in
mammalian cells, which implied a revolution within the field
of molecular cell biology. Inspired by the initial report on
expressed short hair-pin (sh)RNA (Brummelkamp et al.,
2002), driven by RNA polymerase III promoters, we
established this system in our EBV-based shuttle vectors
(Fig. 39A). Due to the stringent replication control of the
EBV-based vectors we could simultaneously express up to
four different derivatives in cells, thereby generating cells
depleted for multiple gene-products. We had previously used
EBV-based vectors for inducible expression of cDNAs under
the control of the hMTIIa promoter and developed protocols
for optimized induced expression and for efficient
suppression of this promoter (Fig. 39A). The present system
of co-transfected EBV-based shRNA vectors and pMEP
expression proved indeed very versatile for semi-stable
depletion of multiple gene-products combined with inducible
expression as depicted in Fig. 39B. This system also
A)
B)
Inducible
promoter
cDNA X
Resistance
EBV Ori
Constitutive
promoter
shRNA Y
Resistance
EBV Ori
Fig. 39. EBV-based shuttle vector system used for manipulation of
multiple gene-products in leukemic cells
12. THE MCAK AND XMAP215/TOGp PROTEINS
ARE NOT IMPORTANT FOR THE CONTROL OF
TUBULIN SUBUNIT PARTITIONING BUT HAVE
ESSENTIAL INTERCONNECTED FUNCTIONS
DURING SPINDLE ASSEMBLY
12.1 Background
Previous studies in Xenopus egg extracts had suggested that
MCAK and XMAP215/TOGp exerts predominant
counteractive activities on the microtubule system throughout
the cell cycle (see section 9.5). It therefore felt important to
document the role of these regulators in intact human cells
and thereby allow a comparison with the regulatory
importance of Op18 and other microtubule regulators.
However, although we found clear-cut evidence for MCAK
and XMAP215/TOGp interplay in human cells, the
functional importance of this interplay appeared completely
different as compared with Xenopus egg extracts. Most
importantly, these two regulators do not have any significant
18
microtubule-content, it is clear that depletion of
XMAP215/TOGp has completely different consequences in
human somatic cell lines and Xenopus egg extract model
systems. I therefore conclude that TOGp does not have a
general role to stabilize microtubule polymers in human cells,
but instead performs a localized counteraction of MCAK at
microtubule minus ends during mitosis (Fig. 40A-B).
role in the regulation of the interphase microtubule systems.
Thus, our initial experiments to understand the significance
of the claimed counteractive activities of MCAK and
XMAP215/TOGp expanded and resulted in the discoveries
presented in Papers I and II. In this thesis I will take the
opportunity to focus on aspects that are not already discussed
in these reports.
Mitosis
Physiological
levels
Interphase
A)
B)
MCAK
TOGp
Overexpressed
12.2 XMAP215/TOGp is not essential to enhance the
polymerization rate in human somatic cells
As outlined in the introduction section, an elegant series of
recent structural and functional characterization has
established XMAP215/TOGp as a prototype for a protein that
has potential to facilitate increased rates of both
polymerization and depolymerization of microtubules. It is
notable that a modest 60% depletion in Xenopus egg extracts
significantly reduced the polymerization rate at least in
interphase cells. It seems reasonable to assume that the high
rate of polymerization is required for the complex process of
spindle formation. If this assumption is correct, it follows that
the TOGp depletion phenotypes in mammalian cells indicate
that this function of TOGp is redundant (note that codepletion of TOGp and MCAK allows bipolar spindle
formation (see Paper I, Fig. 2), which implies that other as
yet unidentified proteins may serve to facilitate the high rates
of polymerization/depolymerization in TOGp/MCAK codepleted cells. The alternative interpretation, namely that
>95% depletion is not sufficient to abolish this function of
TOGp, seems less likely.
C)
Fig. 40. Model of observed interplay between MCAK and TOGp in
human cells
The discrepancy concerning the role of TOGp may
be explained by differences in species, cell types or
experimental system (i.e. Xenopus egg extracts versus human
somatic cell lines). There are several lines of evidence that
microtubules are regulated differently in embryonic and
somatic cells, but a direct comparison within a single species
is lacking. For example, the alteration of microtubule
dynamics at mitotic entry appears very different since there is
an ~7 fold increase in catastrophe-frequency in the
embryonic egg extracts compared to only an ~2 fold increase
in somatic mammalian cells (Belmont et al., 1990; Rusan et
al., 2001). Independent evidence that TOGp is of major
importance for the general microtubule stability in embryonic
cell systems but not in somatic cells has been provided by
studies in the nematode C. elegans and the fruit fly
Drosophila. Thus, the XMAP215/TOGp family member
Zyg-9 appears essential for the stability of meiotic spindle
microtubules (Matthews et al., 1998), while the phenotype of
partial depletion of the Drosophila XMAP215/TOGp family
member Msps in larval brain cells is reminiscent of
phenotypes of somatic mammalian cells (Cullen et al., 1999),
i.e. spindle disorganization without a clear-cut decrease of
microtubule stability.
It may be speculated that an embryonic cell, i.e. an
egg cell with a very large intracellular space, requires spindle
microtubules with higher dynamicity to probe the cellular
space within a reasonable time frame. In Xenopus egg
extracts the large increase in catastrophes at mitotic entry has
been shown to be dependent on MCAK (see Fig 32). In
human cell lines, however, we find no evidence for a general
role of (cytosolic) MCAK for microtubule dynamicity during
mitosis (see below). It seems therefore likely that MCAK is
less active in somatic cells, which would explain why TOGp
mediated counteraction is not important for the general
microtubule stability.
12.3 Differential phenotypes of XMAP215/TOGp and
MCAK depletion in human cells and Xenopus egg
extracts
Analysis of the microtubules in Xenopus egg extracts has
revealed that even a partial depletion (60%) of
XMAP215/TOGp cause a striking increase of catastrophepromotion
(Tournebize et al., 2000). By codepletion/inactivation of MCAK, it was evident from this
report that a prime importance of XMAP215/TOGp was to
counteract MCAK dependent catastrophe-promotion.
Moreover, with an efficiency of depletion approaching 90%,
very short or no microtubules were observed, which
precluded analysis of microtubule dynamics. Thus,
XMAP215/TOGp is certainly a dominant microtubule
stabilizer in this embryonic model system. At a first glance at
data, it also appears reasonable to assume that this dominance
reflects the requirement to counteract MCAK activity, which
seems to provide a major destabilizing activity during both
interphase and mitosis.
Given the dominance by which XMAP215/TOGp
suppresses catastrophe-promotion in Xenopus egg extracts
(see Fig. 32) it was surprising that an essentially complete
depletion of the human homologue TOGp does not produce
detectable alterations in tubulin partitioning during interphase
or mitosis (Paper I, Fig. 6A and our unpublished data).
Furthermore, even though depletion of TOGp resulted in a
dramatic phenotype manifested as dissorganized spindles
(Paper I, Fig. 2), it is notable that co-depletion of MCAK
reversed this phenotype since the spindle poles in double
depleted cells were well organized and at least 75% of all
cells formed bipolar spindles (Paper I, Fig. 2). Thus, since
TOGp-depletion does not alter tubulin partitioning, i.e.
19
abnormality was monoastral spindles, which was found in
~25% of all mitotic cells (Paper I, Fig. 2).
The monoastral spindle phenotype in MCAK
depleted cells has been reported by others (Kline-Smith &
Walczak, 2002). The mechanism behind these monoastral
spindles is unclear but may involve i) failure to destabilize
the interphase microtubule array at mitotic entry, ii) nonsuccessful centrosome separation during prophase, or iii)
failure to repair syntelic microtubule attachment to
chromosomes. While we were unable to detect a significant
overexpression or depletion phenotype in interphase cells, the
first alternative appears less likely. However, since MCAK to
some extent seems to accumulate to the centrosome in
mitotic cells, it seems plausible that a local MCAK mediated
increase of microtubule dynamicity may be of importance for
centrosome separation. It seems also plausible that excessive
syntelic microtubule attachment may cause regression of a
bipolar spindle to an apparent monopolar state.
Taken together, our studies seem very clear in that
MCAK
does
not
exert
significant
global
destabilizing/catastrophe-promoting activities during either
interphase or mitosis. It follows that the important function of
MCAK in mammalian somatic cells is as a locally acting depolymerase that is involved in a regulatory net-work,
required for high fidelity spindle formation. However, future
studies are required to establish whether the apparent
insignificance of globally acting MCAK also applies to
mammalian egg cells with a large cytosolic space and lack of
cell cycle check-point controls.
12.4 The overexpression phenotypes of MCAK and
TOGp reveal an unexpected interplay
The differences in phenotypes of XMAP215/TOGp depleted
Xenopus egg extracts and human cell lines might, as argued
above, be the result of different levels of MCAK activity in
embryonic and somatic cells. It was therefore surprising to us
that the MCAK overexpression phenotype was unaltered by
TOGp-depletion (Paper I, Fig 6C). It was even more
surprising that TOGp overexpression resulted in a significant
decrease of polymeric tubulin and that this phenomenon
depends on endogenous MCAK (Fig. 40C; Paper I, Fig. 7)
While these data are consistent with interplay between TOGp
and MCAK, the dependency on MCAK for the observed
unexpected TOGp mediated microtubule destabilization
reveals that the simple model on counteractive activities (see
Fig. 33) is not correct.
It is notable that ~25-fold overexpression of MCAK
in human cells results in a quite modest destabilization of
both interphase and mitotic microtubules, which appears
surprising since MCAK clearly exerts a potent microtubule
destabilizing activity as analyzed in Xenopus egg extracts and
in vitro (Walczak et al., 1996). It is also notable that the
presence of endogenous TOGp does not influence the MCAK
overexpression phenotype (Paper I, Fig. 6C). The only
evidence for counteractive activities between MCAK and
TOGp that we observed was that overexpressed TOGp to
some extent rescued spindle formation in MCAK
overexpressing cells, which appears consistent with the
model derived from Xenopus egg systems. However, while
this rescue effect was evident on the level of the functionality
of spindles (Paper I, Fig. 5), it was not manifested on the
microtubule polymer content (data not shown).
The result discussed above indicates the presence of
microtubule-stabilizing protein(s) in my cell model system
that efficiently counteracts the documented potent
catastrophe-promoting activity of MCAK. While the identity
of this counteractive protein(s) is unclear, I believe that my
reports on MAP4 mediated counteractions of catastrophe
promoting proteins show that MAP4 at least in certain human
cell types will be of significance (Holmfeldt et al., 2002).
12.6 TOGp provides protection from centrosomal
MCAK activity, which is essential for bipolar spindle
formation
Previous reports have revealed an essential role for
XMAP215/TOGp family members for spindle organization
in Drosophila (Cullen et al., 1999) and human cells (Gergely
et al., 2003), which is manifested by disorganized multipolar
spindles in cells lacking XMAP215/TOGp. Our depletion
studies of TOGp confirmed the multipolar spindle phenotype,
and moreover showed that formation of the multipolar
spindles was dependent on the activity of endogenous MCAK
(Paper I, and Fig. 2). Co-depletion of MCAK not only
suppressed the formation of multipolar spindles but also
restored cell proliferation to a level observed after depletion
of MCAK alone (Paper I, Fig. 1). The demonstrated MCAKdependence for the multipolar spindle phenotype in TOGpdepleted cells was concurrently shown in a study using HeLa
cells (Cassimeris & Morabito, 2004).
A possible mechanism for the formation of
multipolar spindles in TOGp depleted cells may be
nucleation of microtubules from disintegrated centrosomes.
Indeed, Gergely et al. found γ-tubulin, a protein primarily
concentrated at the centrosome, at all spindle poles in
multipolar spindles formed in TOGp depleted cells (Gergely
et al., 2003). However, time-lapse analysis of mitosis in
TOGp depleted cells revealed that chromosomes aligned near
the cellular equator before reverting to a more disorganized
state, which indicate that the multipolar spindles are formed
from disintegrating bipolar spindles (Gergely et al., 2003).
Our analysis of the localization of the centrosomal protein
pericentrin revealed that multipolar spindles, formed in
12.5 Confirmation that MCAK function is important for
spindle fidelity
Previous reports have characterized MCAK as a globally
acting and major catastrophe promoting activity in mitotic
Xenopus egg extracts (Tournebize et al., 2000; Walczak et
al., 1996). More recent studies in mammalian cells have also
implicated MCAK as a locally acting microtubule
depolymerase, which facilitates repair of incorrectly attached
microtubules at kinetochores (see Fig. 23; Kline-Smith et al.,
2004; Maney et al., 1998). Since either of these functions
depends on the depolymerizing/catastrophe-promoting
activity of MCAK, these functional roles of MCAK are not
mutually exclusive.
Our studies on tubulin partitioning during interphase
or mitosis did not reveal any significant effect of >90%
depletion of MCAK (Paper I, Fig. 6A, data not shown).
However, determination of cell growth still revealed about
50% inhibition (Paper I, Fig. 1), which was associated with a
modest increase of the mitotic index and spindle
abnormalities (Paper I, Fig 2). The most prominent spindle
20
from the spindles observed in TOGp-depleted cells (Paper II,
Fig. 1). In addition, using the CaMKII specific drug KN93,
we observed similar types of multipolar spindles. Finally, we
also demonstrated that the phenotype of CaMKIIγ-depletion
was dependent on endogenous MCAK, which firmly
established the significance of this phosphorylation-pathway
(Paper II, Fig. 2).
TOGp depleted cells, only contain two centrosomally
associated microtubule asters and additional acentrosomal
microtubule asters (Paper I, Fig. 3A-B). Furthermore,
through the microtubule minus end marker NuMa we
established that all microtubule asters of the multipolar
spindles were gathered at the microtubule minus ends (Paper
I, Fig. 3C). Taken together, these data indicate that the
dissorganized multipolar spindles in TOGp-depleted cells
evolve by the gradual release of spindle microtubules from
centrosomal microtubule asters, with the subsequent focusing
of non-centrosomal microtubules by dynein molecular
motors.
Given that TOGp appears to be enriched at the
centrosome (Charrasse et al., 1998), we have proposed that
TOGp is essential for the protection of exposed microtubule
minus ends against MCAK mediated destabilization at the
centrosome during mitosis. (Paper I). Thus, according to our
model, removal of TOGp allows access to MCAK at the
centrosome, which causes destabilization of minus-ends and
subsequent release of microtubules as depicted in Fig. 41, II.
This model readily explains why co-depletion of MCAK
suppresses the formation of multipolar spindles and that the
phenotype of MCAK/TOGp co-depleted cells appears
indistinguishable from the phenotype of cells depleted of
MCAK alone, i.e. bipolar spindles with potential missattachments (Fig. 41 (IIIa)) or monoastral spindles (Fig. 41
(IIIb)). In addition, this model is also consistent with my
proposal in section 12.5, namely that MCAK in mammalian
somatic cells primarily acts as a locally acting de-polymerase
involved in regulatory net-works that is required for high
fidelity spindle formation.
Fig. 41. Model depicting the role of kinetochore localized MCAK
during spindle assembly and the regulatory mechanisms that ensure
suppression of MCAK at the centrosome.
Besides the significance of discovering the
importance of negative regulation of MCAK activity, which
may be exerted locally at centrosomes, our findings also
provided a tool that allowed an independent confirmation of
our model of TOGp and MCAK interplay at the centrosome.
Thus, using a system of inducible TOGp expression in cells
depleted of CaMKIIγ, or alternatively, inducible CaMKIIγ
expression in cells depleted of TOGp, I believe that we could
provide formal evidence that our model on the protective
function of TOGp at the centrosome presented in Paper I is in
principle correct (Paper II, Fig. 6). A simplified version on
this model is given in Fig. 41.
12.7 The functions of both CaMKIIγ and TOGp are
essential for protection of spindle microtubules from
centrosome-directed MCAK activity
An objection to the model outlined in section 12.6, as
presented in our first report on MCAK/TOGp interplay
(Paper I), was that the model predicts that excessive MCAK
activity would result in the same phenotype as TOGp
depletion, which according to the literature is not the case.
However, in Paper II we prepared an MCAK derivative that
was expressed at 5-fold higher levels than the previously
used derivatives (Paper II, Fig. 3). Under these conditions our
data clearly demonstrated that excessive MCAK activity has
potential to generate the same phenotype as TOGp depletion,
namely, dissorganized multipolar spindles (Fig. 41 (IV)).
Our studies on CaMKIIγ mediated regulation of
MCAK derived from the observation by our colleague Claire
Walczak that MCAK is phosphorylated by this kinase in
vitro. Ironically, we have in Paper I used a CaMKIIγ-specific
shRNA derivative as a negative control, which seemed
appropriate since this derivative allowed ~60% depletion. My
initial experiment indeed documented that a constitutively
active CaMKIIγ derivative suppresses the microtubuledestabilizing activity of ectopic MCAK (Paper II, Fig. 4).
This evidence prompted us to reassess the phenotype of
CaMKIIγ-depletion using new highly efficient shRNA
derivatives that resulted in >95% specific depletion of
CaMKIIγ. Under these conditions of extensive CaMKIIγ
depletion we observed, to our delight, abundant
dissorganized multipolar spindles that were indistinguishable
13. PHOSPHORYLATION-REGULATION OF Op18
AND MAP4 CONTROLS THE PARTITIONING OF
TUBULIN SUBUNITS IN THE INTERPHASE
MICROTUBULE SYSTEM
13.1 Background
As shown in Papers I and II, the MCAK and TOGp proteins
are not of any significance for tubulin monomer-polymer
partitioning in mammalian cells. Given the original aim to
identify regulators of tubulin partitioning, we carried on by
evaluating the physiological significance of two other
potentially important microtubule regulatory proteins, Op18
and MAP4, which had previously been shown to exert
counteractive activities (Holmfeldt et al., 2002). Both of
these proteins have previously been inferred as significant
regulators of microtubule content (Drewes et al., 1997;
Marklund et al., 1996; Melander Gradin et al., 1997).
While there has been a consensus that both Op18
and MAP4 have the potential to regulate the interphase array,
the literature concerning a potential role of these microtubule
21
kinetochores in the large intracellular space of egg cells
(Andersen et al., 1997; Tournebize et al., 1997). An obvious
argument against this model is that Op18 mouse knockouts
are viable and breeds normally (Schubart et al., 1996).
Moreover, the reports on spindle-defects in K562 cells
partially depleted of Op18 using antisense RNA inhibition
(Iancu et al., 2001; Luo et al., 1994), I am convinced
represent artifacts that derive from the use of anti-sense RNA
to decrease Op18 content.
regulators during spindle assembly has been, to say the least,
confusing (Chang et al., 2001; Iancu et al., 2001). However,
results presented in Paper II (Fig. 2) and Paper III (Fig. 1)
show that depletion of either MAP4 or Op18 has no
detectable phenotypes during spindle assembly. In the case of
Op18, this would be consistent with the previously proposed
phosphorylation-inactivation of Op18 during mitosis (see
Fig. 42; Marklund et al., 1996). Moreover, analysis of cells
partially (Paper I, Fig. 6A) or extensively (Paper III, Fig. 1)
depleted of Op18 clearly established Op18 as the
predominant regulator of microtubule-content in the K562
leukemia cell line. These previous publications from us and
others provide the background to the experimental
approaches and analysis of tubulin partitioning presented in
Paper IV. I will use this thesis as an opportunity to focus on
aspects that are not discussed in this publication.
13.3 The significance of MAP4 and Op18 as
phosphorylation-responsive regulators of tubulin subunit
partitioning
To evaluate the physiological importance of counteractive
MAP4 and Op18 activities, we used the principle genetic
approach described in Papers I and II. We have spent a lot of
efforts in screening targeting sequences for shRNA shuttlevector derivatives. Most of these shRNAs mediated only
partial downregulation of Op18 and MAP4 in the range 50 to
90%. In the case of Op18, 70% depletion was sufficient for a
dramatic increase of microtubule-content (Paper I, 6A).
However, we found that a partial depletion of MAP4 had no
detectable effect and it required extensive screening and
modifications of MAP4 shRNA derivatives to establish a
condition in which depletion exceeded 85% and that was not
associated with non-specific growth inhibitory effects by
shRNA. Under these conditions of optimized extensive
MAP4 depletion, we indeed observed a substantial decrease
of microtubule content without significant effects on cell
growth. Thus, while a partial depletion of Op18 is sufficient
for a clear phenotype, the significance of MAP4 for
microtubule stability is only evident under conditions of
essentially complete depletion. These findings were not
commented on in Paper IV but are still worth considerations
in a discussion of mechanisms by which Op18 and MAP4
exert their counteractive activities.
By a combination of extensive shRNA mediated
depletion and ectopic expression of Op18, we have
established the dose-response of Op18 mediated microtubuledestabilization in K562 cells (Paper III, Fig. 5 and Paper IV,
Fig. 4) and Jurkat T cells (Sellin et al., 2008). These studies
have shown that the microtubule-system rapidly responds to
changes of Op18 levels within the range of expression levels
found in normal and malignant cells. However, by comparing
the microtubule-phenotypes at various levels of MAP4
depletion, we noted that >80% depletion was required to even
detect a decrease in microtubule content. This unpublished
peculiarity of the dose-response of MAP4 depletion, indicates
that the endogenous MAP4 level is in great excess over the
level required for its microtubule stabilizing function.
Op18 has been shown to have potential to rapidly
regulate the microtubule-system by three distinct
mechanisms, namely catastrophe-promotion, tubulinsequestering and by regulating the synthesis of tubulin
heterodimers (see section 9.6). However, the relative
importance of the mechanisms in various cell types remains
unclear. Nevertheless, the demonstrated responsiveness of
cells to variations within the physiological relevant range of
Op18 levels is fully consistent with the conclusion in Paper
IV that phosphorylation-inactivation is a relevant mechanism
for alterations of the microtubule-system in response to signal
transduction events.
13.2 Evidence for the importance of MAP4 and Op18 as
interphase-specific counteractive regulators of tubulin
subunit partitioning
It is shown in Paper IV that MAP4 and Op18 are interphasespecific principal determinants of tubulin subunit partitioning
in three human suspension growing model system (Paper IV,
Fig. 1, and suppl. Fig. 1). It is also shown that MAP4 and
Op18 exert phosphorylation-responsive counteractive
regulation of the microtubule-system as depicted in Fig. 42
(Paper IV, Fig. 2-3 and suppl. Fig 3). Since we have
developed a method to quantitate microtubule polymercontent in distinct cell cycle phases (Holmfeldt et al., 2003b),
we were also able to address the importance of MAP4 and
Op18 during the assembly of the mitotic spindle. Our results
provided a clear-cut demonstration that these microtubuleregulatory proteins are only of significance during the
interphase of the cell cycle (Paper III, Fig 1; Paper IV, Fig.
1). Thus, in all cell types in which Op18 and MAP4 are
expressed at sufficient abundance, it seems reasonable to
assume that these proteins are the principal determinants of
tubulin subunit partitioning of the interphase microtubule
system.
CaMKIV
PKA
Op18
MARK2
MAP4
Fig 42. Model showing the phosphorylation-regulated balance
between MAP4 and Op18 that determines the partitioning of tubulin
during interphase in human cells
Our evidence that MAP4 and Op18 have no
functional role during spindle assembly are consistent with
several previous reports and with that both MAP4 and Op18
appear to become phosphorylation-inactivated during mitosis
(see section 9.7 and 9.11). However, published evidence
suggest that Op18 is only partially phosphorylated in mitotic
Xenopus egg extracts. In this embryonic model system, a
model has been proposed in which local phosphorylationinactivation of Op18 occurs in the vicinity of condensed
chromosomes, which would facilitate microtubule capture of
22
for gene product exchange. Generally this involves the
exchange of the wild type protein with a protein in which the
specific phosphorylated residue is exchange to a similar but
non-phosphorylated amino-acid. In my thesis work, shRNA
based gene product depletion was combined with inducible
expression of mutant derivatives (see section 11). Since
phosphorylation-site deficient Op18 derivatives are
incompatible with cell growth (phosphorylation-inactivation
is essential during mitosis, see Fig. 43), the rapid induction of
the mutated gene product combined with well defined
expression levels is an essential feature of the experimental
system. The versatility of this system for inducible geneproduct exchange is illustrated by the data in Fig. 4 in Paper
IV.
Given the homology of MAP4 to neural classical
MAPs (see section 9.10), it seems reasonable to assume that
MAP4 increases microtubule content by direct stabilizing
interactions with polymers. It seems therefore surprising that
this stabilizing effect only requires a small fraction of the
endogenous MAP4, as evidenced by our unpublished studies
of human suspension growing cell model systems. Since the
MAP4 levels of our hematopoetic cell model systems appear
to be very similar to an epithelial cell line such as HeLa
(Holmfeldt et al., 2002), which implies ~0.1% of total
cellular proteins (Bulinski & Borisy, 1979), our findings are
likely to have general implications. While I can not explain
the apparent “excess” of MAP4, our findings have clear
implications for the potential to regulate the microtubulesystem by posttranslational regulation of MAP4 activity.
Thus, it is difficult to envisage that sub-stoichiometric
phosphorylation-inactivation of MAP4 is of significance for
alterations of the microtubule system. Moreover, the recent
identification of septins as negative regulators of MAP4 (see
section 9.11), makes it difficult to speculate on the
importance of post-translational regulation of MAP4 activity.
Interphase
P
P
Op18
Op18
Op18
Regulatory phosphorylations in
response to signal transduction events
14. IDENTIFICATION OF Op18 AS A MAJOR
SIGNAL TRANSDUCTION RESPONSIVE
REGULATOR OF TUBULIN SUBUNIT
PARTITIONING
14.1 Background
Previous works from our group have shown that expression
of constitutively active derivatives of the signal transducing
kinases CaMKIV and PKA results in increase of tubulin
polymer levels (Melander Gradin et al., 1997; Gradin et al.,
1998; Paper IV, Fig 2A-B). Since both CaMKIV and PKA
have been shown to mediate phosphorylation-inactivation of
overexpressed Op18 (Melander Gradin et al., 1997) (Gradin
et al., 1998), it appeared likely that the increase in tubulin
polymer levels seen in cells expressing constitutively active
CaMKIV or PKA is a consequence of site-specific
phosphorylation-inactivation of endogenous Op18.
Reports from other laboratories have inferred many
protein kinases as negative regulators of Op18 activity (le
Gouvello et al., 1998; Parker et al., 1998; Daub et al., 2001).
It is important to distinguish the role of signal transduction
mediated Op18 phosphorylation and the apparently complete
inactivation of Op18 by multi-site phosphorylation during
mitotic entry. Thus, while Op18 phosphorylation during
interphase may be of importance for cell communication, I
view the complete phosphorylation-inactivation by mitotic
kinase systems as a necessary action to protect the mitotic
spindle from the otherwise deleterious destabilizing activity
of Op18 as depicted in Fig. 43. As argued in section 13.3, the
interphase microtubule system is clearly highly responsive to
alterations within the range of Op18 expression levels
observed in normal and malignant cells. Thus, Op18 has an
obvious potential to function as a phosphorylation responsive
regulator of the microtubule system in cell types in which it
is expressed at sufficient abundance.
Mitosis
P P P
Op18
Active
P P P P
Op18
Stoichiometric
Multi-site
phosphorylation
Fig. 43. Model depicting the role of phosphorylationinactivation of Op18 during interphase and mitosis
Using this system, we first established a causal
relationship between site-specific Op18 phosphorylation and
the previously reported increase in microtubule content in
cells induced to express constitutively active CaMKIV or
PKA (Paper IV, Fig. 5). It should be noted that these
experiments reflect the maximal impact of Op18
phosphorylation since the induced constitutively active
kinases result in stoichiometric Op18 phosphorylation
(Melander Gradin et al., 1997; Gradin et al., 1998). In all
cases investigated, however, Op18 is sub-stoichiometrically
phosphorylated by cell surface receptor mediated activation
of cognate kinase systems (Brattsand et al., 1994; Marklund
et al., 1993).
The T cell antigen receptor activates an array of
kinase cascades (reviewed in Matthews & Cantrell, 2006) but
previous studies indicate that only about half of all Op18
molecules become phosphorylated (Marklund et al., 1993).
Moreover, it has never been shown that T-cell activation is
associated with alterations in microtubule content. In
addition, phosphorylation occurs on two Ser-residues, namely
Ser-16 and Ser-25, and the functional significance of Ser-25
has not been established. It was therefor important to evaluate
the consequences of T cell antigen receptor stimulation for
both tubulin subunit partitioning and the potential role of
Op18 phosphorylation in such a response. In paper IV it is
demonstrated that site-specific phosphorylation of Op18 is
both necessary and sufficient for T-cell activation mediated
microtubule-stabilization (Paper IV, Fig. 6). To my
knowledge, this provides the first formally proven example
of a signal-transduction chain that mediates changes of
microtubule-density.
14.2 Identification of an Op18 dependent signal
transduction chain for microtubule polymerization in
response to T cell antigen receptor triggering
To establish the causal relation between a specific
phosphorylation event and a cell response requires a system
23
T-cell antigen receptor stimulation by an antigenpresenting cell results in the formation of an immunological
synapse between the T-lymphocyte and the antigenpresenting cell. The minus end directed molecular motor
dynein is recruited to the immunological synapse and is
responsible for relocalization of the centrosome towards the
antigen-presenting cell (Combs et al., 2006). Centrosomal
relocalization will position the Golgi apparatus in the
proximity of the antigen-presenting cell and the subsequent
polarized secretion of effector molecules from the Golgi
apparatus is essential for the correct immunological response
(see section 7.2). I believe that the phosphorylationinactivation of Op18 after T-cell antigen receptor stimulation
and the consequent increase in tubulin polymers may be
important for microtubule targeting to the immunological
synapse (Fig. 44). However, the significance of
phosphorylation-inactivation of Op18 for centrosomal
relocalization after T-cell antigen receptor stimulation still
remains unclear and is currently under investigation in our
laboratory.
Selective stabilization by
+ Tip proteins
P
Op18 Tubulin heterodimer
15.2 The relationship between the oncogenic Q18ÆE
substitution of Op18 and leukemia-associated upregulation of the wild-type protein
Based on the pattern of an apparent synergistic action of the
Q18ÆE mutation and the microtubule-stabilizing drug
paclitaxel, it was originally proposed that the mutant form of
Op18 may enhance the stability of microtubules (Misek et al.,
2002). This proposal is clearly refuted in Paper III in which
we show that the Q18ÆE mutation greatly enhances the
microtubule-destabilizing activity of Op18. This increase in
activity is manifested during both interphase and mitosis
(Paper III, Fig. 2, 3 and 5). Interestingly, the consequence of
this general increase of microtubule-destabilizing activity
was found to be exacerbated during mitosis, because the
mutation also provides a partial resistance to
phosphorylation-inactivation (Fig. 45B; Paper III, Fig. 2G).
Failure to inactivate Q18ÆE mutated Op18 during mitosis
was found to be the cause of the observed aneugenic
activities, manifested as spindle abnormalities (Paper III, Fig.
2D-E), polyploidization (Paper III, Fig. 5D) and chromosome
loss (Paper III, Fig. 6).
The aneugenic effects of the Q18ÆE mutant can be
predicted to contribute to chromosomal instability in tumors,
thus promoting tumor progression (see section 8).
Interestingly, the potential destabilization of kinetochoremicrotubules during anaphase by uncontrolled Op18 activity
can also be predicted to generate chromosomal instability
without the requirement of a compromised spindle assembly
checkpoint (Fig. 46). This implies a conceptional difference
to the previously established role of oncogenic mutations of
proteins involved in the spindle-assembly checkpoint
(reviewed in King, 2008).
T-cell receptor
P
P
P
P
P
P
P
P
P
Antigen
presenting
cell
P
P
Fig. 44. Model of the potential physiological significance of
phosphorylation-inactivation of Op18 during T-lymphocyte
activation
15. EXCESSIVE Op18 ACTIVITY DURING MITOSIS
RESULTS IN SPINDLE-DESTABILIZATION AND
CHROMOSOMAL INSTABILITY
15.1 Background
The name “Oncoprotein 18” was coined based on
upregulated protein levels in human lymphoma/leukemias
relative to non-transformed but actively proliferating
lymphocytes (Hailat et al., 1990). As outlined in section 9.8,
it has been proposed that increased Op18 levels may facilitate
tumor invasion and metastasis. The general relevance of this
proposal is questionable since suspension growing human
leukemias are often found to express the most extreme levels
of Op18 (~1% of total proteins; Brattsand et al., 1993).
For a protein to formally qualify to be termed an
“oncoprotein”, it should according to current standards
contain tumor-specific mutations. These oncogenic mutations
may either alter the expression level or the function of the
gene-product. Luckily enough, a recent example of a tumorspecific mutation of Op18 has indeed been identified in a
single clinical isolate of a human esophageal adenocarcinoma
(Misek et al., 2002). This mutation, Q18ÆE, causes a shift in
the mobility on 2D-gels due to the negative charge of
glutamic acid.
A demonstrated tumor-specific mutation provides a
very strong link to tumor-progression (see section 8.1).
However, it was not discussed in the original report how this
single case of a tumor-specific Op18 mutation may relate to
the fact that the wild type Op18 protein is frequently upregulated in an array of human malignancies.
A)
Normal cells
Interphase
Mitosis
P P P P
wt
wt
Active
Inactive
B)
Tumor cells
Interphase
Mitosis
Interphase
P P P P
Q18E
Q18E
Hyperactive
Partially
active
Mitosis
P P
wt
wt
P P P P
wt
wt
I
II
Fig. 45. Two distinct mechanisms that may cause chromosomal
instability as a result of incomplete phosphorylation-inactivation of
Op18 during mitosis
Analysis of cells expressing Op18 at the excessive
levels occasionally observed in leukemias revealed that also
the wild-type Op18 protein under these conditions exert
aneugenic activities (Paper III, Fig. 5-6). The mechanism
behind this phenomenon may reflect that excessive Op18
levels saturate mitotically active kinase systems (Fig. 45B
(I)) and/or that phosphorylation does not imply a complete
inactivation (Fig. 45B (II)). Thus, both the hyperactive
24
significance of Op18 overexpression in human tumors is to
increase chromosomal instability. This because mouse cells
probably have close to “optimal” levels of chromosomal
instability already prior to transformation, which would
imply that any oncogenic alteration that functions to increase
chromosomal instability in human tumors will not be found
in mice.
Q18ÆE mutant and overexpressed wild-type Op18 generates
chromosomal
instability
by exerting
uncontrolled
microtubule-destabilization during both the early and late
phases of mitosis.
16. FINAL CONCLUSIONS
Chromosomal loss (encapsulated
into a ”micro-nucleus”)
-
The essential roles of TOGp and CaMKIIγ involve
protection of spindle microtubules against MCAK
activity at the centrosome (Fig. 47A)
-
Abundantly expressed MAP4 and Op18 are the
principle regulators of tubulin subunit partitioning
-
MAP4 and Op18
counteractive
and
activities (Fig. 47B)
-
Op18 phosphorylation is both necessary and
sufficient for T-lymphocyte activation mediated
microtubule-stabilization, which is a prerequisite for
polarization (Fig. 47C)
-
Aberrant Op18 activity during mitosis results in
chromosomal instability in tumors
Fig. 46. Chromosomal loss caused by destabilization of
kinetochore-microtubules during anaphase
15.3 The significance of life-span of animals for the
demand for increased chromosomal instability during
tumor-progression
It is notable that Op18 overexpression is a common
phenomenon in human malignancies, while few studies have
indicated overexpression in naturally occurring or artificially
induced tumors in mice model systems (concluded by
exhaustive PubMed searches). An obvious difference
between mice and humans is that tumor progression in mice
is a matter of weeks, while it in general is a matter of decades
in humans. This is despite that the cell cycle times are similar
in mammalian cells. It seems therefore reasonable to assume
that the demand for chromosomal stability of somatic cells
depends on the life-span and time-span of the reproductive
cycle of a metazoan. This assumption is indeed supported by
the fact that the extremely short reproductive cycle of mice
(~7 weeks) is associated with a less stringent spindle
assembly checkpoint as compared to humans. Thus, while
microtubule poisoning drugs cause a persistent mitotic arrest
of non-transformed human cells, cultured mouse cells have
an intrinsically high propensity to undergo “mitotic slippage”
and thereby enter a tetraploid pseudo G1 state (shown in e.g.
Cross et al., but incorrectly interpreted as evidence for a p53dependent mouse spindle checkpoint, (Cross et al., 1995))
As outlined in section 8.2, most tumors appear more
or less chromosomally unstable, which precludes correlative
studies on the clinical relevance of our finding that both the
Q18ÆE mutation and overexpression of wild type Op18
contribute to chromosomal instability. However, I believe
that the arguments presented above concerning the time
perspective of tumors originating from either mice or humans
are relevant for the understanding of how dysregulated Op18
may contribute to tumor progression. Thus, I interpret the
apparent absence of convincing reports on Op18
overexpression in mice in favor of a model in which the main
Mitosis
have interphase-specific,
phosphorylation-inactivated
Interphase
Signal
transducing
kinases
A)
C)
T-cell
receptor
B)
PAR-1/MARK
family kinases
MCAK
TOGp
Op18
MAP4
CaMKIIγ
Fig. 47. Main findings of the thesis
17. ACKNOWLEDGEMENTS
After these nine fantastic years, possible the best years of my
life yet, I wish to thank all of you concerned – in private.
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