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Theses & Dissertations
Boston University Theses & Dissertations
2015
The role of the Golgi apparatus in
neuronal polarity
Ash, Tyler Dale
http://hdl.handle.net/2144/16363
Boston University
BOSTON UNIVERSITY
GRADUATE SCHOOL OF ARTS AND SCIENCES
Thesis
THE ROLE OF THE GOLGI APPARATUS IN NEURONAL POLARITY
by
TYLER DALE ASH
B.S., Michigan State University 2013
Submitted in partial fulfillment of the
requirements for the degree of
Master of Arts
2015
Approved by
First Reader
__________________________________________________
Angela Ho, Ph.D.
Assistant Professor of Biology
Second Reader _______________________________________________
Cynthia Bradham, Ph.D.
Assistant Professor of Biology
ACKNOWLEDGEMENT
I would like to thank the many people who helped make this Master’s
Thesis possible. I would like to thank everyone from the Ho lab for their help and
support. I would like to specifically thank my advisor, Dr. Angela Ho, for all of her
guidance and expertise.
I would like to thank the entire faculty involved in my graduate education
for continued support in and out of the classroom. I particularly want to thank Dr.
Ulla Hansen for her counseling from start to finish as my program director, and
Dr. Cynthia Bradham for her career counseling through graduate school.
I am grateful for the support network provided by my fellow graduate
students who have been friends, colleagues, and mentors. I want to thank Greg
Dillon for teaching me everything I know in lab, Andressa Mota for going through
it all with me, and Patrick Stoiber for being the JD to my Turk.
I want to thank my family, old and new, who have always encouraged me
to pursue my dreams, and tolerated my science rambles, as well as jokes.
Most of all I want to thank my loving wife Erin for all her help and support.
For all of the guidance you have given me. For always being there when I
needed someone to talk to, someone to encourage me, and someone to support
me. For providing me with the most conducive work environment through
graduate school. I would not have made it this far without her. Thank you.
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THE ROLE OF THE GOLGI APPARATUS IN NEURONAL POLARITY
TYLER DALE ASH
ABSTRACT
The Golgi apparatus has always been an interesting organelle of study
because of its unique morphology as well as the critical roles it plays in cell
biology. It is situated next to the endoplasmic reticulum and secreted proteins
must pass through the Golgi vesicular pathway for modifications and targeting. In
addition, the Golgi apparatus plays an essential role in establishing cellular
polarity. Cell polarity refers to difference in orientation of cell structures spatially,
and is involved in establishing functionality. The Golgi apparatus establishes cell
polarity in various ways including orienting itself spatially, biasing vesicular
trafficking within the cell, and most importantly through its role as a microtubule
organizing center.
The cytoskeleton provides the structural framework for cells. Microtubules
nucleated from the Golgi-dependent microtubule organizing center result in an
asymmetric cytoskeleton. An asymmetric cytoskeleton is essential to establishing
cell polarity. Neurons require cell polarity to establish the essential structures
such as the axon and dendrites. The Golgi apparatus establishes neuronal
polarity through its extensive network of associated proteins. In this review, we
will discuss the growing evidence supporting the role of the Golgi apparatus in
establishing
neuronal
iv
polarity.
TABLE OF CONTENTS
INTRODUCTION: ..................................................................................................1
AIM: .......................................................................................................................2
BACKGROUND INFORMATION: .........................................................................2
Golgi Apparatus ...............................................................................................2
Golgi Structure and Function ..........................................................................2
Microtubule Organizing Center .......................................................................7
Neurodevelopment ...........................................................................................8
Brain Cells ......................................................................................................8
The Basics of Brain Development ................................................................10
Cerebral Cortex ............................................................................................12
Polarity of Cells .............................................................................................13
Neuronal Migration .......................................................................................15
Secretory Pathway Involvement with Growing Dendrites and Axons ...........18
GOLGI APPARATUS ASSOCIATED PROTEINS: .............................................19
Rab GTPases ..................................................................................................19
Activation of Rab Proteins ............................................................................20
Rab Proteins in Neuronal Polarity ................................................................21
Cytoplasmic Linker Associated Proteins ....................................................22
Structure and Function of CLASPs ...............................................................23
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CLASP2 and Protein 4.1R ............................................................................26
CLASP2 and LL5β ........................................................................................26
CLASPs and the Golgi ..................................................................................27
CLASP2 Regulates Neuronal Polarity ..........................................................29
GRIP and Coiled-Coil Domain Containing Protein 185 ...............................30
Structure of Golgin Proteins .........................................................................31
GCC185 and Rab GTPase Binding Sites .....................................................34
GCC185 and AP-1 Protein ...........................................................................34
GCC185 and CLASPs ..................................................................................35
CONCLUSION: ...................................................................................................38
JOURNAL ABBREVIATIONS: ...........................................................................42
REFERENCES: ...................................................................................................43
CURRICULUM VITAE: ........................................................................................50
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LIST OF FIGURES
Figure 1: Two models for membrane traffic through the Golgi apparatus
6
Figure 2: Schematic diagram of radial-directed cortical neuronal migration
13
Figure 3: Diagram of layering in human cortical development stages
16
Figure 4: Cycling of Rab from active to inactive states
21
Figure 5: Representation of microtubule nucleation and catastrophe
24
Figure 6: CLASPs in the cytoplasm are involved in microtubule nucleation
from the Golgi apparatus ultimately establishing cell polarity
28
Figure 7: Schematic representation of known human golgin domains
32
Figure 8: Schematic representation of GCC185 fragments
37
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LIST OF ABBREVIATIONS
Abbreviation
+TIP
AP1
aPKC
ArfGEF2
Arl
BIG2
CC
CLASP
EB1
FERM
Fz
GAP
GCC185
GDP
GEF
GRASP
GRIP
GSK3β
GTP
PHLDB2
SR
TMD
TOG
TOGL
Vangl2
Full Text
Plus-End Tracking Protein
Adaptor Protein 1
Atypical PhosphoKinase C
ADP-Ribosylation Factor Guanine Nucleotide Exchange
Factor
ADP Ribosylation factor-Like
Brefeldin A-Inhibited Guanine Nucleotide Exchange
Factor
Coiled-Coil
Cytoplasmic Linker Associated Protein
End Binding 1
4.1 Ezrin, Radixin, Moesin
Frizzled
GTPase Activate Protein
GRIP and Coiled-Coil Domain Containing 185 kiloDaltons
Guanosine DiPhosphate
Guanine Exchange Factor
Golgi Reassembly and Stacking Proteins
Golgin-97, Ranbp2α, Imh1p, and P230/golgin-245
Glycogen Synthase Kinase-3 Beta
Gaunosine TriPhosphate
Pleckstrin Homology-Like Domain B2
Serine Rich
Transmembrane Domain
Tumor Over Expressed
Tumor Over Expressed Like
Van Gogh Like 2
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1
INTRODUCTION:
Research of the Golgi apparatus initially focused on the unique structure
of the organelle and its role in protein trafficking. It is a dynamic organelle that
receives vesicles containing proteins from the endoplasmic reticulum. As these
vesicles traverse through the Golgi, resident proteins add post-translational
modifications. The proteins are then trafficked to their destination. Proteins that
pass through the Golgi are often targeted for secretion. Because of this, the Golgi
apparatus is the center of the secretory pathway.
Recent experiments aiming to elucidate the function the Golgi apparatus
plays in cellular polarity have been performed in multiple model organisms
ranging from Drosophila to primates. Polarity of cells provides functionality by
orienting organelles spatially in a targeted direction. Cell polarity is required in
most cell types. The Golgi apparatus and its network of associated proteins are
required in establishing cell polarity. This occurs through Golgi-dependent
nucleation of microtubules. This association with the cytoskeleton allows
orientation of the Golgi during cell polarization.
Neurons are a unique cell type that requires cell polarity to establish
cellular structures essential for neurodevelopment. Neurons have two unique
structures, axons and dendrites, which set them apart from other cell types. The
axon is a long singular projection from the soma, or cell body, that are used to
send information from the neuron. There are multiple dendrites that branch out
2
from the soma. Dendrites receive information from neurons. These unique
structures are required for proper functioning of neurons, as well as for migration
during neurodevelopment. Cell polarity is essential in establishing these dendritic
and axonal projections in neurons. Interestingly, the Golgi apparatus has been
shown to be involved in establishing polarity in various cell types including
neurons.
AIM:
This review aims to detail the role of the Golgi apparatus in establishing
neuronal polarity during neurodevelopment.
BACKGROUND INFORMATION:
Golgi Apparatus
Golgi Structure and Function
The Golgi apparatus is a ribbon-shaped organelle made up of disk-like
structures called cisternae. The resident proteins of the cisternae are responsible
for post-translational modifications such as adding oligosaccharide side chains.
The Golgi apparatus is considered to be divided into three functional and spatial
segments. Structurally, the cis face of the Golgi is the portion adjacent to the
3
endoplasmic reticulum. The middle portion of the Golgi is termed the medial
Golgi. The trans-Golgi is the portion facing the plasma membrane. The transGolgi network is the final portion of the Golgi, which sends off vesicle buds
targeting proteins to their final destination (Glick and Nakano, 2009).
A crucial function of the Golgi apparatus is to transport cargo proteins in
vesicles from the endoplasmic reticulum to target destinations within the cell or to
the cell membrane for exocytosis, and thus is referred to as the secretory
pathway. The directionality from the cis-Golgi face towards the trans-Golgi face is
anterograde movement. Vesicles can also be transported from the trans face
towards the cis face of the Golgi and continue on to the endoplasmic reticulum,
which is called retrograde transport, and is usually involved during endocytosis
from the cell membrane to the Golgi apparatus.
Proteins discovered early in the history of Golgi studies appeared to exist
to maintain the structural integrity of this organelle while vesicles were moving
bidirectionally through the Golgi. There are two common morphologies of the
Golgi apparatus, stacked or ribbon. A Golgi stack is a round or globular-like
shape of the Golgi. A stacked Golgi is usually found in a symmetrical cell where
transport vesicles leave the Golgi in all directions. This requires a more circular
shape for the Golgi because it is continuously sending out and receiving vesicles
to and from all directions of the cell. A Golgi ribbon is a long flat morphology of
the organelle and is associated with an asymmetric cell growth or shape. If one
4
end or portion of a cell is growing or moving then the Golgi will orient itself
towards that region while supplying it with vesicular bound proteins as well as
lipids for the expanding membrane.
Two Golgi Reassembly and Stacking Proteins (GRASP) known as
GRASP55 and GRASP65 were shown to cross-link adjacent cisternae to support
the stacked and ribbon structure of the organelle (Lowe, 2011). When both
GRASP proteins were depleted, the Golgi structure is abolished resulting in
fragmentation of the Golgi apparatus, while singular loss of a GRASP leads to a
reduction in the number of cisternae (Xiang and Wang, 2010).
Intra-Golgi cargo transport is accomplished through the use of coat
proteins. COPII coated vesicles are involved in anterograde movement of Golgi
cisternae, and COPI coated vesicles are involved in retrograde movement of
Golgi cisternae. COPII and COPI coat the budding vesicles to provide structural
support to the vesicle membrane and support fusion to Golgi cisternae using
SNARE complexes (Szul and Sztul, 2011).
There are two current theories regarding trafficking through the Golgi. The
first is the stable compartments model (Figure 1a). In this model, the various
regions of the Golgi (cis, medial, and trans-Golgi) are seen as distinct entities
that maintain a specific set of resident proteins. Vesicles are donated from the
endoplasmic reticulum to the Golgi through COPII vesicles and then COPI
vesicles are responsible for coating vesicles in intra-Golgi transportation. The
5
stable compartments model implies that the Golgi is a relatively static organelle.
The second theory that is most strongly supported by current literature is the
cisternal maturation model (Figure 1b). This model postulates that the Golgi
compartmental proteins common to the sections of the Golgi form de novo,
describing the Golgi as a dynamic organelle. The Golgi proteins carried in the
budding vesicles are kept in the proper region of the Golgi through signal
sequences that target them to their region of function. Clathrin-coated vesicles
bud from the trans-Golgi network towards their targeted destination. The cisternal
maturation model is the currently supported model, and it accounts for the
dynamic range of function by the Golgi apparatus in an ever-changing cell that
has to constantly respond to inter- and intra-cellular changes (Glick and Nakano,
2009; Pelham, 2001).
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Figure 1: Two models for membrane traffic through the Golgi apparatus. (a)
Stable compartments model. (b) Cisternal maturation model (Glick and Nakano,
2009).
Important findings regarding protein localization within the secretory
pathway support the cisternal maturation model. As previously mentioned, there
are sequences that target vesicular proteins to specific locations. One example is
the C-terminal KDEL amino acid sequence in soluble endoplasmic reticulum
proteins. This sequence is recognized by KDEL receptors in post-endoplasmic
reticulum Golgi compartments and shuttles proteins containing the amino acid
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sequence from the Golgi back to the endoplasmic reticulum (Munro and Pelham,
1987;
Pelham
et
al.,
1988).
Another
example
includes
recycling
of
glycosyltransferases within the Golgi (Storrie et al., 1998). The ability, provided
by the KDEL sequence, to localize Golgi processing proteins is crucial for proper
function of the secretory pathway. Without them, resident proteins would quickly
become depleted after multiple vesicles are trafficked away, and Golgi regions
would lose functional processing properties.
Microtubule Organizing Center
The role of the microtubule organizing center in animal cells is to act as
the nucleation site for microtubules. Traditionally, centrioles are credited as the
microtubule organizing center of a cell; however, non-traditional microtubule
organizing centers may refer to other structures such as the pericentriolar matrix,
or centrosomes (Kloc et al., 2014). Recent research identified the Golgi
apparatus as a non-traditional microtubule organizing center that can nucleate
microtubules with a slightly unique function (Zhu and Kaverina, 2013).
Golgi-derived microtubule organizing centers are required for cell polarity.
Centriole-based microtubule organizing centers usually provide a relatively
symmetrical microtubule formation. Non-centrosomal microtubules derived from
a Golgi-based microtubule organizing center have been found to be critical to
vesicular trafficking of Golgi vesicles, as well as the directionality of cellular
8
processes, also known as cell polarity. Cell polarity signifies that a cell is
asymmetrical, adopting an orientation in one direction for a specific function.
Because of this, microtubules are crucial in the function of the Golgi. The plusend of a microtubule refers to the end that is polymerizing. Microtubule minusends interact with the Golgi apparatus along with the motor protein dynactin
enabling the essential motor coordination of Golgi vesicles (Lowe, 2011;
Vaughan, 2005).
Neurodevelopment
Brain Cells
Cell polarity plays an essential role in development of neurons. The
neuron is the most well-known cell type of the brain. A neuron is defined by its
ability to send electrical action potentials through neuronal circuitry and form
connections, referred to as synapses between two neurons. Synapses are the
essential form of communication in the brain and occur through passage of
electrochemical information from one neuron to another. The information can
cause excitation or inhibition of other neurons and is passed through
endogenous neurotransmitters, which are packaged in vesicles in the cell and
are released through exocytosis of the vesicle. There are numerous
neurotransmitters that are usually categorized as peptides, amino acids, or
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monoamines. Neurons have dendrites and axons, which are uniquely extensive
for varying neuronal cell types. Generally, a neuron has one axon that sends
signals to other neurons. There are multiple dendritic branches from a single
neuron and they receive signals sent by axons. The dendritic branches allow one
neuron to simultaneously receive input from multiple neurons (Stiles and
Jernigan, 2010; Tau and Peterson, 2009).
Maturation of white matter and gray matter is essential during proper
development of the brain. Gray matter contains nerve cell bodies and branching
dendrites, and is where the main functions of the brain occur, such as the relay of
somatosensory information. White matter consists of nerve fibers as well as
myelin sheaths and glial cells. The white matter connects different functional
regions of the brain together forming complex networks and is the matter that
makes up the corpus callosum (Dubois et al., 2014).
Glia is another major cell type in the brain. As brain size increases, the
proportion of glia to neurons increase. There are only a few glia in nematode
brains, whereas up to 90% of the human brain consists of glia. Glia were
originally thought to only have a passive role in supporting neurons by providing
a suitable environment. Glia have since been found to have an important function
in assisting informational processing in adulthood, as well as in development
(Allen and Barres, 2009; Barres and Barde, 2000).
10
There are three types of glia: oligodendrocytes, astrocytes, and microglia.
Oligodendrocytes wrap axons in myelin to facilitate faster transfer of action
potential along the axon. Astrocytes provide neurons with a homeostatic
environment by providing energy and substrates for neurotransmitters. Microglia
act as specialized immune cells, which monitor the brain for damage or infection
by engulfing neuronal debris and dead cells. In addition, microglia are involved in
synaptic
remodeling
in
development
by
pruning
unnecessary
synaptic
connections (Allen and Barres, 2009; Barres and Barde, 2000).
The Basics of Brain Development
Neurodevelopment is very complex and has drawn a lot of interest in its
study because of the drastic phenotypic effects that can occur from small
changes in basic cell function. There is a large body of work covering many
levels of neuronal development ranging from the microscopic level to whole
systems analysis (Stiles and Jernigan, 2010; Tau and Peterson, 2009). Research
has advanced markedly in the field of neurodevelopment within the last ten
years; however, with each new insight comes more potential questions, making
the study of neuronal development as important today as it was 100 years ago.
Development of the human brain spans decades to produce a fully
matured brain. In humans, initial development begins in gestational week three
with differentiation of neural progenitor cells, and requires specific environmental
11
and genetic cues to occur properly. The first step is production of neuronal stem
cells, or neural progenitor cells. Through a complex cascade of genetic signaling
molecules, neuronal progenitor cells populate the region called the neural plate.
During neurulation, which is the development of the nervous system, the neural
plate compresses towards the notochord forming the neural tube, the precursor
to the spinal cord. Transcription factors such as bone morphogenetic proteins
and sonic hedgehog are expressed in gradients along the neural tube and lead to
specific activation of developmental genes (Barres and Barde, 2000; Stiles and
Jernigan, 2010; Tau and Peterson, 2009).
Synaptogenesis, the development of synapses, is a dynamic process,
which peaks in humans during postnatal development, and results in a high
density of neuronal synapses. As was mentioned previously, a synapse is the
connection between two neurons where synaptic information is passed. As
synapses are formed, they are concurrently pruned through phagocytosis,
resulting in clearance and necrosis of up to 50% of synapses and neurons,
respectively (Purves and Lichtman, 1980; Rakic et al., 1986; Stiles and Jernigan,
2010; Tang et al., 2014). Despite this drastic reduction in neuronal population;
intellectual capabilities are not compromised, as intelligence does not correlate
with brain weight. The significance of the neuronal circuits, connections, and
quality of synapses is important to intellectual processing and defects in proper
pruning of synapses have been implicated in frontal and parietal lobes of autism
12
spectrum disorder patients (Bourgeron, 2009; DeFelipe, 2011; Peça and Feng,
2012).
Cerebral Cortex
The cerebral cortex is the outer layer of the cerebrum and is composed of
folds and indendations of gray matter making the gyrus and sulcus, respectively.
The extensive folding provides an increase in surface area while maximizing the
available volume of space the brain can occupy. The cortex develops in an inside
out manner. During development, the progenitor cells divide symmetrically until
there is a substantial population of progenitors. Once this occurs, progenitor cells
begin asymmetric cell division producing one renewed progenitor cell and one
dissimilar daughter cell. In the cortex, the primary progenitor cell is a radial glial
cell that undergoes asymmetric cell division to produce another radial glial cell
and a neuron (Miyata et al., 2001; Noctor et al., 2002). The neuron then migrates
from the ventricle towards the pia (radially) along the radial glial projection.
Subsequent divisions of the radial glial cell result in the younger neuron migrating
radially past the older neuron to the outer layer (Figure 2) (Bielas et al., 2004).
Thus functional migration is essential for proper neurodevelopment.
13
Figure 2: Schematic diagram of radial-directed cortical neuronal migration.
Neurons (green) are generated in the ventricular zone and migrate along radial
glial cells (orange) through the subventricular zone, intermediate zone, subplate,
and cortical plate. The cortical plate is established in an inside-out pattern so that
later-generated neurons (progressively darker green shading) destined for more
superficial layers bypass earlier-generated neurons in deeper layers. The
marginal zone includes horizontally oriented Cajal-Retzius cells (red) (Bielas et
al., 2004).
Polarity of Cells
During neurodevelopment, it is important that cells undergo asymmetric
cell division to continue the progenitor cell line and produce neurons. A crucial
component of asymmetric cell division is planar cell polarity. Planar polarity is
described as a uniform orientation of a population of cells within a 2-dimensional
14
space. Planar cell polarity is a common property in epithelial tissue. In the animal
kingdom, planar cell polarity has been widely studied in Drosophila, and the
principles of planar cell polarity in asymmetric cell division are conserved in
mammalian cells (Goodrich and Strutt, 2011; Guilgur et al., 2012). Core proteins
of the planar cell polarity pathway have been shown to be expressed
asymmetrically in the cell including the transmembrane receptors Frizzled (Fz)
and Van Gogh Like 2 (Vangl2) (Goodrich and Strutt, 2011). These two
transmembrane receptors are implicated as core components of the Wnt7a
pathway which is known to promote the planar cell polarity pathway (Wang and
Nathans, 2007).
Using 3D reconstruction of the mitotic spindle, a study in mice described
asymmetric cell division dependence on spindle size asymmetry during
mammalian corticogenesis (Delaunay et al., 2014). Mitotic spindle size
asymmetry is a measure of the volume of microtubule spindles during
metaphase. Studies have found that Vangl2 and the Wnt7a pathway control the
magnitude of spindle size asymmetry. During asymmetric cell division of a radial
glial cell, the larger spindle volume leads to a neuronal cell, and smaller volume
portion of the cell resulted in a radial glial cell. In recent studies spindle size
asymmetry was shown to occur during corticogenesis in the macaque monkey
(Delaunay et al., 2015). Taken together, this suggests that cell polarity is
essential in neurodevelopment.
15
Neuronal Migration
As previously mentioned, once the neuron is produced from asymmetric
cell division of a radial glial cell, it must migrate radially to produce proper
layering of the brain. In Figure 2, the cell architecture/structure of the brain can
be partitioned into six layers within the cortical plate (Figure 3). Neurons migrate
along the radial glial cell until they receive a stop signal. In Figure 3A, the CajalRetzius cells are positioned in yellow just before the pia matter at the extreme
outer boundary of the brain. Cajal-Retzius cells produce the molecular stop
signal, Reelin, a protein recognized by the neurons signaling an end of their
migration (Bielle et al., 2005; Huang, 2009).
Migration defects resulting from dysfunctional Reelin lead to severe brain
malformation such as inversion of the cortical layers. Complete loss of Reelin can
result in a form of lissencephaly (smooth brain). Each layer has its own unique
density of neurons, number of synapses, length and branching of dendrites
and/or axons, as well as inputs and outputs of information (Bielas et al., 2004;
Stiles and Jernigan, 2010; Tau and Peterson, 2009).
Neuronal migration requires a complex coordination in movement of
organelles between the centrioles, the Golgi, and the nucleus. These organelles
are implicated in associations with microtubule and axonal nucleation along with
the motor protein dynactin. Not all neurons migrate in the same fashion. Some
16
produce continuously changing filopodial projections; however, they generally
have a long leading process that resembles a growth cone with dynamic
filopodia. Despite the subtle differences in migration of neurons, it is clearly
established that neuronal migration requires an intense and dynamic
reconstruction of the cytoskeleton in close association with known microtubule
organizing centers (Cooper, 2013).
Figure 3: Diagram of layering through stages of human cortical development
stages. A) Migration of neurons in an inside out pattern with the darker
blue/purple being the older neurons. B) Layering changes through development
from left to right, producing the final layering of the cortical plate shown on the
right (Stiles and Jernigan, 2010).
17
Secretory trafficking regulates growth of the plasma membrane in cell
motility (Etienne-Manneville and Hall, 2003). Mutations that disrupt functional
cytoskeletal reorganization and their interaction with associated proteins have
profound effects on neuronal migration. Mutations in filamin disrupt actin-binding
proteins leading to the human disease periventricular nodular heterotopia, which
is a human disease caused by improper migration of neurons in utero. Individuals
with periventricular nodular heterotopia will usually experience mild seizures in
their teen years and have normal intelligence; however, will rarely have learning
disabilities.
Disruption of vesicle trafficking within the Golgi also causes periventricular
nodular heterotopia, epilepsy, as well as microcephaly (small brain size) from
mutations in the gene ADP-ribosylation factor guanine nucleotide-exchange
factor 2 (ArfGEF2) which codes for the protein brefeldin A-inhibited GEF2 (BIG2).
BIG2 has functional importance in vesicular trafficking from the trans-Golgi
network. Double mutations of ArfGEF2 and/or disruption of BIG2 activity resulted
in loss of vesicular trafficking-dependent proliferation and migration of neurons
during development (Sheen et al., 2004). It is clear from previous research that
defects in proteins implicated in the Golgi vesicular trafficking network such as
Reelin, filamin, and BIG2 have significant effects on neuronal migration (Bielas et
al., 2004).
18
Secretory Pathway Involvement with Growing Dendrites and Axons
Functional neurons must establish an axon as well as branching
dendrites. The dendritic arborization and the aspects of axonal growth have
significance for the establishment of neuronal circuits (Tau and Peterson, 2009).
To produce dendrites and axons, the membrane of the cell must expand greatly
to compensate for the extending filopodia. This is made possible through the
secretory pathway, which is the largest source of additions to the plasma
membrane through vesicular trafficking (van Vliet et al., 2003).
The Golgi apparatus is critical in the extension of dendrites. In the soma, a
polarized Golgi apparatus orients towards the growing dendrite and is required
for polarized dendritic arbors. Small Golgi outposts were also found at branch
points of growing dendrites, but are excluded from axons. Overexpression of
GRASP65 leads to dispersal of Golgi vesicles. Golgi dispersal depletes Golgi
polarization towards the longest dendrite. This results in loss of dendritic polarity,
causing symmetric dendritic arbors, and the longest axon was not formed
(Horton et al., 2005).
Unlike dendrites, axonal growth is not regulated through control of the
secretory pathway. During development, the neuron receives signals from
amacrine cells, unique unipolar neurons, to irreversibly switch from axonal
growth to dendritic, suggesting separate modes of outgrowth. More recent work
identified that when expression of Sar1, a protein involved in vesicle budding
19
from the endoplasmic reticulum to the Golgi, was knocked down using siRNA, a
significant loss of dendritic growth was observed with no noticeable affect to axon
length (Goldberg et al., 2002). This implicates Golgi involvement in dendritic
growth and that axonal growth relies on different cellular and molecular
mechanisms which may help to establish their unique morphologies.
GOLGI APPARATUS ASSOCIATED PROTEINS:
Given the integral role the Golgi apparatus plays in the secretory pathway,
neuronal migration, and a plethora of other physiological cell processes, it is no
surprise there are numerous Golgi associated proteins. This chapter focuses on
proteins that are associated with the Golgi and its role in neuronal polarity.
As previously discussed, there are proteins involved in processing
proteins and lipids as they pass through the secretory pathway. These proteins
will not be discussed in this chapter unless they have a specific role in the Golgi
apparatus relating to neuronal polarity.
Rab GTPases
Rab proteins are notably associated with the Golgi apparatus. Rab
proteins are in the Ras superfamily of small GTPases. This family is categorized
by the function of the small GTPase, meaning it has a catalytic site that can bind
20
and hydrolyze guanosine triphosphate (GTP). Rab proteins are highly conserved
in eukaryotes, and there are more than 50 Rab genes in the human genome
alone. Rabs are transmembrane proteins localized to the cytosolic face of
organelles by Rab escort proteins, and anchored via a prenyl group on the Cterminus. In relation to the Golgi, Rab proteins are involved in trafficking through
the secretory pathway. Once bound by an effector protein, Rab modulates
vesicle formation, membrane fusion, and membrane trafficking. Multiple Rab
proteins are essential in the functions performed by the Golgi apparatus (Liu and
Storrie, 2012, 2015; Stenmark and Olkkonen, 2001).
Activation of Rab Proteins
The activity of Rab proteins, like other Ras superfamily small GTPases,
depends on their binding of GTP or GDP (Figure 4). For example, Rab bound by
GTP will be termed as active Rab. Inactive GDP-Rab is activated by a guanine
exchange factor (GEF), which switches a GDP for GTP. GTP-Rab is active and
can act on its target proteins to influence vesicular trafficking and fusion. To
deactivate Rab, the small GTPase domain of Rab must hydrolyze the γphosphate of the GTP; however, the domain cannot initiate hydrolysis alone.
Rab’s GTPase activity will be activated by a GTPase activating protein (GAP),
which catalyzes cleavage of the tertiary phosphate by the small-GTPase site
21
resulting in GDP-Rab. Inactive Rab can be shuttled by Rab escort protein, or
reactivated by a GEF (Stenmark and Olkkonen, 2001).
Figure 4: Cycling of Rab GTPases from active to inactive states. Active GTP-Rab
is inactivated through GAP catalyzing the Rab small GTPase resulting in
cleavage of the γ-phosphate leaving GDP-Rab. GDP-Rab can be translocated by
REP. GDP-Rab is activated by a GEF switching a GTP for the GDP resulting in
active GTP-Rab (Stenmark and Olkkonen, 2001).
Rab Proteins in Neuronal Polarity
A study in 2004 implicated a mutation of the gene ADP ribosylation factor
guanine nucleotide-exchange factor2 (ArfGEF2) as a cause of the neurological
disorder periventricular nodular heterotopia, which is a result of improper neuron
migration (Bielas et al., 2004; Sheen et al., 2004). ArfGEF2 is a GEF for Rab
proteins. ArfGEF2 is known to localize to the Golgi and be involved in membrane
trafficking. Because of this, a group performed studies of ArfGEF2 involvement in
neuronal migration. They found that the Rab GTPase-dependent pathways have
22
roles in neuronal maturation and migration, and inhibition of Rab5 and Rab11
disrupts neuronal migration. This supports the model that Rab5 dependent
endocytosis of membranes is involved in recycling N-cadherin from the trailing
process to the leading edge. N-cadherins are transmembrane proteins required
for cell adhesion during migration. Rab dependent recycling of N-cadherin from
the trailing process to the leading edge is required to promote continued leading
edge adhesion during neuronal migration (Kawauchi et al., 2010).
The Golgi-associated Rab proteins are known to be integral to Golgi
function, specifically in the Golgi’s ability to organize membrane trafficking (Liu
and Storrie, 2012, 2015). Rab5 has been shown to be integral to Golgi trafficking
of multiple proteins, including those important to neuronal migration (Fukuda et
al., 2013). This information shows that certain Rab proteins are involved in
establishing neuronal polarity through their interactions with the Golgi apparatus.
Cytoplasmic Linker Associated Proteins
Cytoplasmic linker associated proteins (CLASPs) are microtubule linking
proteins in a family of plus-end tracking proteins (+TIPs). CLASPs are highly
conserved across animal, plant, and fungal kingdoms (Galjart, 2005). They are
known to be involved in attaching microtubules to the cell cortex. CLASPs direct
microtubule dynamics, facilitate the relay of information within the cytoskeleton,
and modulate the orientation of growth. Because of the range of functions
23
CLASPs are involved in, they are essential for proper growth in developing
neurons (Akhmanova et al., 2001). There are two mammalian CLASP homologs
known as CLASP1, which is ubiquitous, as well as CLASP2, which is brain
specific. CLASPs are integral in establishing cell polarization and migration
(Beffert et al., 2012; Drabek et al., 2006; Efimov et al., 2007; Stramer et al.,
2010).
Structure and Function of CLASPs
The function of CLASP proteins is elucidated by their structure. The
common domains of CLASP proteins are tumor over expressed (TOG)-like
(TOGL) domains, as well as serine, proline, and arginine rich domains (SR-rich
domains). In human CLASP proteins, there are three TOGL domains that stretch
from the N-terminus to the middle of the protein. TOGL 1-3 are required for
CLASP binding to microtubules. Each TOGL domain has differing affinity for
microtubules. This is thought to provide CLASP proteins with varying levels of
microtubule binding capabilities to increase versatility of CLASPs in vivo
(Akhmanova et al., 2005; Al-Bassam and Chang, 2011).
Structural analysis of TOGL domains showed they are not responsible for
direct binding of CLASPs to microtubules. Instead, C-terminal to TOGL domains
are SR-rich domains that have stretches of positively charged amino acids.
These stretches are relatively unstructured in their exact sequence order;
24
however, they are highly conserved across species. It is thought that the
combined function of TOGL and SR-rich domains allows CLASP to act as a
rescuing protein during microtubule catastrophe, an event in which microtubule
bundles undergo an explosive shrinkage through filaments peeling off of the
microtubule bundle (Figure 5) (Akhmanova et al., 2005; Al-Bassam and Chang,
2011).
Figure 5: Representation of microtubule nucleation and catastrophe. a) GTP
bound β-tubulin is represented in green, and α-tubulin is light blue and they form
a straight dimer. After hydrolysis of the γ-phosphate GDP bound β-tubulin
25
undergoes a conformational change resulting a bend in the dimer. b) During
microtubule assembly GTP tubulin is added at the plus-end of the microtubule. If
the tubulin at the plus-end is dephosphorylated the dimers bend resulting in
microtubule catastrophe. CLASPs binding to microtubules through TOGL
domains provide structural support that can rescue microtubule catastrophe (AlBassam and Chang, 2011).
Beyond interacting with microtubule networks directly, there are structural
components of CLASPs C-terminal region that localize it in the cell. The S-X-I-P
motif is an end binding 1 (EB1) interaction domain that allows EB1 and CLIP-170
binding to CLASP. EB1 proteins are conserved targeting proteins. CLIP-170 is a
microtubule plus-end tracking protein known to modulate dynactin localization,
and microtubule organization and linkage to endosomes. EB1 and CLIP-170 both
target CLASP to the plus end of growing microtubules. CLASP proteins also
have phosphorylation sites in the C-terminus that affect its localization. Glycogen
synthase kinase-3 beta (GSK3β) is a serine-threonine kinase known to affect
localization of CLASP to plus-ends through phosphorylation of CLASPs Cterminus. GSK3β acts as a guide through its regulation of CLASPs for
microtubules dynamics in the sorting process from the trans-Golgi network
(Akhmanova et al., 2005; Al-Bassam and Chang, 2011).
CLASPs have various essential functions in the cell. They are required at
the growing plus-end of microtubules, as well as for anchoring at the Golgi
associated minus-end. CLASPs binding along their length also stabilize
microtubules. These functions are made possible through CLASPs association
26
with various proteins. We will now discuss CLASPs associating proteins relevant
to the Golgi apparatus, neurodevelopment, and neuron function in detail.
CLASP2 and Protein 4.1R
The protein 4.1R was recently found to be involved in localization of the
brain specific CLASP2. Protein 4.1R is part of the large band 4.1 superfamily,
defined by their highly conserved 4.1 ezrin, radixin, moesin (FERM) domain
which functions to bind proteins and lipids (Chishti et al., 1998). Initially, 4.1R
was found to regulate architecture of microtubules, centrosomes, endosomes,
and the nucleus. Later work found that CLASP2 associates with protein 4.1R
independently of microtubules. 4.1R modulates binding of CLASP2 to the plusends of microtubules by reducing the ability of GSK3β to phosphorylate the Cterminal region of CLASP2. Through this mechanism, 4.1R with CLASP2 exhibits
control over microtubule dynamics and organization in the leading edge required
for proper cell polarity that is essential for migration (Ruiz-Saenz et al., 2013).
CLASP2 and LL5β
The protein LL5β is a binding protein of phosphatidylinositol-3,4,5triphosphate. It is expressed by the gene pleckstrin homology-like domain B2
(PHLDB2) and is involved in intracellular signaling. LL5β is localized to the
postsynaptic membrane because of its pleckstrin homology domain, where it
27
binds
CLASP2
bound
microtubules.
Acetylcholine
is
an
essential
neurotransmitter in both the peripheral and central nervous system and is
essential to the somatic nervous system. The acetylcholine receptor accumulates
in neurons by anchoring to the cytoskeleton. LL5β, located at postsynaptic
membranes, binds CLASP2, which captures microtubules that have anchored
acetylcholine receptors. This focuses accumulation of acetylcholine receptors at
postsynaptic membranes. This localization of acetylcholine receptors is required
to capture acetylcholine signaling between neurons, and is organized by the
LL5β and CLASP2 transport system (Basu et al., 2015). The association of LL5β
with CLASP2 anchors the plus-end of microtubules to the plasma membrane,
facilitating the establishment of neuronal polarity.
CLASPs and the Golgi
The functionality of the Golgi apparatus is dependent on CLASPs. Early
on, CLASPs were observed to localize to the Golgi apparatus (Akhmanova etal.,
2001). Later work in human cells by Efimov et al., (2007) established that
CLASPs association is required for microtubule nucleation at the Golgi apparatus
in a centrosome-independent manner. Microtubule structures were ablated using
nocodazole, which results in depolymerization of cellular microtubules.
Microtubule polymerization was then observed forming a centrosomal-dependent
28
network as well as non-centrosomal microtubule network nucleating at the Golgi
apparatus (Efimov et al., 2007).
Nucleation of microtubules at the Golgi apparatus microtubule organizing
center requires gamma-tubulin to initiate polymerization, similar to centrosomal
nucleation (Figure 6). CLASPs are recruited to the trans-Golgi network by the
Golgi associated protein, GRIP and coiled-coiled domain containing 185 kilodaltons (GCC185). CLASPs bind to gamma-tubulin, which acts as a seed to
begin microtubule polymerization. Not only are CLASPs bound at the minus-end
(trans-Golgi network), but they are also required at the plus-end (the growing
microtubule tip) for proper anchored growth of microtubules. If CLASP is only at
the plus-end then treadmilling of microtubules occurs. However, if there is no
CLASP present to anchor microtubules, then there is no microtubule growth
originating from the Golgi apparatus (Efimov et al., 2007).
Figure 6: CLASPs in the cytoplasm are involved in microtubule nucleation from
the Golgi apparatus ultimately establishing cell polarity. M) The role of CLASPs in
Golgi-dependent microtubule nucleation shows that GCC185 attracts CLASPs to
the Golgi, which establishes γ-tubulin to initiate microtubule initiation. N) Golgidependent microtubule nucleation is essential in establishing cell polarity, which
is involved in vesicular trafficking, migration, actin assembly (Efimov et al., 2007).
29
Interestingly, it was found that microtubules nucleated from the trans-Golgi
network are preferentially oriented towards the leading edge of migrating cells.
Centrosomal microtubule networks are symmetrical. This implies a specific
mechanism of Golgi-derived asymmetric microtubule formation in polarized cells.
These findings support the idea that the Golgi apparatus is a crucial microtubule
organizing center for cell polarity and migration, and that CLASPs are required
for this function (Efimov et al., 2007).
CLASP2 Regulates Neuronal Polarity
Brain specific CLASP2 is involved in cytoskeletal rearrangements required
for neuronal polarity when forming dendritic and axonal projections. As was
previously discussed, axonal and dendritic growth occur through two different
mechanisms; however, both require dynamic rearrangement of the cytoskeleton
to establish the drastic polarization of neurons (Craig and Banker, 1994). Until
recently, it was unclear how microtubule rearrangements occurred during
neuronal development of synapses.
Expression of CLASP2 is enriched in neuronal growth cones and
increases throughout neuronal development. When CLASP2 expression is
knocked down, neurons developed stunted dendritic branching as well as shorter
axons. In the presence of abundant CLASP2, neurons develop multiple axons
30
and have increased arborization of dendrites. It also resulted in a condensed
Golgi, which is expected given the extensive relationship between CLASP2 and
the Golgi apparatus. The overexpression of CLASP2 also resulted in an increase
in synaptic vesicle release, a component of vesicular trafficking. This lines up
with previous knowledge of CLASP2 involvement with the Golgi apparatus.
CLASP2 directs neuronal polarity through its association with microtubule
dynamics (Beffert et al., 2012).
The microtubule plus-end tracking protein CLASPs are crucial in neuronal
polarity. CLASPs are known to be highly conserved across species, indicating
that they perform an essential function, and are known to have an extensive
association with the Golgi apparatus and Golgi associated proteins. There are
two mammalian homologs, CLASP1 and CLASP2, both of which are known to be
integral in establishing polarity. CLASP2 is exclusively expressed in the brain,
and aberrant regulation of CLASP2 results in faulty dendritic arborization and
axonal projection of neurons. Taken together, this signifies that CLASP2 is an
essential protein in establishing Golgi directed neuronal polarity.
GRIP and Coiled-Coil Domain Containing Protein 185
GRIP and coiled-coil domain containing protein 185 kilo-daltons (GCC185)
is a golgin protein. Golgins are a family of proteins containing coiled-coil domains
that are peripherally associated with the Golgi apparatus and involved in
31
tethering events during fusion of membranes. They also provide organizational
and structural support to Golgi cisternae. Depletion of golgins leads to a loss of
the normal Golgi ribbon structure resulting in fragmented stacks of Golgi (Derby
et al., 2007; Munro, 2011; Yoshino et al., 2003, 2005). Golgi vesicular trafficking
is reliant on the trans-Golgi golgin, GCC185 for structural support, organization,
membrane fusion, and protein recruitment. GCC185 has an intricate relationship
in Golgi processes (Brown et al., 2011; Derby et al., 2007; Efimov et al., 2007;
Hayes et al., 2009; Lin et al., 2011; Matsui et al., 2015). Because of this,
GCC185 is essential in establishing Golgi-dependent cell polarity.
Structure of Golgin Proteins
Golgins are ubiquitously expressed within the cell and are conserved
across multiple species. They have been described as tentacle-like projections
from the Golgi apparatus providing tethering sites for other binding partners
associated with the Golgi. The golgin family of proteins is grouped on the basis
that they are bound to the Golgi periphery and share two common structural
domains. A schematic representation of known golgins can be seen in Figure 7.
The first requirement is that golgins have a C-terminal domain that attaches them
to the Golgi apparatus, which varies depending on if the golgin is associated with
cis-Golgi, medial-Golgi, or trans-Golgi cisteranae (Munro, 2011).
32
Figure 7: Schematic representation of known human golgin domains. Each line is
a schematic of a human golgin protein with domains shown in different colors.
The domains are represented as the approximate proportion of the protein based
on structural analysis. Gray: predicted coiled-coil domains, yellow: GRIP domain,
purple: GRAB domain, green: ALPS domain, red: transmembrane domain (TMD)
(Munro, 2011).
Golgins also have a coiled-coil domain that is structurally comprised of
alpha-helices coiled together. This results in a rope-like structure that comprises
the majority of golgin proteins. The coiled-coil domains are not generally
conserved between golgins, and much of the space within the domain is
unstructured leading researchers to believe these stretches of the domain allow
golgins increased flexibility through hinge or extension regions (Munro, 2011).
33
Golgins that are localized to the trans-Golgi have a C-terminal (golgin-97,
RanBP2α, Imh1p, and p230/golgin-245) GRIP domain. GCC2, the human
homolog of GCC185, is a GRIP domain containing trans-Golgi golgin (Figure 7).
The GRIP domain is comprised of an approximately 80 amino acid conserved
motif that localizes GCC185 to the trans-Golgi. There has been much debate on
whether or not the GRIP domain is localized to the Golgi through association with
the small G protein ADP-ribosylation factor-like 1 (Arl1), with research supporting
both sides. Some GRIP domain proteins have been shown to be involved with
cargo export from the trans-Golgi network (Munro, 2011).
Cis-Golgi golgins do not have one universal method of attaching to the
Golgi apparatus. GM130 has a domain on the carboxy terminus that binds to the
PDZ domain, a common attachment domain found in proteins involved in
signaling pathways, of the Golgi structural protein GRASP65. Another cis-Golgi
golgin, GMAP-210 utilizes a GRAB domain to attach to the Golgi, which functions
similar to the GRIP domain discussed previously. The golgins that attach to the
cytoplasmic surface of medial cisternae do so through anchoring into the
membrane via their transmembrane domain (Munro, 2011).
Establishment of neuronal polarity requires the Golgi apparatus during
axonal growth and dendritic arborization. Golgins play an essential role in the
Golgi apparatus by acting as tethering sites for Golgi associated proteins. The
trans-Golgi golgin GCC185 is required for the association of multiple proteins
34
with the Golgi apparatus, including CLASPs. We will discuss the association
between GCC185 and relevant Golgi associated proteins, as well as its role in
establishing neuronal polarity.
GCC185 and Rab GTPase Binding Sites
Vesicular tethering at the Golgi apparatus is crucial for trafficking vesicles,
and processing secreted and membrane bound proteins. The structure and
function of the Golgi apparatus is sensitive to the loss of golgin proteins because
of the role they play in tethering cisternae. Cells depleted of GCC185 have an
accumulation of vesicles in the cytoplasm because they lose the ability to fuse to
the trans-Golgi network (Reddy et al., 2006). GCC185 has binding sites for up to
14 Rab GTPases, which act in affiliation with Arl1 GTPases to localize GCC185
to the trans-Golgi network. Through Rab binding GCC185 is able to maintain
normal Golgi structure and stack morphology, and functional loss of GCC185
results in fragmentation of the Golgi (Hayes et al., 2009). This establishes the
model that GCC185 is a vesicular tether at the trans-Golgi network.
GCC185 and AP-1 Protein
Mannose 6-phosphate receptors are transmembrane glycoproteins that
bind newly synthesized acid hydrolases in the trans-Golgi network, and
participate in their delivery to lysosomes Ghosh et al., 2003). After mannose 6-
35
phosphate receptors deliver the cargo to the lysosomes they need to be recycled
back to the trans-Golgi network. Cells depleted of GCC185 accumulate mannose
6-phosphate receptors in transport vesicles (Reddy et al., 2006). Clathrin adaptor
proteins bind cytoplasmic domains of cargo proteins and recruit clathrin coating
to induce vesicle budding. Clathrin adaptor protein 1 (AP-1) binds the
cytoplasmic domain of mannose 6-phosphate receptors. Retrograde transport of
mannose 6-phosphate receptors occurs by attachment of AP-1, which has a
binding site on GCC185. The golgin protein, through its GRIP localization
domain, shuttles the complex to the trans-Golgi network ultimately recycling the
mannose 6-phosphate receptors (Brown et al., 2011).
The AP-1 mediated vesicle tethering by GCC185 shows that the golgin
plays an essential role in vesicular trafficking. As discussed previously, the Golgi
apparatus needs to orient vesicular trafficking to establish neuronal polarity.
Taken together, this indicates the GCC185 function in vesicular trafficking, plays
a role in Golgi-dependent establishment of neuronal polarity.
GCC185 and CLASPs
Up to this point, we have discussed how GCC185 can be involved in
vesicular trafficking and structural organization of the Golgi apparatus through
association with other proteins. In regards to the role the Golgi apparatus plays in
microtubule organization of cell polarity, the most important proteins GCC185
36
interacts with are CLASPs. As was previously established, CLASPs are required
for Golgi structure and organization, and ultimately for establishing neuronal
polarity. In this section, we will discuss how GCC185 and CLASP interaction is
required for normal Golgi function and establishing cellular polarity through its
control over microtubule dynamics.
GCC185 is involved in recruiting the plus-end microtubule binding
CLASPs to the trans-Golgi network in a similar fashion to how GCC185 tethers
AP-1 to recycle mannose 6-phosphate receptor vesicles to the Golgi (Efimov et
al., 2007; Hayes et al., 2009). This interaction acts to bridge together the transGolgi network with CLASP, which organizes Golgi-derived microtubules to
maintain structural support and organization, and establish cell polarization and
motility (Efimov et al., 2007; Miller et al., 2009).
ADP-ribosylation factor-like 4A (ARL4A) is a GTPase that acts as a
positive modulator of the GCC185-CLASP interaction. ARL4A interacts with the
coiled-coil 2 (CC2) sub-region of GCC185 (Figure 8), in a GTP-dependent
manner. Depletion of ARL4A or deletion of the ARL4A interacting region CC2 on
GCC185 abolishes the interaction between GCC185 and CLASP resulting in
fragmentation of the Golgi. Taken together, this data suggests that binding by
ARL4A to GCC185 facilitates the interaction between GCC185 and CLASPs (Lin
et al., 2011).
37
Figure 8. Schematic representation of GCC185 fragments. GCC185 is a
representation of the full length protein. CC1, 2, and 3 are predicted CC domains.
Full length GCC185 was fragmented into the various CC domains to determine
which domains are required for protein-protein interactions. CC: coiled-coil, CTD:
C-terminal domain, C270: C-terminal 270 amino acid fragment (Lin et al., 2011).
The cell polarity protein complex comprised of PAR3, PAR6, and atypical
phosphokinase-C (aPKC) is essential in several aspects of cell polarity. PAR6
acts as an adaptor protein holding aPKC and PAR3 together. PAR3 interacts
with the brain specific CLASP2, and negatively regulates its interaction with
GCC185 in epithelial cells. PAR3 complexes with CLASP2 by binding to the
middle of the protein in the serine-rich region, and escorts aPKC to
phosphorylate the C-terminal region of CLASP2 between amino acids 900-1000.
Knockdown of either PAR3 or aPKC resulted in hyper accumulation of CLASP2
to the trans-Golgi network. This means that phosphorylation of CLASP2 by aPKC
38
inhibits the binding of GCC185 and CLASP2.
The aberrant accumulation of
CLASP2 at the trans-Golgi network resulted in fragmentation of the Golgi
apparatus. PAR3 and aPKC regulation of CLASP2-GCC185 interaction controls
Golgi organization, and ultimately cellular polarity (Matsui et al., 2015).
Taken together this data suggests that PAR3 escort’s aPKC, via the
PAR3, PAR6, aPKC complex, to CLASP2 where aPKC phosphorylates
CLASP2s C-terminal region. Once phosphorylated CLASP2 is targeted to the
plus-end of microtubules. This data concurs with results discussed previously
that found GSK3β phosphorylation of the C-terminal region of CLASPs localize
them to the plus-end of the microtubule structure. Loss of aPKC or PAR3 activity
results in CLASP2 accumulation at the trans-Golgi network. These data suggest
that the cell polarity complex regulates the interaction between GCC185 and
brain specific CLASP2, modulating Golgi-dependent cellular polarity.
CONCLUSION:
The Golgi apparatus plays an essential role in establishing neuronal
polarity through its function as a microtubule organizing center, and control of the
secretory pathway. During development, the cortex requires proper migration of
neurons to establish layering. To migrate, neurons send out a leading process
that adheres to the extracellular matrix through binding of N-cadherins. This
39
provides leverage for locomotion of the cell. The Golgi apparatus is required for
recycling of N-cadherin from the trailing edge to the leading edge.
The Golgi apparatus also orchestrates trafficking of proteins and lipids
towards the leading edge by establishing cell polarity. This occurs on two levels.
First, the Golgi orients itself from the endoplasmic reticulum toward the leading
edge. This results in a bias in vesicular trafficking towards the edge, increasing
the total membrane in that region for expansion of filopodial projections, as well
as proteins required for cell movement and adhesion.
The second, and likely most important method of establishing cell polarity
is through establishing a non-centrosomal microtubule organizing center. The
Golgi apparatus establishes the microtubule organizing center through its
association with CLASPs. CLASP proteins are required at the trans-Golgi
network to anchor the Golgi to the cytoskeleton, as well as attract γ-tubulin,
which acts as the seed for growing microtubule networks. CLASPs also maintain
established microtubules by binding along their length and stabilizing them to
prevent microtubule catastrophe. At the growing plus-end of microtubules,
CLASPs bind to attach microtubules to the plasma membrane. If CLASPs ability
to perform any of these functions is modified or lost, it results in loss of the Golgi
structure and function, catastrophe for the cytoskeleton, and loss of cell polarity.
There are multiple supporting proteins that associate with CLASPs and
are required for its proper function. The trans-Golgi network specific golgin
40
GCC185 tethers CLASPs to the Golgi anchoring the cytoskeleton. The PAR
complex negatively regulates brain specific CLASP2 interactions with GCC185.
PAR3 binds directly to CLASP2, bringing the aPKC in range to phosphorylate the
C-terminus of CLASP2 localizing it to the plus-end of microtubules. CLASPs
have also been shown to be phosphorylated at the C-terminus by GSK3β
targeting it to the plus-end of microtubules. This suggests that unphosphorylated
CLASPs are localized to the trans-Golgi network. Loss of function to any of a
number of CLASP-associating proteins has implications on Golgi organization,
cytoskeletal structure, and cell polarity.
The brain specific CLASP2 is required for the Golgi apparatus to establish
neuronal polarity. Polarity in neurons is essential in establishing proper dendritic
arborization and axonal projection. Without these neuron specific structures,
neurons are unable to migrate properly during development or establish
synapses to relay information. Loss of CLASP2 results in decreased dendritic
arborization and axon length; while over expression of CLASP2 results in
multiple axons being formed, as well as increased dendritic branching. Both of
these phenotypes result in improper neurodevelopment signifying the critical role
CLASP2 plays in Golgi regulated neuronal polarity.
The Golgi apparatus plays a crucial role in establishing neuronal polarity.
Future studies in this area should aim to elucidate the exact mechanisms of how
the Golgi apparatus modulates neuronal polarity in primary neurons or in vivo.
41
Much of the work on cell polarity up to this point has been performed in nonneuronal cells. For example, the work on GCC185 interaction with brain specific
CLASP2 being modulated by the PAR complex was performed in COS-7 (kidney)
cells expressing exogenous CLASP2. It is clear that CLASP2 plays an essential
role in establishing neuronal polarity, and that CLASP2 has an intimate
relationship with the Golgi apparatus. The next steps should be to perform similar
studies with CLASP2 and associated proteins, such as GCC185, in primary cell
cultures and in vivo, to further elucidate the mechanism of Golgi-dependent
neuronal polarity.
42
JOURNAL ABBREVIATIONS:
Abbreviation
Annu. Rev. Cell Dev. Biol.
Annu. Rev. Neurosci.
Biochim. Biophys. Acta.
Cell Mol. Life Sci.
Cell Rep.
Cold Spring Harb.
Perspect. Biol.
Curr. Opin. Cell Biol.
Curr. Opin. Neurobiol.
Dev. Cell
Front. Cell Neurosci.
Front. Neuroanat.
Genes Dev.
Histochem. Cell Biol.
Int. Rev. Cell Mol. Biol.
J. Cell Biol.
J. Cell Sci.
J. Leukoc. Biol.
J. Neurosci.
Mol. Cell Neurosci.
Mol. Biol. Cell
Nat. Cell Biol.
Nat. Genet.
Nat. Rev. Mol. Cell Biol.
Neuropsychol. Rev.
Plant Physiol.
Prog. Biophys. Mol. Biol.
Trends Biochem.
Trends Cell Biol.
Journal Name
Annual Review of Cell and Developmental
Biology
Annual Review of neuroscience
Biochimica Et Biophysica Acta
Cellular and Molecular Life Sciences
Cell Reports
Cold Spring Harbor Perspectives in Biology
Current Opinion in Cell Biology
Current Opinion in Neurobiology
Developmental Cell
Frontiers in Cellular Neuroscience
Frontiers in Neuroanatomy
Genes & Development
Histochemistry and Cell Biology
International Review of Cell and Molecular
Biology
Journal of Cell and Biology
Journal of Cell Science
Journal of Leukocyte Biology
Journal of Neuroscience
Molecular and Cellular Neuroscience
Molecular Biology of the Cell
Nature Cell Biology
Nature Genetics
Nature Reviews Molecular Cell Biology
Neuropsychology Review
Plant Physiology
Progress in Biophysics and Molecular Biology
Trends in Biochemical Sciences
Trends in Cell Biology
43
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50
CURRICULUM VITAE:
Tyler D. Ash, 810-814-0593, [email protected], 2350 Nadine Drive, Sterling Heights, MI 48825
Education:
Boston University, Boston, Massachusetts
• Master of Science in Molecular Biology, Cellular Biology, and Biochemistry
• Research performed in Molecular Biology of the Neuron Lab
Michigan State University, East Lansing, Michigan
• Bachelor of Science in Microbiology
• Lyman Briggs Residential College for the Study of Science
• Minor: History, Philosophy, and Sociology of Science
May 2015
May 2013
Research Experience:
Graduate Student, Dr. Angela Ho, Cellular and Molecular Neuroscience Laboratory
Boston University, Boston, Massachusetts
December 2013 – February 2014, July 2014 – May 2015
• Mastered various molecular biology techniques including confocal microscopy,
cloning, shRNA knockdown of targeted proteins, infection and transfection of
cell cultures, western blot, immunohistochemistry, immunocytochemistry, coimmunoprecipitation , and aseptic subculturing techniques
• Worked with mice as models for development
• Presented research data as well as journal papers in lab meetings and journal club
Laboratory Rotation, Dr. Tarik Haydar, Laboratory of Neural Development
Boston University, Boston, Massachusetts
• Performed confocal microscopy, cloning, shRNA protein knockdown,
western blot, immunocytochemistry, and subculturing techniques
• Established N2A cell line from mouse neuroblastoma in the lab
May – June 2014
Laboratory Rotation, Dr. Aaron Beeler, Medicinal Chemistry Laboratory
March – April 2014
Boston University, Boston, Massachusetts
• Performed pull-down assays to identify protein binding partners for drug targets
• Exposed to the process of organic synthesis
Laboratory Rotation, Dr. Horacio Frydman, Host-Microbe Interactions in
Stem Cell Niche Laboratory Boston University, Boston, Massachusetts September – November 2013
• Dissected reproductive organs of Drosophila
• Performed confocal microscopy and immunohistochemistry
Research Assistant, Dr. Mark Whalon, Alternative Pesticides
Laboratory, Michigan State University, East Lansing, Michigan
• Wrote portions of federal grant proposals, reports, and policy
papers
• Performed insect dissections, database management, in vitro
bio-assays, grant budget management, and field work
June 2010 – August 2013
51
Research Assistant, Dr. Cindy Arvidson, Pathogenesis of Neisseria
gonorrheae Laboratory Michigan State University, East Lansing,
Michigan
September 2011 – May 2013
• Research funded for $1,000 by the National Science Foundation
• Performed DNA extraction and transformations
• Trained an undergraduate student to perform DNA extractions,
gene transformations, and other general lab duties
Research Intern, Dr. Douglas Luckie, Genetic Disease Research
Laboratory, Michigan State University, East Lansing, Michigan
• Pursued, presented, and defended results on a research
project aimed at developing a PCR-based diagnostic assay for
a mutation known to cause sickle cell anemia
• Gained experience with PCR, DNA sequence analysis, primer
design, agarose gel electrophoresis with both TBE and rapid
Lithium Borate
January 2011 – May 2011
Lab Training Completed:
Responsible Conduct of Research Training
IACUC-Mouse Handling
Biosafety Level 2
Office of Radiation, Chemical and Biological Safety
February 2015
August 2014
September 2011
June 2010
Honors/Awards:
First place award at University Undergraduate Research and Arts Forum (UURAF)
Michigan State University
• Cell Biology, Genetics, and Genomics for poster presentation
of Detection of 1510 BP Band Using PCR and Gel
Electrophoresis to Diagnose E6V Sickle Cell Anemia in Human
Genomic DNA
Lyman Briggs College National Science Foundation S-STEM Scholarship
• Awarded $18,000.00 of funding over three years to
undergraduates at Michigan State University in Lyman Briggs
College with an interest in working in a research related
scientific field in the future who have maintained a minimum
3.0 GPA
Lyman Briggs College National Science Foundation STEM Research Funding
• $1,000.00 of funding was provided in support of research through the
Pathogenesis of N. gonorrheae lab to work with gene regulation
Activities:
Study Abroad: History of Science in Florence and Rome, Italy
• Studied the interactions between Galileo and the Roman Catholic
Church during the transition from geocentrism to heliocentrism
• Examined how scientific discoveries influence societies and cultures
Spring 2011
August 2010
July 2012
May – June 2012
52
Skills:
• Skilled in grant writing and budget management
• Proficient in Zen microscopy software, R statistical package, Adobe
InDesign and Photoshop
Professional Works:
Title
Authorship
Type of Work
Functional Ecology
Indicators to Help Defend
Michigan Apple Production
Practices, 2010 Summary
Mark Whalon,
Jeanette Wilson, Tyler
Ash
Poster presented at the
Great Lakes Expo, 2010
USDA Risk Avoidance and
Mitigation Program in
Michigan Tart Cherries: Pest
Status, Economics, and
Ecological Effects in the
Transition from
Organophosphates to
Reduced Risk IPM
Detection of 1510 BP Band
Using PCR and Gel
Electrophoresis to Diagnose
E6V Sickle Cell Anemia in
Human Genomic DNA
Mark Whalon,
Jeanette Wilson, Tyler
Ash, Alex Johnson
Poster presented at the
Entomological Society of
America Conference, 2010
Tyler Ash, Jon
Walters, Kristina
Knirk, Nicole Arcy
Poster presented at
University Undergraduate
Research and Arts Forum
(UURAF) at Michigan State
University, 2011
Functional Ecology
Indicators to Help Defend
Michigan Apple Production
Practices, 2011 Summary
Mark Whalon, Tyler
Ash
Poster presented at the
Great Lakes Expo, 2011
US Tart Cherries: Pesticide
Policy and the Precautionary
Principle, 22: (4) 177-182.
Outlooks on Pest
Management
Acknowledged
Journal Publication, 2011
Risk Avoidance and
Mitigation Program in Tart
Cherries: a Seven Year
Project
Multiple authors
including Tyler Ash,
can be provided upon
request
United States Department
of Agriculture (USDA)
Funded Grant Report, 2012
Tart Cherry Pest
Management in the Future:
Development of a Strategic
Plan
Multiple authors
including Tyler Ash,
can be provided upon
request
Policy Document submitted
to the United States
Environmental Protection
Agency (EPA), 2012