Download MB207_15 - MB207Jan2010

MB 207 Molecular Cell Biology
The Cytoskeleton
The eukaryotic cytoskeleton
 The cytoskeleton is a cellular
"scaffolding" or "skeleton" contained,
as all other organelles, within the
cytoplasm.
 It is a dynamic structure that provide
structural support to the cell. Besides
that, cytoskeleton also functions in cell
motility and regulation.
 It is a fibrous network made of three
families of protein molecules, which
assemble to form 3 main types of
filaments.
Three main types of fibers in the cytoskeleton
• Each structural element of the cytoskeleton is formed by the
polymerization of different kind of subunit.
eg. Microtubules are composed of protein tubulin.
Microfilaments are polymers of protein actin.
• In addition, each type of cytoskeletal filament has a number of
proteins associated with it.
eg. Accessory proteins facilitate in the structural and functional diversity of
cytoskeletal elements.
The cytoskeleton is dynamically assembled and disassembled
•
•
•
•
•
Each type of the cytoskeletal element is
constructed from smaller protein subunits.
→ repetitive assembly of large numbers of the
small subunits.
These subunits are small that they an diffuse
rapidly within cytoplasm whereas assembled
filaments cannot.
All the three types of cytoskeletal filaments selfassociate using a combination of end-to-end and
side-to-side protein contacts.
The cytoskeletal ‘polymers’ are held together by
weak non-covalent interactions.
→ Assembly and disassembly can occur rapidly.
Cells can undergo structural organization,
disassembling filaments at one site and
reassembling them at another site far away.
Level of organization affects thermal stability
Microtubules (MT)
• Classified into two general groups, cytoplasmic microtubules and
axonemal microtubules.
→ organization and structural stability.
• Cytoplasmic microtubules (loosely organized):
i) Animals
- required to maintain axons.
- maintain polarized shape.
ii) Plants
- govern the orientation of cellulose microfibrils deposited during the growth of cell
walls
iii) Others
- form the mitotic and meiotic spindles.
- facilitate the movement of vesicles and other organelles.
• Axonemal microtubules (highly organized)
Nucleation
- associated with cellular movement (ie. Cilia, flagella and the basal bodies to which
these appendages are attached).
• MT are polarized structures composed of α- and β- tubulin heterodimer
subunits assembled into linear protofilaments.
→ noncovalent binding.
• A single MT is comprised of 10-15 protofilaments (usually 13 in mammalian
cells) that associate laterally to form a 24nm wide hollow cylinder.
• Different polymerization rates at two ends:
→ In each protofilament, the heterodimers are oriented with their β-tubulin
monomer pointing towards the faster-growing end (plus end) and their α-tubulin
monomer exposed at the slower-growing end (minus end).
• Each tubulin subunit has a GTP-binding domain at N-terminus, a domain in
the middle to which MT poison colchicine can bind and a third domain at the Cterminus that interacts with MT-associated proteins.
• MT can form as singlets, doublets or triplets.
→ Doublets: cilia and flagella
Triplets: basal bodies and centrioles
Arrangement of protofilaments in singlet, doublet, and triplet
microtubules
• Singlet composed of a 13 protofilaments tubule (A).
• Doublet composed of a singlet (A tubule) and one additional B
tubule consisting of 10 protofilaments appended to its side.
• Triplet composed of A tubule plus additional B and C tubules of 10
protofilaments.
Microtubule assembly
• Assemble by polymerization of tubulin dimers.
• Addition and loss only at the ends.
• Addition of GTP-bound tubulin is favored over GDP-bound.
• Assembly and disassembly depend on critical concentration of αβ
subunits.
Tubulin dimers
Oligomers
Protofilament
Sheets of
protofilaments
Closing
microtubule
Elongating
microtubule
Assemble by polymerization of tubulin dimers
• At the start of the nucleation process, several dimers can aggregate into
clusters called oligomers.
• One an MT has been nucleated, it grows by addition of subunits at either end
to form linear chains of tubulin dimers called protofilaments.
• The protofilaments can then associate with each other side by side to form
sheets.
• Sheets containing 13 or more protofilaments can close into a tube, forming a
microtubule.
• Elongation of the microtubule continues by the addition of tubulin subunits at
one or both ends.
Addition of tubulin dimers occurs more quickly at the plus ends of
microtubules
•
•
•
•
•
One end can inherently grow or shrink much faster than the other.
Rapidly growing end of the microtubule is called plus end and the other end
is the minus end.
Growth rates depending on critical concentrations of free tubulins.
→ [tubulinsfree] is higher than the [critical] for the plus end but lower at the
minus end, assembly will occur at the plus end while disassembly takes
place at the minus end.
Simultaneous assembly and disassembly → treadmiling
Assembly: Relatively smooth ends; some protofilaments are longer
(elongate unevenly)
Disassembly: Ends are splayed; frayed appearance
Drugs that affect the assembly of microtubules
• Colchicine
→ binds to tubulin monomers, strongly inhibiting their assembly into
mirotubules and fostering the disassembly of existing ones.
• Vinblastine and vincristine
→ promote tubulin aggregation inside the cell.
• Nocodazole
→ inhibits MT assembly (effects are more readily reversible when the drug is
removed)
¤ antimitotic drug – disrupt mitotic spindle of dividing cells, blocking the further
progress of mitosis.
 Taxol
→ binds tightly to microtubules and stabilizes them, causing much of the
free tubulin in the cell to assemble into microtubules and arrests dividing
cells in mitosis.
GTP cap and its role in dynamic instability of microtubules
• Stable when capped with GTP-tubulins and unstable when capped with
GDP-tubulins.
• Each tubulin heterodimer binds two GTP molecules.
→ α-tubulin binds one GTP;
→ β-tubulin binds to the other GTP which can be hydrolyzed to GDP
sometime after the heterodimer is added to an MT.
Microtubule-organizing enters (MTOC)
• serves as a site at which MT assembly is initiated and acts as an anchor for one end
of these MTs.
• minus ends are anchored in the MTOC and plus ends extend toward the cell
membrane.
• MTOC in animal cells: centrosome
Microtubule-associated proteins (MAPs)
• Increase MT stability and affect the density of bundles of MTs.
• Stabilizing MAPS
→ Tau: causes microtubules to form tight bundles in axons.
→ Map2: causes the formation of looser bundles of MTs.
→ +-TIP proteins: stabilize plus ends.
Microtubule destabilizing MAPs
→ stathmin/Op18: increases depolymerization by binding tubulin dimers.
Microtubule severing MAPs
→ catastrophins: promoting the peeling of subunits from their ends.
Accessory proteins regulate filament dynamics
Microfilament (MFs)
• smallest cystoskeletal filaments with a diameter of about 7nm.
• important in mediating cell movement and maintaining cell shape.
→ found at sites of attachment of cells to one another and to the extracellular
matrix
• building block of MFs is actin.
→ G-actin: individual actin molecules.
→ F-actin: polymerization of G-actin to
form microfilaments
• Two major groups of actin:
→ muscle-specific actins (α-actins)
→ nonmuscle actins (β- and γ-actins)
G-actin monomers polymerize into F-actin microfilaments
• F-actin filaments that form are composed of two linear strands of
polymerized G-actin wound around each other in a helix, with roughly 13.5
actin monomers per turn.
• Subunits assembled in a head-to-tail manner hence protofilaments have
polarity.
+ end: fast growing (barbed)
- end: slow growing (pointed)
• As G-actin monomers assemble into mirofilament, ATP bound to them
is slowly hydrolyzed to ADP.
→ Ends of growing MF tend to have ATP-F-actin whereas the bulk of
the
MF is composed of ADP-F-actin.
Actin-binding proteins regulate polymerization, length and
organization of microfilaments
Polymerization
• Growth of MFs depend on [ATP-bound-G-actin]
• Large amount of free G-actin is not available for assembly into filaments.
→ bound by protein thymosin β4
• Profilin transfer G-actin monomers from thymosin β4 complex to the end of growing
filament only when free filament ends available.
Filaments capping
• Capping prevents further addition or loss of subunits, thereby stabilizing it.
• Example: i) CapZ: binds to the plus ends of actin filaments.
II) tropomodulins: binds to the minus ends of actin filaments, preventing loss of
subunits from the pointed ends of F-actin.
Crosslinking actin filaments
• filamin: long molecule consist of two identical polypeptides
joined head to head, with an actin-binding site at
each tail.
→ joining two MFs together where they intersect.
Severing actin filaments
• gelsolin: breaking MFs and capping newly exposed plus
ends, thereby preventing further polymerization.
→ can be regulated by polyphosphoinositides.
Promoting actin branching and growth
• Arp2/3 complex: helps branches to form by nucleating new branches on the sides of
existing filaments.
Intermediate filaments (IF)
 Size is intermediate filaments: 10 nm outer diameter
 Polypeptides (constituent of polypeptides vary, six major classes) have an
alpha-helical core and flanking globular domains
 Dimers form a coiled-coil, with amino terminal at the same end
– Dimers may be homo- or heterodimers
 Dimers can associate with each other in an antiparallel fashion to form the
tetramers (the protofilaments for IF assembly and are antiparallel)
 Tetramers is the soluble subunit of IF.
 8 protofilaments pack together laterally to form the IF assembly (10nm rope
like filament, having 16 dimers in cross section)
 Accessory proteins e.g. filaggrin and plectin help to bundle IF as well as link
them to other filaments as well as motor proteins
 Intermediate filament structure depends on the lateral bundling and twisting
of coiled coils
IF structure ad assembly
•
General functions: Provide mechanical strength and resistance to remove stress.
Various types of IF in vertebrate cells
TYPES OF IF
COMPONENT POLYPEPTIDES
CELLULAR LOCATION
Keratins type I
Acidic keratins
epithelia
Keratins type II
Neutral/basic keratins
Epithelia
Vimentin-like
IFs
Vimentin
During embryogenesis
Desmin
Muscle
Glial fbrllary acidic protein
Astrocytos
Peripherin
Neurons
Neurofilaments
Internexin
Neurons
Lamins
Lamin
Nucleus, almost ubiquitous
Nestins
Nestin
Neuro-epithelial stem cells
Others
Filensin, phakinin
lens
Keratin defects in the skin
Motor proteins
1. Cellular functions
2. Structure and organization
3. Cytoskeletal motor proteins
3.1 Actin motors
3.1.1 Myosin
3.2 Microtubule motors
3.2.1 Kinesin
3.2.2 Dynein
4. Diseases associated with motor protein
Motor Proteins
•
Motor proteins attach to microtubules
or microfilaments and to their cargo
(vesicles, organelles, chromosomes,
other cytoskeletal filaments). Motor
proteins are the driving force behind
most active transport of proteins and
vesicles in the cytoplasm.
•
Functions:
– Carry membrane organelles or
secretory vesicles to their
appropriate locations in cell
– Cause cytoskeletal filaments to
slide against each other ,
generating force that drives
phenomenon such as muscle
contraction, ciliary beating and cell
division
•
Structure:
Most eukaryotic motor proteins consist
of 2 distinct domains:
Motor head domain
Tail domain
• With ATPase
function, the proteins
carries out the
movement by binding
to a specific site on
the substrate and
changing
conformation.
• Can either form
fibers (muscle myosin)
or attach to cargo.
• Example
chromosomes during
anaphase of mitosis
(kinesin) or vesicles
during endocytosis
(dynein)
• Binds adaptor
proteins that allow for
stable interactions with
the cargo to be moved
along the substrate.
Families of motor proteins:
a. Myosins (along microfilaments or
actin)
-is responsible for muscle
contraction
b. Kinesins (along microtubules)
-moves cargo inside cells away
from the nucleus along
microtubules
c. Dyneins (along microtubules)
-produces the axonemal beating of
cilia and flagella and also
transport cargo along microtubule
towards the cell nucleus
Myosins
• More than 20 known classes of myosins.
• General structure:
¤ have at least one polypeptide called heavy chain with a globular head group at one
end attached to a tail of varying length.
¤ Globular head binds to actin and uses energy of ATP hydrolysis to move along an
actin filament.
¤ structure of tail region varies among different kinds of myosin, giving myosin
molecules the ability to bind to various molecules or cell structures.
¤ tail structure also determines the ability of myosins to bind to other identical myosins
to form dimers or large arrays.
¤ small polypeptides, light chain, bound to the globular head group which often play a
role in regulating the activity of the myosin ATPase.
• Functions:
¤ muscle contraction (muscle myosin II)
¤ cell movement (nonmuscle myosin II)
¤ phagocytosis (myosin VI)
¤ vesicle transport or other membrane-associated events (myosin I, V)
Microtubule motors
Dynein
Kinesin
Kinesins
• At least 10 families.
• General structure:
¤ A globular head region that attaches to microtubules and is involved in hydrolysis of
ATP.
¤ A coiled helical region.
¤ A light chain region that is involved in attaching kinesin to other proteins and
organelles.
• Movement:
¤ One of the two globular heads moves forward to make an attachment to a new βtubulin subunit.
¤ This is followed by detachment of the trailing globular head which can now make an
attachment to a a new region of the MT.
¤ This movement is coupled to the hydrolysis of ATP bound at specific sites within the
heads which resulted kinesin moves toward the plus end of an MT in an ATP-dependent
manner.
• Function:
¤ moving and localizing substances within cells.
Dyneins
• Consist of two types:
¤ Cytoplasmic dyneins
→ contains two heavy chains that interact with MTs, two intermediate chains, two
light intermediate chains and various light chains.
→ moves toward the minus ends of MTs.
→ associated with protein complex, dynactin.
→ involved in shaping endomembrane system and vesicle transport.
¤ Axonemal dynein
→ basal body consists of nine sets of tubular structures arranged around its
circumference, ‘9+2’ pattern (nine outer doublets of tubules and two additional
microtubules in the center).
→ Each outer doublet consists of one complete MT (A tubule) and one incomplete
MT (B tubule).
→ Sidearms that project out from each of the A tubules which is responsible for
sliding MTs within the axoneme past one another to bend the axoneme.
→ Radial spokes project inward from each of the 9 MT doublets for slidingof adjacent
doublets into the bending motion that characterizes the beating of these appendages.
• Functions: Motility (Cilia and flagella)
Diseases associated with motor protein defects
a. Kinesin deficiencies have been identified as cause for Charcot-Marie-Tooth
disease and some kidney diseases.
b. Dynein deficiencies can lead to chronic infections of the respiratory tract as
cilia fail to function without dynein.
c. Defects in muscular myosin predictably cause myopathies, whereas defects in
unconventional myosin are the cause for Usher syndrome and deafness.