Chapter 16: Cytoskeleton
Know the terminology:
Actin, microfilament, thin filament, tubulin,
microtubule, intermediate filament, microtubuleassociated protein, flagella, cilia, pseudopodia,
lamellapodia, fillipodia, MTOC, linker proteins,
accessory proteins, motor proteins, myosin,
dynein, kinesin, capping, cross-linkers, MAPs,
focal contacts, cadherins
What is the cytoskeleton?
… a network of microtubules
(MT), microfilaments (MF),
intermediate filaments (IF),
and their accessory proteins
used in conjunction with
motor proteins.
Chapter 16: Cytoskeleton
Outline:
I. Cytoskeletal elements and their roles
II. Cytoskeleton structure and assembly
III. Regulation of assembly
IV. Molecular motors
I. Roles of the
cytoskeleton
Its capacity to undergo
assembly and disassembly
is central to its many
structural and functional
roles.
I. Roles of the cytoskeleton
Cell structure
•Cell shape (microvilli)
•Internal organization (membrane networks)
•Physical robustness (erythrocyte deformability)
Cell function
•Cell movement (lamellapodia, flagella, cilia)
•Organismal movement (muscles)
•Intracellular traffic (vesicle and organelle traffic)
•Signal transduction
II. Cytoskeletal structure
Cytoskeleton is composed primarily of 3 types of
polymers
Microfilaments (~5nm diameter)
Intermediate filaments (~10nm)
Microtubules (~25nm)
Increasing size
Polymer structure
Polymers are constructed from repeating monomers
Microfilaments (mainly polymers of actin)
Intermediate filaments (diverse subunits)
Microtubules (mainly polymers of tubulin)
How are these analogous to muscles, ligaments,
and bones of a musculoskeletal system?
Microfilaments
Most organisms have multiple isoforms of actin
-most cells have β, γ
-muscles have α
Very conserved structure between isoforms and
across species, but subtle variations are important
Actin polymers form microfilaments that are used
to build fibers and networks
Microfilaments: Fibers and networks
Microfilaments
Microtubules
Most organisms have multiple isoforms of both α
and β tubulin (Other tubulins (γ) are involved in MT
assembly)
Very conserved structure between isoforms and
across species but subtle variations are important
Microtubules are organized to form an intracellular
network, radiating from the MT organizing center
(MTOC)
Also the basis of cilia and flagella
Microtubules: MTOC
Microtubules: Flagella
Intermediate filaments
Composed of many different types of subunits
Each subunit is a tetramer
Filaments can be bundled together or cross-linked
via accessory proteins
Types include keratins (hair, scales, nails, claws)
and neurofilaments
Polymer structure
Polymers provide structural stability
Polymer structure
Polymers provide structural stability
Polymer structure
Polymers provide structural stability
Tubulin polymerization and microtubules
Tubulin is a dimer composed of:
α−tubulin & β−tubulin
Both proteins are GTP-binding proteins
-the GTP in α−tubulin is part of its structure
-the GTP in β−tubulin can be hydrolyzed
Tubulin monomers join end to end to form
protofilaments
Protofilaments join side by side to form
microtubules
Polymerization of tubulin to produce
microtubules
Polymerization of actin to produce
microfilaments
Actin is a monomeric protein ATP/ADP binding
protein
Nucleotide hydrolysis
The NTPs in the monomers can be hydrolyzed to
NDPs (Tubulin GTP to GDP, Actin ATP to ADP)
Monomers with NDP are more likely to dissociate
than are monomers with NTP.
Monomers hydrolyze
NTP to make NDP, but
exchange NDP for NTP
(they do not function
as ATP/GTP
synthases)
Nucleation
2 monomers can join
together to begin
the formation of a
polymer (nucleation)
This first step is
the slowest step in
polymer formation
Polarity
The growing polymer has a plus (+) end.
While monomers can bind to either end, they are
much more likely to:
-bind to the + end
-leave the minus (-) end
Assembly and disassembly
Assembly and disassembly occur simultaneously
The direction of growth and rate of growth are
controlled
At a Critical concentration (Cc) the rates of
assembly and disassembly are equal.
The plus and minus ends have different Cc values
Polarity, hydrolysis, and critical
concentrations
Capping effects on polymerization
If the plus end of
the polymer has a
series of monomers
with NTPs, this is
referred to as a
“cap”
A capped polymer is
more likely to grow.
Capping effects
A capped microtubule
is stable
GTP hydrolysis
accelerates
disassembly
Summary
Cytoskeletal proteins are polymers of repeating
subunits.
Many structural and functional roles within cells
Assembly and disassembly important to their
actions
They have intrinsic polarity, tending to grow from
the + end.
III. Regulation of cytoskeleton assembly:
1. Nucleation sites
2. Elongation proteins
3. Stabilizing proteins
4. Capping proteins
5. Cross-linking proteins
6. Severing proteins
Many of these
proteins are
regulated by protein
kinases and protein
phosphatases
Signal transduction
pathways often act
through changes in
the cytoskeleton
Regulation of cytoskeleton assembly:
1. Nucleation sites
1. Microtubule organizing center (MTOC)
A structure around paired centrioles, near the
nucleus
Acts as nucleating site for MT growth
Binds the minus ends of outwardly directing MT
MTOC
MTs anchored by γ-tubulin
and accessory proteins that
form ring structures
MTOC
Regulation of cytoskeleton assembly:
1. Nucleation sites
2. Actin related proteins (ARP)
Structurally similar to actin
Binds minus end to stabilize it, allowing growth only
at plus end
Can also bind other actin filaments to create
network
ARPs
ARPs
Regulation of cytoskeleton assembly:
2. Elongation control
1. Both microtubules and microfilaments can bind
proteins that alter the ability of monomers to
incorporate into the polymer
Actin-profilin assembles faster than actin alone
Actin-thymosin assembles slower than actin alone
Stathmin sequesters tubulin to reduce the
concentration of free monomers
Actin elongation
Tubulin elongation
Regulation of cytoskeleton assembly:
3. Stabilization control
Many microtubule associated proteins (MAPs) bind
to the sides of microtubules to stabilize or
destabilize (example: tau protein)
Many proteins bind to microfilaments to stabilize
or destabilize the filament (example: cofilin)
Regulation of cytoskeleton assembly:
3. Stabilization control
Regulation of cytoskeleton assembly:
4. Capping proteins
Both microfilaments and microtubules have proteins
that can bind the ends to alter the ability to
assemble or disassemble
Actin: tropomodulin, CapZ
Microtubules: γ-tubulin
Regulation of cytoskeleton assembly:
5. Cross-linking proteins
Assembly of MT and MF into 3 dimensional
networks requires proteins that interconnect
strands
Actin cross-linking proteins
Red regions are actin binding sites
Actin cross-linking proteins
Actin cross-linking proteins
Regulation of cytoskeleton assembly:
6. Severing proteins
Rapid disassembly of the cytoskeleton is triggered
by proteins that cut MT and MF, allowing depolymerization
Katanin cuts MT, using the energy of ATP
hydrolysis
Gelsolin cuts MF in response to high Ca2+
Interactions with the cell membrane
Cytoskeletal elements (actin stress fibers) are
connected to the cell membrane at:
-focal contacts which connect the cell to a surface
-cadherins, which connect cells to other cells
Actin remodeling and motility
Platelets are activated in response to Ca2+ causing
the actin cytoskeleton to break apart then reform,
with extensions (filipodia and lamellapodia)
Actin remodeling and motility
Cytoskeleton and signal transduction
Summary
Extracellular signals trigger changes in the
cytoskeleton by altering the activity of proteins
that bind MT and MF
Molecular motors
1. What are the molecular motors?
2. How do they catalyze different types of
movement?
Molecular motors
Enzymes that use the hydrolysis of ATP to provide
the energy to move along cytoskeletal “tracks”
Each specific type of motor protein moves:
-on a specific type of track
-in a characteristic direction (toward + or - end)
Myosin moves along actin (toward + end)
Movement along microtubules uses kinesin (mostly
toward +) and dynein (mostly -ve)
Basic structure of molecular motors
Basic structure of molecular motors
Catalytic (ATPase) head hydrolyses ATP causing a
change in three dimensional structure.
A tail that interacts with other proteins such as:
-other motors (e.g. muscle thick filament)
-vesicle membrane proteins
-organelles proteins
-cell membrane proteins
Neck, connects head to tail, and may possess
accessory proteins that modify the properties of
the motor protein
Molecular motors:Myosin
Myosin (II) is composed of:
•2 myosin heavy chains
•4 myosin light chains
Myosin diversity
All eukaryotes have
myosins and there are
multiple isoforms within
each class
Diverse classes that
differ in structure (e.g.,
neck length) catalytic
properties (e.g., ATPase
rates, unitary
displacement, duty
cycle)
Myosin classes
Myosin: Unitary displacement
Length of the neck
influences “step size”
(=unitary displacement)
Longer neck allows longer
steps in each catalytic
cycle
Myosin: Duty cycle
Myosin: Duty cycle
Duty cycle: Proportion of time in a contractile
cycle where myosin is attached to actin
microfilament
Vesicle myosins have long duty cycles (~50%)
-allows myosin to remain in contact with the actin
microfilament
-why doesn’t the vesicle fall off the microfilament
when the myosin detaches?
Muscle myosins have short duty cycles (attached to
the actin filament only ~5% of the time)
Kinesin
Arose from same
ancestral protein as
myosin
Similar structures
(head, neck, tail)
Molecular motors
Dynein
Intracellular movement
Motor proteins carry diverse intracellular
particles and vesicles along cytoskeletal tracks
•Organelles
•Secretory vesicles
•Transport vesicles
•Pigment granules
Intracellular movement
Cells control color by changing the dispersal of
pigment granules, mediated by motor proteins
Intracellular movement
Cells control color by changing the dispersal of
pigment granules, mediated by motor proteins
Intracellular movement
•Pigment granules (shown in yellow) bind both
dynein and kinesin
•When kinesin is inactivated, dynein carried the
pigment toward the minus end (MTOC)
•When kinesin is activated, it carries the kinesin
away from the center, toward the plus end
RNA localization
Some RNA binding proteins can be carried along
the cytoskeleton by motor proteins, control where
the protein will be made
Muscle contraction
Actin filaments in contractile apparatus are “thin
filaments”-like microfilaments except actin
isoform (α) and stabilized structures are
integrated into a lattice
Muscle contraction
Myosin arranged in bundles, with catalytic heads
protruding toward surrounding thin filaments
Muscle contraction
Actin binding
proteins control
interaction
between actin and
myosin
Cilia and flagella
Similar structures of
9 microtubule
doublets and 2
microtubule singlets
Cilia and flagella
Dynein (attached to a microtubule) walks along
opposing microtubule to generate a bend
Summary
•Motor protein genes are ancient, with large gene
families for most motor proteins
•Molecular motors use ATP hydrolysis to induce
conformational changes that allow the motor to
walk along a cytoskeletal filament
•Tail regions of motor proteins bind diverse cargo
(organelles, vesicles, particles)
•Diversity in motor activity can be obtained by
altering the nature of the cytoskeleton and the
activity of the motor protein