Cytoskeleton and Cell Movement
Ch. 12 Cytoskeleton
Student learning outcomes:
1*. Explain structure/ function of cytoskeleton filaments:
• Actin (myosin & cell movement)
• Intermediate filaments
• Microtubules (& microtubule motors)
2*. Describe different monomers, associated proteins;
Explain filaments involved in cell movement,
plus additional proteins, energy requirements.
3. Describe tools to probe cytoskeleton:
microscope, mutant proteins, inhibitor molecules
4. Describe some diseases due to defects in cytoskeleton
Introduction
Cytoskeleton of eukaryotic cells
• Network of protein filaments throughout cytoplasm.
• Structural framework for cell shape, positions of
organelles, organization of cytoplasm.
• Dynamic structure, continually reorganized as
cells move and change shape
• Movement of cells,
internal transport of organelles
Fig. 4.31 Immunofluorescence to
detect actin (blue), tubulin (yellow)
Structure and Organization of Actin Filaments
1. Actin filaments (microfilaments – 7 nm diameter)
• Polymerize to actin filaments
• (flexible fibers, up to several µm in length)
• Organized into bundles, 3-D networks
• Actin is 375 amino acids (43 kd), highly conserved protein
• Abundant (5-10% cell protein)
• Mammals have 6 actin genes:
• 4 expressed in muscle cells,
• 2 in non-muscle cells
Prokaryotic ancestor:
MreB, structure for
rod-shaped bacteria
Fig. 12.1 Actin
Structure and Organization of Actin Filaments
Actin monomer (globular [G] actin) - tight binding
sites mediate head-to-tail interactions with 2 other
monomers, form filaments (filamentous [F] actin).
Polarity of filaments:
• All monomers
oriented in same
direction
• Important in
assembly,
• In direction of
myosin movement
relative to actin.
Fig. 12.2
Reversible Polymerization of Actin Filaments
Actin reversible polymerization:
Nucleation: first step of polymerization:
• trimer is formed,
• monomers added to either end.
Reversible polymerization
Rate monomers are added is proportional
to concentration.
Polymerization requires ATP, but not
hydrolysis of ATP
Fig. 12.3
Structure and Organization of Actin Filaments
Treadmilling:
•
•
•
•
Barbed end of filament grows faster
Actin-ATP associates with barbed ends; ATP later hydrolyzed
ADP-actin dissociates from pointed end
Treadmilling at intermediate concentrations of monomers
Cytochalasins bind to
barbed ends, block
elongation: inhibit
cell division.
Phalloidin binds to
filaments, prevents
dissociation. (label
with fluorescent dye)
Fig. 12.4 Actin
Structure and Organization of Actin Filaments
Actin-binding proteins
•
•
•
•
regulate assembly, disassembly
diverse group of proteins
act in diverse ways
Have ABD domains
Activities of these
proteins controlled by
cell signals (Chapt.
15) → remodel
cytoskeleton
Fig. 12.5 Actin
Figs 12.6,7 Initiation of actin filaments, branches
• Formin nucleates filaments to start chains, long unbranched
• Formin tracks at barbed end
• Tropomyosin stabilizes
long filaments;
• Movement requires filaments
actively turn over, branch
• Arp2/3 complex nucleates
branches near barbed end
Figs. 12.6,7 Actin
Fig 12.8 Effects of ADF/cofilin and profilin on actin filaments
ADF/cofilin (actin depolymerizing factor) proteins modify
existing filaments:
• enhance dissociation of actin/ADP monomers from pointed
end, (remain bound to monomers, prevent reincorporation).
• ADF/cofilin can also sever actin filaments
Profilin stimulates exchange of bound ADP for ATP
so monomers available for reassembly
Fig. 12.8 Actin
Fig 12.9 Actin bundles and networks
Actin bundles— filaments cross-linked in closely packed
parallel arrays.
Actin networks—filaments cross-linked in 3-D meshwork
arrays (semisolid gels)
• Actin-bundling proteins are rigid
(68-102 kd), cross-link, have 2 ABD
• Network proteins are large (280 kd)
flexible, have 2 ABD
Fig. 12.9 Actin bundles,
networks
A is macrophage surface
Structure and Organization of Actin Bundles
Parallel bundles —same polarity
• barbed ends at plasma membrane
• 14 nm apart
• Fimbrin in intestinal microvilli
Contractile bundles —
• more widely-spaced (40 nm)
• α-actinin (102 kd dimer) cross-links
• motor protein myosin binds
Networks:
• protein filamin (280 kd) flexible cross-links
• Filamin dimer V-shaped molecule,
• Actin-binding domains (ABD) each arm.
Figs. 12.10,11 Actin
bundles, networks
Structure and Organization of Actin Filaments
Actin filaments associate with plasma membrane:
• 3-D network.
Network and associated proteins (cell cortex)
determine cell shape, involved in movement.
Red blood cells (erythrocytes)
model system of cortical cytoskeleton
• no nucleus or organelles,
• easy to isolate plasma membranes,
associated proteins
• lack other cytoskeletal components
Fig. 12.12
Structure and Organization of Actin Filaments
Red blood cell:
Spectrin: major structural protein
• member of calponin family of actin-binding proteins
(other members include a-actinin, filamin, fimbrin, dystrophin)
•
tetramer of two polypeptides, α and β (220, 240 kd);
• ends associate with short actin filaments.
Fig. 12.13
Fig 12.14 erythrocyte cortical cytoskeleton binds to plasma membrane
• Spectrin binds short actin chains
• Ankyrin links spectrin-actin network to plasma membrane
by binding to spectrin and transmembrane protein (band 3).
• Protein 4.1 binds spectrin-actin junctions to transmembrane
protein glycophorin.
Fig. 12.14* red blood
cell membrane
Structure and Organization of Actin Filaments
Cytoskeleton linking proteins in other cells:
Dystrophin, (427 kD), a calponin, links actin filaments to
transmembrane proteins of muscle cell membranes.
• Transmembrane proteins link to extracellular matrix,
to maintain cell stability during muscle contraction.
• Muscular dystrophy, X-linked inherited disease,
results in progressive degeneration of skeletal muscle.
• Dystrophin absent or abnormal in patients with
Duchenne’s or Becker’s muscular dystrophy
Structure and Organization of Actin Filaments
Actin bundles attach to plasma membrane
Specialized regions of plasma membrane:
• Contact adjacent cells, extracellular matrix,
• other substrata (surface of culture dish).
Focal adhesions: cells attach to culture dishes:
• Actin bundles (stress fibers) cross-linked by a-actinin
• Transmembrane Integrins
• Extracellular matrix proteins
• Tropomyosin stabilizes
• Talin, vinculin link
Fig. 12.15,16 actin bundles
fibroblasts in culture; focal
adhesions
Fig 12.17 Attachment of actin filaments to adherens junctions
Adherens junctions attach epithelial cell-cell
• Continuous beltlike structure (adhesion belt) around each cell
• Transmembrane proteins cadherins bind to cytoplasmic
catenins, anchor actin filaments to plasma membrane
• (b-catenin also signaling molecule transcriptional activator)
Fig. 12.17
Figs. 12.18, 19 microvilli
Actin filaments support protrusions
from cell surface
• Microvilli on cells for absorption
Ex. microvilli of epithelial cells of intestine form
layer on apical surface (brush border)
~1000 microvilli per cell; increases surface area
Each microvillus:
• Closely packed parallel bundles
20 to 30 actin filaments.
• Filaments cross-linked by fimbrin, villlin.
• Actin bundles attach to plasma membrane
by calcium-binding protein calmodulin
in association with myosin I. Figs. 12.18,19
Fig 12.20 cell surface projections for phagocytosis and movement
Pseudopodia: phagocytosis, movement of amoebae
Lamellipodia broad, sheetlike extensions at the
leading edge of fibroblasts.
Microspikes or filopodia, thin projections from cell
Fig. 12.20: A, macrophage and tumor cell; B, amoeba, C, tissue culture cell
Fig 12.21 Structure of muscle cells
2. Myosin and cell movement:
• Myosin - prototype molecular motor —converts
chemical energy (ATP) to mechanical energy
• Muscle contraction model: actin-myosin interactions
and the motor activity of myosin molecules
• Muscle fibers (50 um diam)
– Fused muscle cells
• Myofibrils (cytoplasm)
– Thick myosin filaments
– Thin actin filaments
• Sarcomeres
– Individual contractile units
Fig. 12.21: Muscle cell
Fig 12.22 Structure of sarcomere
Sarcomere structure
• Dark bands reflect presence
or absence of myosin
• Actin filaments attached at
barbed ends to Z disc, includes
cross-linking protein α-actinin
Fig. 12.22 EM
of muscle
• Titin (extremely large, 3000 kd); single titin molecules extend
from M line to Z disc; act like springs on myosins
• Nebulin filaments associate with actin; regulate assembly of
actin – act as rulers of length
Fig. 12.23
Fig 12.24 Sliding filament model of muscle contraction
Sliding filament model of muscle contraction
• During contraction, sarcomere shortens, bringing Z discs
closer together.
• No change in width of A band;Actin moves into A band (H zone)
Molecular basis: reversible binding of myosin to actin filaments:
myosin motor drives filament sliding.
Fig. 12.24
Fig 12.26 Organization of myosin thick filaments
Muscle Myosin (Myosin II):
• 500 kd (4500 aa)
•
•
2 heavy chains – coil
2 pairs of light chains:
– regulatory, essential
Thick muscle filaments:
• Several hundred myosins, parallel staggered array.
• Globular heads bind actin, form cross-bridges between
thick and thin filaments.
Figs. 12.25,26
• Orientation of filaments reverses at M-line.
Fig 12.27 Model for myosin action
Model of myosin action:
• Reversible conformation of myosin – binds actin, ATP
• ATP hydrolysis powers dissociation of actin-myosin complex
• Sliding
Fig. 12.27
Fig 12.28 Association of tropomyosin, troponins with actin
Skeletal muscle contraction triggered by nerve impulses:
• Release of Ca2+ from sarcoplasmic reticulum (ER)
• Increased [Ca2+] in cytosol affects actin binding proteins:
• tropomyosin and troponin complex
• Binding of Ca2+ to troponin C shifts complex, allows
contraction by exposing myosin binding sites
Fig. 12.28
Fig 12.29 Contractile assemblies in nonmuscle cells
Nonmuscle cells have similar contractile assemblies:
•
•
•
•
Actin and Myosin II
Contraction by sliding actin filaments relative to one another.
Ex, stress fibers and adhesion belts (Figs. 12.16, 17)
Cytokinesis after mitosis (Fig. 12.30)
– Membrane-bound myosin under membrane
Figs. 12.29,30
Fig 12.31 Regulation of myosin by phosphorylation
Contraction in nonmuscle cells, smooth muscle:
• Regulated by phosphorylation of a myosin light chains.
• Catalyzed by myosin lightchain kinase (MLCK)
• Regulated by Ca2+-binding protein calmodulin.
Fig. 12.31
Other Non-muscle myosins
Oother non-muscle myosins:
Myosin I - smaller than myosin II (110 kd);
globular head acts as molecular motor;
short tails bind other structures
• Movement of myosin I along actin
filament transports cargo, such as vesicle
12 other nonmuscle myosins (III - XIV)
Myosin V is two-headed myosin
• transports organelles, other cargo
(intermediate filaments) toward barbed ends.
Fig. 12.32,33
Fig 12.34 Cell migration
Cell locomotion:
• Extensions of plasma membrane driven by dynamic
properties of actin cytoskeleton
•
•
•
•
Amoeba,
Migration of embryonic cells,
White blood cells into tissues
Cells for wound healing
Inhibition of actin polymerization
blocks formation of
cell surface protrusions.
Fig. 12.34
Intermediate Filaments
3. Intermediate filaments - 8-11 nm diameter
• Not directly
involved in cell
movements
• Mechanical
strength, scaffold
for localization of
cell processes
• Nuclear lamina
Intermediate Filaments
Common structure:
assembly
Figs. 12.36, 37
See also Fig. 9.4
Intermediate Filaments
Intermediate filaments
• Not distinct ends
• More stable, not dynamic
behavior of actin filaments
• Phosphorylation can regulate
assembly and disassembly
(ex. nuclear lamins
disassemble in mitosis)
• Network in cytoplasm
• Associate with other elements
of cytoskeleton → scaffold
Fig. 12.38;
Network of keratins (Ab)
Intermediate Filaments
Intermediate filaments function in contacts,
such as epithelial cells
Desmosomes— junctions to adjacent cells.
• Keratin filaments attach to dense protein
plaques on cytoplasmic side.
• Attachments mediated by plakins;
• Transmembrane cadherins link cells
Hemi-desmosomes— junctions to
underlying connective tissue (Fig. 12.39)
• Keratin filaments attached to
different plakins (e.g. plectin)
• Transmembrane integrins link
to extracellular matrix.
Fig. 12.38;
Desmosome
Fig 12.40 EM of plectin bridges between intermediate filaments,
microtubules
• Plakins link intermediate
filaments to other
cytoskeleton elements
• Ex. Plectin binds actin
filaments, microtubules,
forms bridges between them
and intermediate filaments.
• Increases mechanical
stability of cell.
Fig. 12.40 Plectin, green; Ab yellow,
IF blue, microtubules, red
Fig 12.41 Experimental demonstration of keratin function
Transgenic mice with mutant keratin 14:
evidence for importance of intermediate filaments
• Truncated keratin disrupted formation of normal
keratin cytoskeleton → severe skin abnormalities.
Fig. 12.41;
Normal skin (top)
TG skin (lower)
Intermediate Filaments & disease
Human diseases - disorders of intermediate filaments:
• Epidermolysis bullosa simplex (EBS) patients develop skin
blisters from cell lysis after minor trauma; keratin gene
mutations
• Amyotrophic lateral sclerosis (ALS) - progressive loss of
motor neurons, muscle atrophy and paralysis.
Abnormalities of neurofilaments (NF-L, NF-H)
• Hutchinson-Gilford (Progeria) causes premature aging,
involves mutations affect Lamin A protein (Chapt. 9)
Fig 12.42 Structure of microtubules
4. Microtubules - rigid hollow rods 25 nm
• Dynamic structures undergo continual assembly,
disassembly: cell movements, cell shape.
• Globular protein (55 kd) tubulin
• Tubulin dimers of α-tubulin and β-tubulin
encoded by related genes
• 13 protofilaments
• hollow core
• 25 nm diameter
• Polarity: - end, + end
• GTP
Fig. 12.42
Fig 12.43 Treadmilling, role of GTP in microtubules
Treadmilling of Microtubules:
• Tubulin dimers with GTP bound to β-tubulin associate
with the growing end.
• After polymerization, GTP hydrolyzed to GDP, makes
tubulin less stable; dimers at minus end disassociate.
Fig. 12.43
Fig 12.44 Dynamic instability of microtubules
Rapid GTP hydrolysis results in dynamic instability:
• High concentrations tubulin-GTP: dimers added more rapidly
than GTP is hydrolyzed, and microtubule grows.
• Low concentration of tubulin-GTP: GTP at plus end is
hydrolyzed, dimers are lost.
Fig. 12.44
- End + end
Fig 12.45 Intracellular organization of microtubules
Most microtubules extend from centrosome (animals)
Need rapid remodeling, as for mitosis and spindle formation:
Drugs colchicine and colcemid affect microtubule assembly;
experimental tools, cancer treatments
Vincristine and vinblastine
inhibit microtubule polymerization,
inhibit rapidly dividing cells,
cancer chemotherapy
Taxol stabilizes microtubules,
blocks cell division.
[plant cells do not have centrosome;
microtubules extend from nucleus]
Fig. 12.45
Fig 12.46 Growth of microtubules from centrosome
Centrosome is microtubuleorganizing center:
• Minus ends of microtubules anchored:
colcemid disassemble microtubules
– after drug is removed, microtubules grow
•
- Key protein is special γ-tubulin, initiates
microtubules formation
Paired centrioles: perpendicular;
amorphous pericentriolar material.
• Centrioles are cylindrical;
• 9 triplets of microtubules;
• Lots of other proteins
• Centrioles also in basal bodies of
cilia, flagella
Fig. 12.46 Ab to tubulin;
Fig. 12.47,48 centrioles
Fig 12.49 Stability of microtubules in nerve cells
Stability of microtubules:
• Modulated by modifications of tubulin, e.g. phosphorylation
• Microtubule-associated proteins interact:
• Stabilize by capping ends of microtubules
• Disassemble by sever microtubules, depolymerize
• Track +-end (bind tubulin/GTP) and focus cell location
• MAPs are also regulated by phosphorylation:
Ex. Nerve cells: (axons, dendrites):
• microtubules organized differently
• distinct MAPs each type:
• MAP-1, -2, tau; MAP-4
Fig. 12.49
Microtubule Motors and Movement
5. Microtubule motors and movement:
2 families of motor proteins (kinesins and dyneins) power
movements involving microtubules: Position vesicles, organelles,
beating cilia, flagella, mitosis
Kinesin 1: 380 kd (2 heavy chains, 2 light chains)
• head binds ATP, microtubule; tail binds vesicles; move to + end
Dynein: 2000 kd
(2-3 heavy chains, other chains)
• head binds ATP, microtubule;
tail binds organelles; move to – end
• Both use ATP energy
Fig. 12.50
Microtubule Motors and Movement
Video-enhanced microscopy to study movements:
• Especially vesicles, organelles along giant squid axon
• Nerve cell axons may extend more than meter in length;
• Ribosomes only in cell body and dendrites, so proteins,
membrane vesicles, etc. transported from cell body to axon
• Kinesin carries secretory vesicles
with neurotransmitters from Golgi
to terminal branches of axon.
• Cytoplasmic dynein transports
endocytic vesicles back to cell body
Fig. 12.49
Fig 12.51 Transport of vesicles along microtubules
• Microtubules usually oriented with - end
in centrosome; + end extends toward
periphery.
• Members of kinesin and dynein families
transport cargo in opposite directions
• Microtubules, associated motor proteins
position organelles in cell.
• Ex.: ER extends to periphery of cell in
association with microtubules, which
involves kinesin I.
• Drugs that depolymerize
microtubules cause ER
to retract toward cell center.
Figs. 12.51, 52
Microtubule Motors and Movement
Cilia and flagella
• microtubule-based projections of plasma membrane
• responsible for movement of many eukaryotic cells.
Similar structure:
Cilia beat back-and-forth.
Flagella longer, wavelike
pattern of beating
[Some bacteria have flagella - very
different structure: protein filaments
projecting from cell surface]
Figs. 12.51, 52
Paramecium, Cilia, sperm
Fig 12.54 Structure of axoneme of cilia and flagella
Axoneme of cilia and flagella: 250 nm diameter
• Microtubules in “9 + 2” pattern: central pair, 9 outer doublets.
• Complete A and partial B (10–11 protofilaments) fused to A
• Nexin links microtubules; 2 arms of dynein to each A tubule
Movement of cilia and flagella
•
•
•
•
Sliding outer microtubule doublets relative to one another
Powered by motor activity of axonemal dyneins.
Dynein bases bind A tubules,
Head groups bind adjacent B tubules
Fig. 12.54, 56
Fig 12.55 Electron micrographs of basal bodies
Minus ends of microtubules anchored in basal body,
• Similar structure to centriole: 9 triplets of microtubules.
• Basal bodies initiate growth of axonemal microtubules,
• Anchor cilia and flagella to surface of cell.
Fig. 12.55
Microtubule Motors and Movement
Mitosis reorganizes microtubules
• Interphase microtubules disassemble
• free tubulin subunits reassembled to form
mitotic spindle
• Centrosome duplicate to form 2
microtubule-organizing centers
(poles)
4 types microtubules in spindle:
• Kinetochore
• Chromosomal
• Polar
• Astral
Fig. 12.57, 58
Fig 12.59 Anaphase A chromosome movement
Chromosome movement in Anaphase:
Anaphase A — chromosomes move toward poles
along kinetochore microtubules, which shorten
Motor proteins
Directed to
Minus end help
Kinesins help
depolymerize
microtubules
Fig. 12.59
Fig 12.60 Spindle pole separation in anaphase B
Anaphase B: spindle poles separate,
accompanied by elongation of polar microtubules.
• polar microtubules slide against one another,
pushing spindle poles apart.
• Plus-end–directed kinesins
cross-link polar microtubules
move them toward the plus end
• Cytoplasmic dynein anchored
moves along astral microtubules
in minus-end direction.
Fig. 12.59
Review questions
Review Questions
1. Briefly compare/contrast the 3 types of filaments
2. Involvement of ATP, GTP in cytoskeleton filaments
3. Explain the nature of actin/myosin movement
4. Explain microtubule motors and movement