cytoskeleton and cell movement ch. 12 cytoskeleton student learning outcomes : 1*. explain...

Cytoskeleton and Cell Movement

Ch. 12 CytoskeletonStudent 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

IntroductionCytoskeleton 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 forrod-shaped bacteria

Fig. 12.1 Actin


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


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


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 filamentsactively 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 filamentsProfilin 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, networksA is macrophage surface

Structure and Organization of Actin BundlesParallel 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


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


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


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


Fig. 12.15,16 actin bundles fibroblasts in culture; focal adhesions

Actin bundles attach to plasma membraneSpecialized 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 -actinin• Transmembrane Integrins • Extracellular matrix proteins • Tropomyosin stabilizes• Talin, vinculin link

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• (-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 absorptionEx. microvilli of epithelial cells of intestine formlayer on apical surface (brush border) ~1000 microvilli per cell; increases surface areaEach 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 sarcomereSarcomere structure• Dark bands reflect presenceor 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.• Orientation of filaments reverses at M-line. Figs. 12.25,26

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


Common structure:assembly

Figs. 12.36, 37See also Fig. 9.4


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 → scaffoldFig. 12.38; Network of keratins (Ab)

Intermediate FilamentsIntermediate filaments function in contacts, such as

epithelial cellsDesmosomes— 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 microtubule-organizing 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


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 depolymerizemicrotubules cause ER to retract toward cell center.

Figs. 12.51, 52


Cilia and flagella • microtubule-based projections of plasma membrane• responsible for movement of many eukaryotic cells.

[Some bacteria have flagella - very different structure: protein filaments projecting from cell surface]

Similar structure:Cilia beat back-and-forth.Flagella longer, wavelike pattern of beating

Figs. 12.51, 52 Paramecium, Cilia, sperm

Fig 12.54 Structure of axoneme of cilia and flagella

Fig. 12.54, 56

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 12.55 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


Fig. 12.57, 58

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 12.59 Anaphase A chromosome movement

Chromosome movement in Anaphase:Anaphase A — chromosomes move toward poles

along kinetochore microtubules, which shortenMotor 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

cytoskeleton and cell movement ch. 12 cytoskeleton student learning outcomes : 1*. explain...

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