Cytoskeleton: Microtubules, Actin, and Intermediate Filaments

Cytoskeleton

Made of large multimeric protein fibers in eukaryotic cells; provides structure, mechanical support, intracellular transport, and cell motility.

Microtubules

Composed of tubulin, 25nm in diameter; involved in cilia and flagella.

Microfilaments

Composed of actin, 7 nm in diameter; involved in muscle fibrils.

Intermediate filaments

Variable in composition, 8-12 nm in diameter.

Dynamic assembly

The cytoskeleton is dynamically assembled and disassembled.

Structure of microtubules

Straight hollow cylinders, built from 13 protofilaments.

Protofilament

Linear chain of a-tubulin and b-tubulin heterodimers.

GTP molecules

Each dimer binds 2 GTP molecules; one a-tubulin (nonexchangeable) and one b-tubulin (exchangeable by being hydrolyzed to GDP after incorporation).

MTs polarity

Plus end has rapid growth/shrinkage (dynamic); minus end is slower, anchored by MTOC.

MT Assembly

Polymerization of MTs requires GTP and Mg2+.

Critical concentration

Tubulin concentration at which assembly equals disassembly.

Dynamic instability

MTs alternate between growth and shrinkage.

GTP-tubulin cap

Stabilizes growth of microtubules.

Catastrophe

Loss of GTP cap leading to rapid depolymerization.

Rescue

Re-addition of GTP-tubulin after catastrophe.

Treadmilling

Occurs when addition at the plus end equals loss at the minus end.

Cytoplasmic MTs

Dynamic, maintain cell shape, transport vesicles, position organelles, form spindles.

Axonemal MTs

Stable, organized structures in cilia, flagella, and basal bodies.

Centrosome

Major MTOC in animals, contains two centrioles surrounded by pericentriolar material.

Antimitotic Drugs

Colchicine and Nocodazole bind b-tubulin, inhibit assembly; Taxol stabilizes MTs, prevents disassembly.

Tau

MT associated protein that forms tight bundles in axons.

MAP2

MT associated protein that forms looser bundles in dendrites.

Cytochalasins

Drugs that cap + ends of actin filaments, stopping addition.

Latrunculin A

Sequesters monomers of actin.

Phalloidin

Stabilizes actin filaments, prevents depolymerization.

Thymosin B4

Prevents G-actin polymerization.

Profilin

Promotes actin-ADP to ATP exchange, encourages growth.

Long filaments

Promoted by formins (move with growing end)

Regulators of actin dynamics

Rho family of GTPases

Rho

Stress fibers

Rac

Lamellipodia

Cdc42

Filopodia

Controlled by

GEFs, GAPs, and GDIs.

Cell cortex

Dense actin gel beneath membrane

Microvilli

Core bundle of F-actin crosslinked by fimbrin and villin, attached to the membrane via myosin 1 and calmodulin.

Terminal web

Base of microvillus, network of myosin 2 and spectrin connecting to IFs.

Intermediate Filaments (IFs)

8-12 nm diameter, fibrous not globular

Most stable

Least soluble cytoskeletal elements

Tension bearing function

Provide mechanical strength

Abundant in cells under stress

Skin, muscle, neuron

Not present in

Plant cytosol.

Monomer

Long a-helical rod

Dimer

Two monomers in parallel, coiled coil.

Tetramer

Two dimers, staggered antiparallel, no polarity

Final filament

8 protofilaments thick

Assembly does not require

ATP OR GTP.

Class 1-2

Acidic and basic keratins (epithelia, hair, nails)

Class 3

Desmin (muscle), vimentin (mesenchymal)

Class 4

Neurofilaments (neurons)

Class 5

Lamins (nuclear lamina)

Class 6

Nestin (developing neurons)

Keratins

Skin structure

Spectraplankins (plectin)

Crosslink MTs, MFs, and IFs which maintains cytoskeletal integrity.

Three broad functions of motility

Movement of the entire cell or organism through the environment, movement of the environment past or through a cell, movement of components within the cell.

Cytoskeleton as a scaffold

Microtubules and microfilaments serve as tracks for motor proteins.

Motor proteins

Convert chemical energy (ATP) into mechanical work.

Kinesins

Move toward the plus end of microtubules (away from centrosome)

Function in

Anterograde transport

Dyneins

Move toward the minus end of microtubules (toward centrosome)

Cytoplasmic dynein

Two heavy chains (AAA+ ATPase ring), two intermediate chains, two light intermediate chains, two light chains

Fast axonal transport of vesicles

About 2 μm/sec

Cilia and flagella

Motile appendages built on microtubules.

Core structure

Axoneme, surrounded by plasma membrane and anchored to a basal body.

Basal body

A modified centriole with nine triplet microtubules.

Transition zone

Links basal body to axoneme.

9 + 2 arrangement

9 outer doublets + 2 central microtubules (motile cilia/flagella)

9 + 0 arrangement

Primary (non-motile) cilia used for signaling

Outer doublet structure

Each outer doublet has an A tubule (complete 13 protofilaments) and a B tubule (incomplete 10-11 protofilaments)

Axonemal dynein arms

Located on A tubules, they slide adjacent doublets

Nexin links and radial spokes

Convert sliding of doublets into bending

Cilia

Short (2-10 μm), numerous, move with oar-like strokes parallel to surface

Flagella

Long (10-200 μm), few per cell, wave-like motion generating force parallel to length

Intraflagellar transport (IFT)

Kinesin moves material to flagellum tip (plus end) and dynein moves material back to base (minus end)

Motor Protein

Myosin Family, ATP-dependent, actin-based motors that generally move toward the plus end of actin filaments

Myosin structure

Globular head with actin-binding and ATPase activity, neck (lever), tail (cargo or filament attachment)

Myosin I

Single-headed; links actin to membranes (e.g., in microvilli)

Myosin II

Double-headed, forms thick filaments for contraction with two heavy chains and one essential light chain and one regulatory light chain

Comparison: Kinesin vs Myosin

Kinesin operates on microtubules, moves toward plus end, over long distances with few molecules; Myosin II operates on actin, also moves toward plus end, over short distances with large arrays working together

Muscle fiber

Long, multinucleate cell containing many myofibrils

Myofibrils

Divided into repeating sarcomeres (between Z lines)

Sarcomere composition

Thin filaments: actin, tropomyosin, troponin; Thick filaments: myosin II

A bands

Dark bands representing thick filaments

I bands

Light bands representing thin filaments

Z line

Anchors actin plus ends

Sliding-Filament Model

Thin filaments slide past thick filaments without changing length; myosin heads form cross-bridges with actin, pulling toward Z lines

Cross-Bridge Cycle

Myosin head (ADP + Pi) loosely binds actin; Pi release tightens bond; power stroke occurs with ADP release; ATP binding causes detachment; ATP hydrolysis re-***** the head

Regulation of Skeletal Muscle Contraction

At rest, tropomyosin blocks myosin-binding sites on actin; calcium binding to troponin C exposes binding sites, allowing contraction

Cardiac Muscle

Found in heart; responsible for rhythmic contractions with cells joined by intercalated discs for electrical coupling

Smooth Muscle

Involuntary muscle with slow, sustained contractions regulated by Ca²⁺ binding to calmodulin and activating myosin light-chain kinase (MLCK)

Microfilament-Based Motility in Nonmuscle Cells

Actin and myosin drive movement in many nonmuscle cells through processes like cell crawling and amoeboid movement

Cytoplasmic streaming (cyclosis)

Actomyosin-driven flow of cytoplasm within cells (especially in plants).

Epithelial tissue

Sheets of polarized cells with distinct apical and basal surfaces.

Connective tissue

Loosely organized, embedded in extracellular matrix (ECM).

Adhesive Junctions

Anchor the cytoskeleton of one cell to that of another, providing mechanical strength.

Adherens junctions

Connect to actin filaments.

Desmosomes

Connect to intermediate filaments.

E-cadherin

Best-studied type of cadherin; has an extracellular domain with five repeats that 'zip together' with cadherins from adjacent cells.

Desmosomal cadherins

Include desmocollins and desmogleins.

Cell Adhesion Molecules (CAMs)

Members of the immunoglobulin superfamily (IgSF) important in transient cell-cell contacts during development and immune responses.

N-CAM

Neural cell adhesion molecule; forms homophilic interactions.