Module 7: The Cytoskeleton

What is the cytoskeleton and what are its roles in the cell?

• Network of protein-based filaments

• Provides cell shape and structural support

• Dynamic, not static → enables:

  • Cell movement

  • Cell growth

  • Cell differentiation

• Composed of 3 filament types:

  • Actin filaments

  • Microtubules

  • Intermediate filaments

How does the cytoskeleton contribute to specialized cell structures and shape?

• Crucial for maintaining cell shape, especially in differentiated cells

• Examples:

  • Microtubules → structure in cilia

  • Actin filaments → shape/function in microvilli (epithelial cells)

• Neuron example:

  • Microtubules (green) → support axon

  • Actin (red) → shapes growth cone

Why is the dynamic nature of the cytoskeleton important?

• Essential for cell movement

• Supports processes like:

  • Cell migration

  • Cell division

• Example:

  • Ovarian cancer cell expressing actin-GFP

  • Shows real-time actin filament dynamics during movement

How is the dynamic nature of microtubules shown during mitosis?

• Seen in cell division (mitosis)

• Example:

  • Breast cancer cell expressing Tubulin:GFP

  • Visualizes microtubule spindle formation over time

• Demonstrates the structural reorganization of cytoskeleton during mitosis

What are the 3 types of cytoskeletal fibres and how are they labeled in cells?

• Types: Actin filaments (microfilaments), Microtubules, Intermediate filaments (IFs)

• Defined by: Diameter and subunit type

• Actin labeling:

  • Phalloidin (fluorescent toxin from death cap mushroom)

    • High specificity & affinity to actin

    • Stabilizes actin filaments

  • Antibody to actin

  • Actin:GFP fusion

• Microtubule labeling:

  • Antibody to tubulin

  • Tubulin:GFP fusion

• Intermediate filament labeling:

  • Antibody to filament-specific subunit

  • GFP fusion proteins

• All 3 fibres:

  • Found in all eukaryotic cells

  • Form overlapping but distinct structures

  • Can be seen together in cells via fluorescent labeling

What are the structural differences among the 3 cytoskeletal fibres?

• All fibres are polymers made of protein subunits

• Actin filaments (microfilaments):

  • Thinnest

  • Made of monomeric actin

• Microtubules:

  • Thickest

  • Made of α- and β-tubulin dimers

• Intermediate filaments (IFs):

  • Medium thickness

  • Made from varied proteins, depending on cell type

How are the 3 cytoskeletal fibres distributed in epithelial cells?

• Actin (red):

  • Located at apical surface

  • Shapes microvilli

• Intermediate filaments (blue):

  • Span the entire cell for structural support

  • Composed of lamin proteins

    • Also form nuclear lamina (supports nucleus)

• Microtubules (green):

  • Form networks for intracellular transport

• Distribution is cell-type specific, with each fibre type occupying unique regions

What are the motor proteins associated with cytoskeletal filaments?

• Motor proteins move along actin filaments and microtubules

• No motor proteins are associated with intermediate filaments

• Actin-based motors:

  • Myosin proteins

• Microtubule-based motors:

  • Kinesin

  • Dynein

• General motor protein structure:

  • Head domain binds to cytoskeletal filament

  • Tail domain binds to cargo

• Movement powered by:

  • ATP hydrolysis → drives protein “walking” or stepping

What are common actin-based cell movements and structures?

• Actin density is highest at cell periphery

• Functions of actin filaments:

  • Microvilli formation

  • Contractile bundles in muscle (sarcomeres)

  • Filopodia & lamellipodia for cell migration

  • Contractile ring in cytokinesis

• Actin structural organization in cells:

  • Contractile stress fibers – throughout the cell

  • Gel-like network – at the cell cortex

  • Tight parallel bundles – in filopodia

What is F-actin and what is its structure?

• F-actin = filamentous actin

• Made of:

  • Two helical strands of polymers

  • Each strand is a chain of G-actin (globular actin) monomers

• Diameter: 5–9 nm

• Structure features:

  • Strands spiral around each other

What does it mean that actin filaments are polar, and how is this polarity visualized?

• Actin filaments are polar → each end behaves differently

• Polarity visualized using myosin head decoration in electron microscopy

  • Myosin binds in one direction, showing filament orientation

• Plus-end (barbed end):

  • Grows faster

  • Higher actin subunit addition

• Minus-end (pointed end):

  • Grows slower or may shrink

• Polymerization is more active at the plus-end

What is G-actin, and how does it contribute to actin filament polarity?

• G-actin = globular actin monomer

• Has 4 structural domains

• ATP-binding site located in cleft between domains 2 & 4

• Each monomer is polar

• Filament polarity arises from monomer orientation

  • ATP-binding pockets face the minus end.

  • Inside the filament, these pockets are hidden, except at the very end

    • Only the last few monomers at the minus end have their ATP-binding sites partially exposed.

How does actin filament polymerization work and what role does ATP play?

• F-actin = filamentous actin (polymerized G-actin)

• Actin filaments are dynamic

  • Undergo both polymerization and depolymerization

• Plus-end:

  • More growth (polymerization > depolymerization)

• Minus-end:

  • More shrinkage (depolymerization > polymerization)

• ATP-bound actin:

  • Adds to the plus-end

  • Actin has ATPase activity → hydrolyzes ATP to ADP + Pi

• Most filament = actin-ADP

  • ADP is trapped inside filament (not released)

• In cytosol:

  • Free actin-ADP → releases ADP → recharges with ATP

What is critical concentration in actin dynamics, and what proteins regulate actin polymerization?

• Critical concentration (Cc):

  • Point where polymerization = depolymerization

  • [Actin] > Cc → filament grows

  • [Actin] < Cc → filament shrinks

• Cc differs at plus- & minus-ends → different filament dynamics

• Regulatory proteins:

  • Profilin:

    • Binds actin-ATP

    • Promotes ATP binding

    • Activates monomer → accumulates at plus-end

  • Thymosin:

    • Binds actin monomers → inhibits polymerization

    • Thymosin-actin dimers accumulate at plus-end and creates storage buffer of actin monomers

  • Capping proteins:

    • Block ends of actin filaments→ inhibit growth or shrinkage

What is actin treadmilling, and why is it important?

• Treadmilling

  • Polymerization at plus-end = depolymerization at minus-end

  • No net length change, but filament moves forward

• Important for:

  • Cell movement

  • Cytoskeletal remodeling

How does actin filament reorganization enable cell movement?

• Reorganization of actin filaments pushes cell membrane outward

• Forms two structures:

  • Lamellipodia = broad, fan-like membrane expansions

  • Filopodia = thin, finger-like projections

• Steps in movement:

  • Leading edge forms

  • Lamellipodia spread (fan like expansions)

  • Filopodia extend

  • Cell moves in direction of leading edge

• All require dynamic remodeling of actin

What are myosin motor proteins, and how do they interact with actin filaments?

• Myosins = actin-based motor proteins

• Use ATP hydrolysis to "walk" along actin filaments

• Most move toward plus-end

• (3/8) Key families:

  • Myosin I, II, V → found in most eukaryotic cells

• Structure:

  • Head (motor) domain at N-terminus:

    • Binds actin

    • Hydrolyzes ATP

  • Tail domain:

    • Varies to carry different cargo or interact with different targets at different rates

What is the structure of Myosin II, and what is its role?

• Myosin II = muscle contraction motor

• Structure:

  • 2 heavy chains → form coiled-coil tail

  • 4 light chains (2 types) → regulate activity

• Motor domain (head):

  • Seen in EM images as globular heads

  • Located at top of tails

  • Responsible for movement along actin

How is Myosin II activated and what is its function in cells?

• Activation:

  • Myosin Light Chain Kinase (MLCK) phosphorylates myosin light chains

  • Triggers tail extension & activates actin-binding domains on motor heads

• Assembly:

  • 15–20 myosin II proteins form a bipolar thick filament

• Function:

  • Myosin II doesn't carry cargo

  • Generates contractile forces essential for many cell processes

What is the structure of a Myosin II thick filament?

• Bipolar filament:

  • Motor heads on both ends

  • Bare zone in the center (no motor heads)

• Function of structure:

  • Allows motor heads to interact with actin on both sides

What role does Myosin II play in skeletal muscle contraction and sarcomere structure?

• Myosin II + actin = sarcomere (basic unit of striated muscle)

• Sarcomere components:

  • Z-discs anchor plus-end of actin filaments

  • CapZ (plus-end capping), Tropomodulin (minus-end capping)

  • Nebulin: stabilizes parallel actin filaments

    • Between parallel actin fibers there are myosin thick filaments

  • Titin: spring-like protein anchors myosin to Z-discs

• Muscle contraction:

  • Myosin interacts with actin → Z-lines pulled closer together

• “Striated muscle”:

  • Refers to striped appearance of sarcomeres under microscope

How does muscle contraction occur at the sarcomere level?

• Myosin heads pull actin toward the center of the sarcomere

  • Due to cyclical association of actin with motor heads

• Shortening of sarcomere but no change in filament length, just sliding past each other

• Each myosin head:

  • Binds ATP → hydrolyzes it to ADP

  • "Walks" toward plus-end of actin

• Shortening of sarcomere = muscle contraction

• Calcium-dependent process:

  • Ca²⁺ allows myosin binding sites on actin to be exposed

• Relaxation:

  • Ca²⁺ is removed

  • Myosin heads along thick filament release actin thin filaments

  • Thick and thin slide past and sarcomere elongates → muscle relaxes

What are the steps in the myosin ATPase cycle that power muscle contraction?

• Chemical energy (ATP) → Mechanical movement (contraction)

• Myosin motor cycle (5 steps):

  1. Myosin binds actin (tight)

  2. ATP binds myosin → releases from actin

  3. ATP → ADP + Pi → myosin changes conformation (relaxed)

  4. Pi released → myosin re-binds actin (strongly)

  5. ADP released → myosin changes conformation → actin is pulled left (back to step 1)

• Cycle repeats with each new ATP during muscle contraction

• Each ATP = small movement (few nm) along actin

What is the role of Myosin V in cells?

• Myosin V powers intracellular cargo transport along actin

• Example: moves melanosomes in melanocytes

  • Melanosomes (membrane-enclosed organelles) = vesicles with melanin pigment

• Melanocytes in the skin have dendrites that connect to keratinocytes

• Melanin is transferred to keratinocytes → protects DNA from UV (tanning process)

• Melanosomes (melanin-filled vesicles) are transported:

  • By microtubules (long-range)

  • By Myosin V (final delivery along actin filaments to cell membrane)

• Myosin V ensures proper distribution of pigment at the apical surface

• Loss of Myosin V function → dilute phenotype in animals

  • Pigments not properly delivered → lighter/diluted fur color

How is myosin movement studied in vitro?

• Myosin proteins are fixed (by tails) to microscope slides

• Fluorescent actin filaments + ATP are added

• Movement of actin = visual indicator of myosin activity

• ATP cycle (binding → hydrolysis → Pi/ADP release) powers motion

• Movement is caused by myosin motor heads cycling through conformations

• Seen under a microscope as fluorescent actin motion

What affects the speed of different myosin proteins?

• Myosin speed = 0.2 to 60 µm/sec, varies by type

• Influenced by:

  1. ATPase rate (how fast ATP is hydrolyzed in myosin head)

  2. Actin binding time (how long myosin stays bound due to affinity)

• Myosin II:

  • Binds actin only ~5% of the cycle → faster movement

• Myosin V:

  • Binds actin ~90% of the cycle → slower but stable movement

What determines the step size of myosin, and how does myosin V move along actin filaments?

• Step size of myosin depends on lever arm length

  • Longer lever → bigger step during power stroke

• Myosin V lever is 3× longer than myosin II

• Step size:

  • Myosin II: ~7 nm

  • Myosin V: ~36 nm

• Myosin V movement:

  • Moves in hand-over-hand fashion

  • Trailing head detaches → swings forward → becomes leading head

  • Moves toward the barbed/plus end of actin filament

• Efficient for cargo transport due to large steps and strong actin binding

What is the structural organization of microtubules?

• Microtubules = hollow tubes made of 13 protofilaments

• Protofilaments arranged in a circular pattern → strong tube wall

• Basic unit = α/β-tubulin dimers

• Protofilaments are staggered → dimers appear to spiral like a spring

• Structure visible via electron microscopy

How do α and β tubulin subunits interact with GTP?

• Both α & β tubulin bind GTP

  • α-tubulin: GTP is tightly bound, never hydrolyzed or exchanged

  • β-tubulin: GTP is hydrolyzed to GDP, then exchanged back to GTP

• Tubulin dimers are added/removed as α-β pairs

• α-β-GTP dimers: high affinity for microtubules

• α-β-GDP dimers: low affinity → more likely to dissociate

How are microtubules polarized and what drives their growth/shrinkage?

• Microtubules are polar:

  • Plus-end: fast-growing

  • Minus-end: slow-growing

• Dimer orientation:

  • β-subunit toward plus-end

  • α-subunit toward minus-end

• Growth ("rescue"): α-β-GTP dimers added to plus-end

• Shrinkage ("catastrophe"): α-β-GDP dimers released

• GTP hydrolysis happens after polymerization → most of microtubule = α-β-GDP

• GTP cap (or α-β-GTP) on plus-end promotes growth; loss of cap → shrinkage

  • 4× slower dissociation rate than α-β-GDP

  • Due to higher affinity between α-β-GTP dimers and their neighbors

What is EB1 and how does it affect microtubule growth?

• EB1-GFP is a plus-end binding protein

• Prevents premature catastrophes

• Acts as a positive regulator of microtubule growth

• Visualized in live cells using RFP-tubulin (red) and EB1-GFP (green)

• Localizes specifically to growing plus-ends of microtubules

What is dynamic instability in microtubules?

• Plus-end undergoes oscillation between growth and shrinkage

  • Growth = polymerization = rescue

  • Shrinkage = depolymerization = catastrophe

• Maintained by concentration of free α-β-GTP dimers that allows polymerization

• Ensures constant remodeling of microtubule structure

What are MAPs and what roles do they play in microtubule function?

• MAPs = proteins that regulate microtubule assembly/disassembly

• Functions:

  • Bundle formation (cross-linking)

  • Stability and rigidity control

  • Regulation of assembly rate

• Two functional groups:

  • Stabilizers: Tau, EB1

  • Destabilizers: Catastrophin

What role does γ-tubulin play in microtubule formation?

• γ-tubulin = nucleation factor (not part of microtubule body)

  • Present in small amounts

• Forms γ-TuRC (γ-tubulin ring complex) with other proteins

• Nucleates minus-end of microtubule

• Acts as a template for plus-end growth

• Caps the minus-end → growth occurs only at the plus-end

What is the MTOC and how does it organize microtubule growth?

• MTOC = Microtubule Organizing Center

• In animal cells → called centrosome, located near the nucleus

• Contains:

  • Two centrioles

  • Pericentriolar material (PCM) with γ-TuRC complexes

• γ-TuRC nucleates minus-ends

• Plus-ends grow outward toward cell periphery

What is the role of the MTOC during mitosis?

• MTOC = Microtubule Organizing Center, duplicates in mitosis

• Forms the mitotic spindle

• Microtubules nucleated from γ-TuRC at each MTOC

• Plus-ends grow outward

  • Some anchor the spindle to cell cortex

  • Others extend inward to attach to chromosomes

• Microtubules guide sister chromatid separation

• Spindle is dynamic – built and disassembled based on microtubule instability

How do microtubules assist in chromosome movement during mitosis?

• Tubulin visualized via antibodies or GFP constructs

• DAPI stains DNA (nucleus = blue)

• In interphase: microtubules fill the cytoplasm

• In metaphase:

  • Replicated chromosomes align at the spindle equator

  • Spindle microtubules attach to centromeres

• In anaphase:

  • Spindle poles move apart

  • Microtubules shorten → chromatids pulled to poles

What drugs affect microtubule dynamics and how do they work?

Colchicine (meadow saffron or autumn crocus):

• Inhibit polymerization.

• Binds to free αβ-tubulin dimers.

• Bound dimers can still join a growing microtubule.

  • BUT they block further addition or removal of tubulin → microtubules can't grow or shrink.

• Effect on mitosis: Cells arrest in metaphase (chromatids can't separate).

Taxol (Paclitaxel):

• Binds to β-tubulin in the microtubule.

• Increases stability by preventing depolymerization (shrinkage).

• Effect on mitosis: Microtubules are too stable → spindle can't form properly, mitosis is inhibited.

• Used as a cancer treatment (from Pacific yew tree).

Vinblastine & Nocodazole:

• Cause rapid depolymerization of microtubules.

• Disrupt spindle formation → inhibit mitosis.

What are the two main types of motor proteins that move along microtubules, and in which directions do they move?

• Kinesin:

  • Moves toward the plus end of microtubules

  • Transports cargo toward the cell periphery

• Dynein:

  • Moves toward the minus end of microtubules

  • Transports cargo toward the cell center/MTOC

What is the structure of kinesin, and how does it function?

• Structure:

  • Tetramer = 2 heavy chains + 2 light chains

  • N-terminal globular motor domains bind microtubules

  • Heavy chains = motor activity

  • Light chains = cargo binding (via variable tails)

• Function:

  • Motor domains use ATP hydrolysis for movement

  • Transports vesicles and organelles to plus ends of microtubules

  • Moves cargo away from MTOC → toward cell edge

  • Tail regions determine cargo specificity

How does kinesin move along microtubules in a “hand-over-hand” fashion?

• Mechanism:

  • Two motor heads (dimers) take turns moving

  • At least one head always remains attached to the microtubule

  • Cycle stages:

    • Lagging head: bound to ATP → hydrolyzes ATP → ADP + Pi → releases microtubule

    • Leading head: bound to ADP → exchanges for ATP → tightens microtubule binding

    • Conformational change in neck swings lagging head forward

  • This resets the cycle for continuous forward movement

• Key concepts:

  • Movement is ATP-driven

  • Coordination between heads ensures continuous stepping

How can kinesin movement be studied using plastic beads in vitro?

Nomarski microscope assay:

• Plastic beads (1 μm) tethered to kinesin

• Kinesin walks along a microtubule fixed to a dish

• Microtubules made of purified tubulin

• Bead motion visualized in real time

• Movement rate: 0.5 μm/second

What is a gliding mobility assay and how does it measure kinesin movement?

• Setup:

  • Kinesin proteins are anchored to a glass slide by their tails

  • Fluorescent microtubules are added to the solution

• Action:

  • Kinesins move the microtubules across the slide

  • Microtubule movement is visualized with a fluorescence microscope

What is dynein and how does it function in cells?

• Dynein = minus-end directed motor

  • Moves toward MTOC, away from cell edge

• Two types:

  • Cytoplasmic dynein: moves organelles and vesicles

  • Axonemal dynein: powers cilia and flagella movement (e.g., sperm cells)

• Structure:

  • 2 identical heavy chains

  • Multiple intermediate + light chains

• Movement:

  • ATP hydrolysis enables movement (described as a “strange walk”)

How does dynein move cargo using its power-stroke mechanism?

• Steps of dynein power-stroke:

  1. ATP binds → motor head releases microtubule

  2. ATP hydrolysis → dynein-ADP+Pi reattaches to microtubule

  3. Pi release → triggers power-stroke via linker arm (moves cargo)

• Within a dimer:

  • Dyneins alternate power-strokes for continuous movement

• Each stroke moves cargo ~8 nm toward minus end

• Electron microscopy:

  • Confirms visual progression of power-stroke steps

How do motor proteins transport cargo along microtubules in cells like neurons?

• Cargo uses both kinesin and dynein motor proteins

• Movement direction depends on which motor is active

• Minus-end anchored at MTOC near the nucleus

• Plus-end extends toward cell membrane/synapse

• Example: Neurotransmitter vesicles move along axons

  • From cell body ➝ synapse

  • Can move in both directions on microtubule “highways”

How is cargo direction decided when two motor proteins pull in opposite directions?

• Dynein pulls to minus-end, kinesin to plus-end

• "Tug-of-war" model:

  • Motors compete to pull cargo

  • Final direction = winner of the battle

• Regulatory proteins control which motor is active

  • Respond to internal cellular signals

What is an example of a single vesicle reversing direction on a microtubule?

• Fluorescently-labeled vesicle tracked in cell

• Initially moves toward minus-end

• In 9th frame, reverses direction to move toward plus-end

• Shows dynamic, bidirectional transport on microtubules

How do molecular motors regulate melanosome movement and skin color in fish?

• Melanosomes = pigment-filled organelles

• Transport affects skin color change in response to signals

• Dynein:

  • Moves melanosomes to center (minus-end)

  • Cell appears lighter

• Kinesin:

  • Moves melanosomes to periphery (plus-end)

  • Cell appears darker

• Switch between directions controlled by:

  • cAMP as a secondary messenger in signal transduction pathways

Applied Lecture

Cytoplasmic streaming in plants

What is cytoplasmic streaming?

Directed flow of cytosol and organelles around cells, especially prevalent in plant cells (can be seen in fruit fly and c.elegan embryos), aiding nutrient and metabolite delivery.

Example: Chloroplast movement in Elodea.

Who first reported cytoplasmic streaming and when?

• Bonaventura Corti in 1774, studying Nitella and Chara green algae

• Occurs from algae to angiosperm (higher order) flowering plants

• Seen more in aquatic plants > land plants

• Primitive + essential

What cell structures are involved in rotational streaming in Nitella?

• Internodal and leaf cells

• Actin filaments near the cell membrane at these regions, chloroplasts arranged in rows, and a central vacuole

• Function: Circling chloroplasts help make energy for aquatic plants, where light sources are not as available.

What composes the microfilament bundles in internodal cells and their suggested identity?

Each microfilament is composed of twisted subunits, suggested to be actin filaments.

How does cytochalasin affect cytoplasmic streaming?

• A fungal metabolite that binds actin filaments and blocks polymerization, showing streaming velocity depends on actin microfilaments.

• Control: DMSO since cytochalasin cannot be injected directly

• Experimental Condition: DMSO + cytochalasin

How is cytoplasmic streaming powered?

• Myosin motor proteins move along actin filaments using ATP hydrolysis, enabling high velocities (up to 70 μm/s in Chara algae)

• Good for large cells diffusion is slow

• High velocity due to high ATPase activity and fast ADP dissociation

What is special about myosin XI (higher order plants)?

• Similar structure to Myosin V (humans)

• It has two ATP-converting head domains and a tail binding cargo

• Moves at 5 μm/s (10x faster than human myosin V, but slower than myosin in Chara and Nitella plants)

What influences cytoplasmic streaming in plants and algae?

Streaming is usually constant (primary streaming) but can be induced in higher aquatic plants by light or chemicals that change ATP availability, affecting chloroplast position.

How does pH affect cytoplasmic streaming rate?

Maximal streaming occurs at pH ~7, with decreased rates in acidic or alkaline conditions.

How does temperature affect cytoplasmic streaming velocity?

Velocity increases linearly with temperature.

What happens when myosin XI is knocked out in plants?

Growth defects, reduced cell size, and delayed flowering, suggesting cytoplasmic streaming is important for development.

What was done to study cytoplasmic streaming effects on plant development?

Motor domains of Arabidopsis myosin XI were replaced with fast (Chara) or slow (human myosin Vb) myosins.

*Myosin XI is same in algae and higher order plants, but velocity differs.

How were chimeric myosins validated?

Using in vitro sliding filament assays to measure velocity; transformed plants carried organelles normally but at altered speeds.

How did high-speed myosin XI affect Arabidopsis growth?

• Plants grew taller with larger leaves compared to wild-type and low-speed myosin plants

• Shoot height + leaf size + dry weight of modified plants differed from wild type 35 days after planting

Did chimeric myosins affect leaf number?

No change in leaf number, but high-speed myosin plants had greater shoot height and dry weight.

*Significant stars are comparing modified to wild type

How did cell size change with high-speed and low-speed myosin XI?

• High-speed myosin XI cells were 47% larger; low-speed myosin XI cells were 20% smaller than wild-type

• No significant difference in number of cells

What did in vitro gliding assays reveal about myosin XI variants?

Wild-type, high-speed, and low-speed myosins have distinct velocities affecting filament movement.

How does myosin XI speed affect cytoplasmic streaming in plants?

High-speed myosin XI plants show the fastest cytoplasmic streaming due to faster myosin motor domain “walking” on actin.

What role does cytoplasmic streaming play in plant cell size and development?

• It regulates cell size by enabling efficient material transport

• Especially important in large aquatic plant cells (Elodea and Chara) that have limited diffusion ability

• Slower in land plants (larger number of small cells)