cell lecture 9 (m2)

Motility and Contractility

• Motility occurs at different levels:

  • Tissue Level: Movement of cells within tissue.

  • Cellular Level: Movement of the entire cell.

  • Subcellular Level: Movement of components within cells.

• Contractility: Specialized form of motility involving the shortening of muscle cells.

  • Driven by motor proteins attaching to scaffolding of microtubules and microfilaments.

Cytoskeleton and Its Role in Motility

• Cytoskeleton Components:

  • Microtubules (MTs):

    • Provide rigid tracks for organelle and vesicle transport.

    • Associated motor proteins walk along MTs to enable movement.

  • Microfilaments (Actin):

    • Involved in muscle contraction and cell movement.

  • Intermediate Filaments:

    • Primarily structural support.

Motor Proteins

• Motor proteins convert chemical energy (ATP) into physical movement.

• They do this by altering their shape and attaching to scaffolds (cytoskeletal filaments).

• They undergo a cycle of:

  • ATP binding

  • ATP hydrolysis

  • ADP release
    (This cycle powers their movement.)

Structural Elements of Motor Proteins

• Common structural domains:

  • Filament binding domains → Attach to microtubules or actin.

  • ATP-dependent motor domains → Generate movement.

  • Connective structures → Link different parts of the motor.

  • Cargo-binding domains → Bind and transport cellular materials.

• Diagram Breakdown:

  • Kinesin-I: Moves cargo along microtubules.

  • Cytoplasmic dynein: Transports cargo in the opposite direction of kinesin.

  • Myosin V: Moves along actin filaments.

Microtubule-Based Movement

• Microtubule-associated motors:

  • Dyneins:

    • Move toward the minus-end of microtubules.

    • Involved in cilia and flagella movement.

  • Kinesins:

    • Move toward the plus-end of microtubules.

    • Important for mitosis and intracellular transport.

• Examples of Motor Proteins:

  • Cytoplasmic dynein → Moves cargo toward the minus end.

  • Axonemal dynein → Enables cilia and flagella movement.

  • Kinesin-1 → Moves cargo toward the plus end.

  • Kinesin-3 → Helps transport synaptic vesicles.

  • Kinesin-5 → Facilitates mitotic spindle movement.

Overview of Cellular Motility

• Microtubule-based motility (tubulin-based):

  • Uses kinesin and dynein for directional transport.

  • Examples:

    • Fast axonal transport.

    • Endomembrane transport.

    • Cilia/flagella movement.

    • Mitosis.

• Microfilament-based motility (actin-based):

  • Uses myosin for movement.

  • Examples:

    • Muscle contraction.

    • Cell crawling.

Microtubule-Based Movement

• Microtubules serve as tracks for movement inside the cell.

• Motor proteins (dynein and kinesin) transport cargo along microtubules.

Cellular Movements: Cilia and Flagella

• Functions:

  • Locomotion in single-celled organisms.

  • Apply force to fluids in multicellular organisms.

• Examples:

  • Cilia on tracheal cells move mucus.

  • Flagella in sperm enable movement.

Structural Differences Between Cilia and Flagella

• Cilia:

  • Size: 2-10 μm long.

  • Number: Many per cell.

  • Movement: Oar-like beating pattern.

  • Force direction: Perpendicular to the cilia.

• Flagella:

  • Size: 10-200 μm long.

  • Number: Typically one (or few) per cell.

  • Movement: Symmetrical, undulating motion (sometimes helical).

  • Force direction: Parallel to the flagellum.

Axoneme Structure (Core of Cilia and Flagella)

• The axoneme is a bundled microtubule structure found in cilia and flagella.

• It:

  • Projects outward from the cell.

  • Is anchored at the basal body.

• Basal bodies:

  • Structurally similar to centrosomes.

  • Composed of 9 triplet microtubules.

Primary Cilia vs. Motile Cilia

• Primary cilia:

  • Found on most cells.

  • Serve as sensory organelles.

  • Have a 9+0 structure (lacking a central pair of microtubules).

  • Important for development (defects cause deafness, left-right asymmetry issues).

• Motile cilia:

  • Have a 9+2 structure.

  • Contain axonemal dynein for movement.

Flagellar Axonemes

• Characterized by the 9+2 microtubule arrangement:

  • 9 outer doublets of microtubules.

  • 2 central microtubules.

• Each outer doublet consists of:

  • A tubule (complete, 13 protofilaments).

  • B tubule (incomplete, 10 or 11 protofilaments).

• Dynein sidearms project from A tubules, allowing movement.

Axoneme Movement

• Axonemal dynein links adjacent microtubule doublets.

• Dynein movements cause microtubules to slide against each other.

• Radial spokes convert this sliding into a bending action.

• Alternating dynein activation allows for:

  • Paddling motions in cilia.

  • Rotational movement in flagella.

Movement Through the Cytoplasm

• Kinesin is involved in ATP-dependent transport toward the plus (+) ends of microtubules (MTs), called anterograde transport.

• Dynein moves particles (cargo) toward the minus (-) ends of MTs, called retrograde transport.

Examples:

• Fast axonal transport.

• Endomembrane transport.

Microtubule-Based Movement

• Fast axonal transport moves vesicles & organelles along MTs.

• Proteins & neurotransmitters made in the cell body must be transported to the nerve ending.

• Diffusion is too slow for this process.

• Organelles move at rates of ~2 µm/sec (~80 minutes per cm).

• Kinesin takes ~250 steps per second.

• Humans walk ~1.7 steps per second for comparison.

Endomembrane Transport

Inside the cell, vesicles constantly move between different compartments like the ER (endoplasmic reticulum), Golgi, and plasma membrane. This is how proteins and lipids get transported to where they’re needed.

• Anterograde transport (forward movement) → Kinesin moves vesicles from the ER → Golgi → Plasma Membrane (outward).

• Retrograde transport (backward movement) → Dynein brings materials from the Plasma Membrane → Golgi → ER (inward).

The centrosome (also called the Microtubule Organizing Center, MTOC) helps establish directionality for these movements.

How Kinesin Walks

Kinesin is the motor protein responsible for forward transport. It works as a heterotetramer—meaning it’s made of four parts:

• 2 heavy chains (the motor part, which "walks").

• 2 light chains (which help bind cargo).

How it works:

• The motor domains (at the base) bind ATP and hydrolyze it, using that energy to step forward along the microtubule.

• It moves in a step-wise fashion, walking 8 nm at a time and binding only to β-tubulin subunits.

• It’s very efficient, converting 60-70% of ATP energy into movement.

Kinesin’s Walking Cycle

Each step of kinesin’s movement follows this cycle:

1. One head (heavy chain) binds ATP, preparing for movement.

2. ATP binding causes a shape change, making the other head swing forward.

3. The trailing head moves to the front, finds a new binding site, and releases ADP.

4. The new trailing head hydrolyzes ATP, resetting the cycle.

This continues as kinesin "walks" along the microtubule, carrying its cargo wherever it needs to go.

Microfilament-Based Movement & Contractility

Microfilaments (actin filaments) are essential for movement in cells. Unlike microtubules, which provide long-range transport, actin filaments contribute to localized movement and contractility.

• Microfilament-based movement:

  • Moves cellular components in some cells.

  • Involved in cell crawling via lamellopodia (flat, sheet-like protrusions).

  • Plays a major role in muscle contraction through actin-myosin interactions.

• Myosin V walking on actin:

  • Just like kinesin moves on microtubules, Myosin V moves cargo along actin filaments.

  • This movement is crucial for intracellular transport.

Actin-Based Motility in Nonmuscle Cells

• Actin filaments are needed for movement in most animal cells.

• Cell crawling involves:

  1. Protrusion extension at the front.

  2. Attachment to the substrate using focal adhesions.

  3. Tension generation to pull the cell forward.

Generating Force and Movement

• Cells extend protrusions (lamellipodia, filopodia) at their leading edge.

• Actin retrograde flow moves microfilaments backward as the protrusion extends.

• This results from actin polymerization at the front and retraction at the back.

Cell Attachment

• Cells need adhesion to a substrate to move.

• New adhesions form at the front, while old contacts at the rear break.

• Integrins play a key role in forming these focal adhesions.

Integrins – Transmembrane Proteins for Attachment

• Integrins are cell surface proteins that attach to the extracellular matrix.

• Inside the cell, integrins connect to actin filaments via linker proteins.

• The focal adhesions they form help transmit mechanical signals between the cell and its environment.

Translocation & Detachment

• Contraction at the rear of the cell squeezes it forward and detaches old attachments.

• This contraction is controlled by actin-myosin interactions.

• Movement occurs through a balance of forming new attachments and breaking old ones.

Chemotaxis – Directional Cell Movement in Response to a Chemical Gradient

• Directional migration happens when a cell senses a chemical gradient and moves toward it.

• When a cell moves in response to a chemical gradient it is called chemotaxis

• Chemoattractants → Cells move toward higher concentrations of signaling molecules.

• Chemorepellants → Cells move away from lower concentrations.

Rho Family GTPases – Regulation of the Cytoskeleton

• Rho proteins regulate cytoskeletal changes in response to signals.

• Activated Rho → Forms stress fibers.

• Activated Rac → Forms lamellipodia (broad protrusions for crawling).

• Activated Cdc42 → Forms filopodia (thin, finger-like protrusions).

Motor Proteins

• Motor proteins use ATP to generate movement.

• They undergo a cycle of ATP binding, hydrolysis, and ADP release to move along cytoskeletal filaments.

• The key motor proteins include kinesins (move along microtubules), dyneins (also microtubule-based but with different directionality), and myosins (actin-based movement).

Motility & Contractility

• Movement in cells is powered by two main cytoskeletal systems:

  • Microtubule-based movement: Kinesins and dyneins transport vesicles, participate in mitosis, and move flagella/cilia.

  • Microfilament-based movement: Actin filaments and myosin motor proteins drive processes like cell crawling and muscle contraction.

Myosin-Based Movement

• Myosins travel along actin filaments to transport molecules or induce changes in cell shape.

• Different types of myosins exist with distinct roles:

  • Myosin II: Major player in muscle contraction.

  • Myosin V: Involved in vesicle transport.

  • Myosin VI: Moves toward the minus end of actin filaments (unlike most other myosins).

Myosins

• All myosins share common structural features:

  • Heavy chains: Contain motor domains for ATP hydrolysis and actin binding.

  • Globular head domains: Bind to actin and generate force.

  • Tail domains: Determine the function (e.g., transport cargo, interact with membranes, etc.).

• Myosin II is the most studied type because it drives muscle contraction.

Myosin ATPase Activity

• Myosins use ATP hydrolysis to generate mechanical movement.

• ATP binding and hydrolysis cause conformational changes that drive the sliding of actin filaments.

Myosins vs. Kinesins

• Both myosins and kinesins convert ATP into movement.

• Differences:

  • Kinesins operate along microtubules, transport cargo over long distances, and work alone or in small groups.

  • Myosins operate along actin filaments, function in large arrays, and move over shorter distances.

Skeletal Muscle Structure

• Skeletal muscles are made up of muscle fibers, which are long, multinucleated cells formed by the fusion of smaller muscle cells.

• Each muscle fiber contains myofibrils, which are bundles of contractile proteins responsible for muscle contraction.

• Myofibrils are made up of repeating contractile units called sarcomeres, which contain thick (myosin) and thin (actin) filaments.

Myofibril Structure

• Myofibrils have a highly organized structure.

• Thin filaments (actin) are arranged alternately with thick filaments (myosin II) in a hexagonal pattern.

• The myosin heads bind to actin filaments and use ATP to generate force for contraction.

Sarcomere Structure

• Sarcomeres are the functional contractile units of muscle, creating the striated appearance of skeletal and cardiac muscle.

• Each sarcomere is 2-3 μm in length when relaxed.

• Important regions of the sarcomere:

  • A-band: Dark band where thick filaments (myosin) are present.

  • I-band: Light band where only thin filaments (actin) are found.

  • H-zone: Central region with only thick filaments.

  • Z-line: Defines the boundary of the sarcomere.

  • M-line: Center of the sarcomere where thick filaments are anchored.

The Sarcoplasmic Reticulum (SR)

• The SR is a specialized type of endoplasmic reticulum found in muscle cells.

• It surrounds myofibrils and works closely with T-tubules, which help conduct electrical signals into the muscle.

• The SR stores and releases calcium ions (Ca²⁺), which are essential for muscle contraction.

The Sarcoplasmic Reticulum & T-Tubules

• The SR is located beneath the sarcolemma (muscle cell membrane).

• T-tubules extend deep into the muscle cell to carry action potentials.

• Calcium release from the SR is triggered when an electrical signal arrives at the neuromuscular junction, allowing muscle contraction to occur.

The Proteins in the Sarcomere

• The sarcomere is the fundamental contractile unit of striated muscle.

• This electron micrograph shows a hexagonal arrangement of thick filaments (myosin) and thin filaments (actin).

• The myosin heavy chain globular domains extend outward from the thick filaments, interacting with actin filaments during contraction.

Thick Filaments

• Thick filaments are primarily composed of myosin II.

• These filaments have 15 nm fibers formed by the staggered assembly of tail domains of myosin molecules.

• The globular domains (heads) of myosin extend outward in a repeating pattern and interact with actin filaments to generate force.

Thin Filaments

• Thin filaments are primarily composed of F-actin (filamentous actin), which forms a coiled-coil structure with tropomyosin.

• Troponin complex (Tn) regulates muscle contraction by controlling myosin interaction with actin.

  • TnT binds tropomyosin.

  • TnI acts as an inhibitor.

  • TnC binds calcium to trigger contraction.

Other Sarcomere Structural Proteins

• Several key proteins stabilize and anchor sarcomere components:

  • CapZ stabilizes the thin filaments at the Z-line.

  • α-actinin anchors actin to the Z-line.

  • Tropomodulin binds the free minus end of actin filaments.

  • Myomesin anchors thick filaments at the M-line.

  • Titin acts as a molecular spring, maintaining alignment.

  • Nebulin stabilizes the thin filament assembly.

The Sliding-Filament Model of Muscle Contraction

• This model explains how muscle fibers contract.

• The thick filaments (myosin) pull the thin filaments (actin) inward, shortening the sarcomere.

• ATP-dependent myosin movement is responsible for this sliding action.

• Key changes during contraction:

  • The H zone and I band both shorten.

  • The A band remains constant.

  • The Z lines move closer together, resulting in muscle contraction.

The Contraction Cycle

1. The Contraction Cycle Overview:

   • The contraction cycle describes how myosin and actin interact to generate muscle contraction.

   • Myosin heads go through a series of binding and unbinding events with actin filaments, powered by ATP.

2. Step-by-Step Process:

   • Cross-Bridge Formation: Myosin, in a "cocked" state (bound to ADP and inorganic phosphate, Pi), binds to the actin filament.

   • Pi Release & Power Stroke: The release of Pi causes a conformational change in myosin, generating the "power stroke," pulling the actin filament toward the sarcomere center.

   • ADP Release: Myosin remains bound to actin until ADP is released.

   • ATP Binding & Myosin Detachment: ATP binds to myosin, leading to its detachment from actin.

   • ATP Hydrolysis & Myosin Reset: ATP is hydrolyzed back into ADP + Pi, resetting myosin into its "cocked" position, ready for another cycle.

3. Significance:

   • This cycle repeats as long as calcium ions (Ca²⁺) and ATP are available.

   • It is fundamental to muscle contraction in skeletal, cardiac, and smooth muscle.

Regulation of Contraction by Calcium

Calcium ions (Ca²⁺) play a crucial role in regulating muscle contraction by controlling the accessibility of myosin-binding sites on actin.

1. Ca²⁺-Regulation & ATP Dependence

   • Myosin can function as long as ATP is present, meaning that the contraction cycle can continue as long as ATP is available to drive it.

   • The cycle is regulated by Ca²⁺, which unblocks myosin-binding sites on actin via its interaction with troponins.

2. Calcium Release from the Sarcoplasmic Reticulum (SR)

   • The sarcoplasmic reticulum (SR) is the main internal calcium store in muscle cells.

   • When stimulated, Ca²⁺ is released from the SR into the cytoplasm to initiate muscle contraction.

3. Low vs. High Calcium Concentrations

   • Low Ca²⁺: The troponin-tropomyosin complex blocks myosin-binding sites on actin, preventing contraction.

   • High Ca²⁺: Calcium binds to troponin C (TnC), which causes a conformational change in the troponin complex, pulling tropomyosin away from the binding sites on actin. This allows myosin to bind to actin and initiate contraction.

• How does calcium get into the cytoplasm?

  • Calcium (Ca²⁺) is stored in the sarcoplasmic reticulum (SR), a specialized endoplasmic reticulum in muscle cells.

  • The Ryanodine Receptors (RyR), a class of intracellular calcium channels, mediate the release of Ca²⁺ from the SR into the cytoplasm.

  • These channels are found in excitable tissues like muscles and neurons.

  • In muscle cells, calcium release from the SR is necessary for contraction.

• Process of Ca²⁺ release into the cytoplasm

  • Electrical stimulus triggers the process.

  • A voltage-sensitive protein in the T-tubule undergoes a conformational change.

  • This change opens the RyR channels, allowing stored Ca²⁺ to flow from the SR into the cytosol.

  • Calcium binds to troponin, initiating the contraction cycle.

• How does calcium get back into the ER (Sarcoplasmic Reticulum)?

  • After muscle contraction, calcium must be removed from the cytoplasm for relaxation.

  • When the electrical stimulus stops, RyR channels close.

  • Ca²⁺ ATPase (SERCA pump) actively transports calcium back into the SR.

  • Lower calcium levels in the cytoplasm allow troponin to return to its resting state, blocking the myosin-binding sites on actin and leading to muscle relaxation.

Summary: The Steps in Muscle Contraction

1. Nerve impulse releases acetylcholine (ACh) at the neuromuscular junction, depolarizing the muscle cell membrane.

2. Voltage-gated Ca²⁺-channel complexes (RyR) in the sarcoplasmic reticulum (SR) release Ca²⁺ into the cytoplasm.

3. Ca²⁺ binds to troponins, which removes tropomyosin from the myosin-binding sites on actin.

4. Actin and myosin interact – myosin heads pull actin filaments, causing sarcomere contraction.

5. Ca²⁺ is pumped back into the SR via Ca²⁺-ATPase, allowing relaxation.

Cardiac and Smooth Muscle

• Cardiac muscle is similar to skeletal muscle in that it is striated but differs in structure and function.

• Mononuclear cells are electrically coupled through intercalated discs, which contain gap junctions.

• Electrical coupling allows cardiac cells to contract in a synchronized manner.

Cardiac Muscle Function

• The pacemaker region of the heart controls the heart rate.

• A depolarization wave initiated by the pacemaker spreads through the heart, triggering contraction.

• Voltage-gated Ca²⁺ channels allow a small calcium influx, which stimulates a larger release of calcium from the sarcoplasmic reticulum via ryanodine receptors (RyR).

Smooth Muscle Overview

• Smooth muscle is responsible for involuntary contractions, meaning you don’t consciously control it (e.g., muscles in your intestines, blood vessels, bladder, uterus).

• Compared to skeletal or cardiac muscle, smooth muscle contractions are slower but last longer.

• Smooth muscle cells don’t have the visible striations seen in skeletal muscle, making them more similar to non-muscle cells in appearance. They are elongated and taper at both ends.

Structure & Function

• Smooth muscle is non-striated and found in the walls of hollow organs (e.g., blood vessels, intestines, bladder).

• It contracts to help with functions like moving food through the digestive tract or regulating blood pressure.

• Instead of sarcomeres (seen in skeletal muscle), smooth muscle has dense bodies that anchor contractile proteins (actin and myosin).

• Intermediate filaments help maintain the structure and shape of the muscle cell.

Smooth Muscle Contraction Mechanism

1. A nerve impulse or hormonal signal triggers the entry of calcium (Ca²⁺) into the cell from the extracellular space.

2. Calcium binds to calmodulin, activating it.

3. The calcium-calmodulin complex then activates myosin light-chain kinase (MLCK).

4. MLCK phosphorylates the myosin light chain, allowing myosin to interact with actin, forming cross-bridges.

5. This cross-bridge formation leads to muscle contraction.

Muscle Relaxation

• After contraction, calcium levels drop, leading to inactivation of MLCK.

• Myosin light-chain phosphatase removes the phosphate from myosin, allowing the muscle to relax.