CH 17

Chapter 17: Cell Organization and Movement I - Microfilaments

17.1 Microfilaments and Actin Structures

• Actin is a highly conserved and abundant eukaryotic cell protein.
• Cells assemble diverse structures of actin filaments for different functions.
• G-actin reversibly assembles into polarized F-actin filaments:
  • Composed of two protofilaments, in which the actin subunits are all oriented in the same direction.
  • Protofilaments are wound around each other to form a helix with the actin nucleotide-binding site exposed on the (-) end of each protofilament.

Cytoskeleton Components

• Cells reversibly and dynamically assemble each type of filament from specific subunits.
• A single cell can have all three filament systems in its cytoskeleton.
• Each filament system has a distinct organization in cells.

Regulation of Cytoskeleton Function by Cell Signaling

• Signals from soluble factors, other cells, and the extracellular matrix influence cytoskeleton organization and movement.
• These signals affect cell shape, movement, and contraction, as well as the organization and movement of organelles.

Examples of Microfilament-Based Structures

• Microvilli
• Cell cortex
• Adherens belt
• Filopodia
• Lamellipodium/leading edge
• Stress fibers
• Moving endocytic vesicles
• Contractile ring
• Phagocytosis

Structures of Monomeric G-actin and F-actin Filaments

• G-actin has an ATP-binding cleft and binds Mg^{2+}.
• F-actin filaments are composed of G-actin subunits and have (+) and (-) ends.

17.2 Dynamics of Actin Filaments

• Actin filament polymerization:
  • In vitro mechanism
  • Kinetics
  • Critical concentration
• Regulation of actin filament polymerization and stability by G-actin and F-actin binding proteins.

In Vitro G-actin Polymerization Phases

• Nucleation: G-actin molecules associate to form a nucleus.
• Elongation: Monomers add to both ends of the nucleus, with faster growth at the (+) end.
• Steady State: Equilibrium between assembly and disassembly.

Actin Treadmilling

• ATP-actin subunits assemble faster at the (+) end (lower Cc) than at the (-) end (higher Cc), resulting in treadmilling at steady state.

Regulation of Filament Turnover by Actin-Binding Proteins

• Profilin (cycle 1):
  • Binds to ADP-G-actin opposite the nucleotide-binding cleft, opening the cleft and catalyzing the exchange of ADP for ATP.
  • Sterically blocks ATP-G-actin assembly on the filament (-) end but allows assembly onto the filament (+) end.
  • ATP-G-actin-profilin complex assembly on the (+) end dissociates profilin to interact with another ADP-G-actin.
  • ATP-G-actin-profilin cannot initiate spontaneous F-actin polymerization.
• Cofilin (cycle 2):
  • Fragments ADP-actin filament regions, enhancing overall depolymerization by making more filament (-) ends.
• Thymosin-β4 (cycle 3):
  • Provides a buffered reservoir of ATP-G-actin for polymerization.
  • Sequesters G-actin at high concentration and releases it at low concentration to polymerize.
  • It is an abundant protein in cells and can buffer a substantial amount of actin in a cell.

Filament Capping Proteins

• Capping proteins block assembly and disassembly at filament ends.
• CapZ binds to the (+) end, where c^+ = 0.12 \mu M.
• Tropomodulin binds to the (-) end, where c^- = 0.6 \mu M.

17.3 Mechanisms of Actin Filament Assembly

• Functionally different actin-based structures are nucleated by formins and Arp2/3 complexes.
• Arp2/3-dependent actin polymerization:
  • Moves pathogenic bacteria and endocytic vesicles within cells.
  • Pushes the leading edge membrane forward in moving cells.
• Toxins affect the dynamics of actin polymerization.

Actin Nucleation by Formin FH2 Domain

• Two major classes of actin-nucleating proteins, regulated by signaling pathways, nucleate actin assembly.

Regulation of Formin Activity

• Formin activity is regulated by an intramolecular interaction.
• Rho GTP binds to the RBD domain, activating formin.
• Profilin-ATP-actin binds to the FH1 domain, promoting actin polymerization at the (+) end.

Actin Nucleation by the Arp2/3 Complex

• The Arp2/3 complex requires an activating factor.
• The complex binds to the side of existing actin filaments to initiate new filament growth.

Regulation of the Arp2/3 Complex

• Regulation by WASp and PI(4,5)P2.
• Wiskott-Aldrich Syndrome protein (WASp).
• Cell division cycle protein (CDC42).

Listeria Movement

• Listeria uses the power of actin polymerization for intracellular movement.
• Actin tail formation propels Listeria through the cytoplasm.
• Arp2/3 complex, capping protein, and cofilin are involved in this process.
• ActA protein on Listeria surface recruits Arp2/3 complex.

Arp2/3-Dependent Actin Assembly During Endocytosis

• Actin assembly drives membrane invagination during endocytosis.
• Endocytosis assembly factors and nucleation promoting factors (e.g., WASP) are involved.

Phagocytosis and Actin Dynamics

• Leukocyte phagocytosis and degradation of a bacterium:
  • Step 1: Opsonization – Bacterium is coated by specific antibodies to a cell-surface protein.
  • Step 2: The leukocyte surface Fc receptor binds the Fc region of the bacterium-bound antibodies.
  • Step 3: Fc receptor-antibody binding signals the cell to activate Arp2/3 complexes, which assemble an actin filament network that moves the cell membrane around the opsonized bacterium. Fusion of the membrane projections pinches off a phagosome into the cytoplasm.
  • Step 4: Fusion of lysosomes with the phagosome delivers enzymes that degrade the bacterium.

17.4 Organization of Actin-Based Cellular Structures

• Proteins of different lengths and flexibilities and F-actin-binding sites organize different actin filament structures with specific functions.
• Actin filaments are attached laterally and end-on to membranes.
• Defects in actin filament organizations and membrane attachment cause human diseases.

Actin Cross-Linking Proteins

• Fimbrin: Microvilli, filopodia, focal adhesions.
• α-actinin: Stress fibers, filopodia, muscle Z line.
• Spectrin: Cell cortex.
• Filamin: Leading edge, stress fibers, filopodia.
• Dystrophin: Linking membrane proteins to actin cortex in muscle.

Lateral Attachment of Microfilaments to Membranes

• Long spectrin spokes intersect at hubs composed of short actin filaments (~14 subunits long), which are stabilized by tropomyosin and the (-) end capping protein tropomodulin.
• Ezrin, a member of the ezrin-radixin-moesin (ERM) family, activated by phosphorylation, links actin filaments laterally to the microvillar plasma membrane.

17.5 Myosins: Actin-Based Motor Proteins

• Myosin superfamily protein structure:
  • Common head and specific tail domains.
• Crossbridge cycle converts ATP hydrolysis energy to mechanical work on actin filaments.
• Myosin class-specific step sizes and processivity support different functions.

Structure of Myosin II

• Myosin II consists of:
  • Two heavy chains.
  • Two essential light chains.
  • Two regulatory light chains.
• The head domain contains actin-binding and nucleotide-binding sites.
• The tail domain mediates dimerization and interaction with cargo.
• Myosins move along actin filaments by converting energy released by ATP hydrolysis into mechanical work.

Sliding-Filament Assay

• The sliding-filament assay is used to detect myosin-powered movement.

Myosin Superfamily in Humans

• All myosins have a similar head and neck motor domain, but each class has a structure/function-specific tail domain.

Three Common Classes of Myosins

• Class I:
  • Step size: 10-14 nm.
  • Function: Membrane association, endocytosis.
• Class II:
  • Step size: 8 nm.
  • Function: Contraction.
• Class V:
  • Step size: 36 nm.
  • Function: Vesicle/organelle transport.

ATP-Driven Myosin Movement Along Actin Filaments

1. Myosin binds ATP, and the head is released from actin.
2. Hydrolysis of ATP to ADP + Pi causes the myosin head to rotate into a "cocked" state.
3. The myosin head binds to the actin filament.
4. "Power stroke": Release of Pi and elastic energy straightens myosin and moves the actin filament.
5. ADP is released, ATP binds, and the head is released from actin.

Myosin II Neck Domain and Movement Rate

• The length of the myosin II neck domain determines the rate of movement.

Myosin V Step Size

• Myosin V has a step size of 36 nm, with each head stepping hand-over-hand in 72-nm steps.

17.6 Myosin-Powered Movements

• Myosin II contraction:
  • Skeletal sarcomere contractile unit.
  • Actin thin filament-myosin II thick filament structure stabilized by thin and thick filament associated proteins.
  • ATP hydrolysis drives sliding filament sarcomere contraction.
  • Skeletal muscle contraction: thin filament Ca^{2+} regulation.
  • Smooth/nonmuscle cell contraction: thick filament Ca^{2+} regulation.
• Myosin V regulation and cargo transport.

Structure of the Skeletal Muscle Sarcomere

• Z disk: on each end of a sarcomere, shared with adjacent sarcomeres.
• I bands: region of actin thin filaments, which are anchored to the Z disk by their (+) ends and extend from both sides of the Z disk into adjacent sarcomeres, that is not overlapped with myosin thick filaments.
• A band: myosin thick filaments interdigitate with thin filaments attached to the Z disks in each half sarcomere.

Sliding-Filament Model of Contraction in Skeletal Muscle

• During contraction, actin filaments slide past myosin filaments, shortening the sarcomere.
• This process requires ATP and Ca^{2+}.

Accessory Proteins in Skeletal Muscle

• Titin: long elastic molecules.
• Calcium is required by two proteins, troponin and tropomyosin, that regulate muscle contraction by blocking the binding of myosin to filamentous actin.

Sarcoplasmic Reticulum and Ca^{2+} Regulation

• The sarcoplasmic reticulum regulates the level of free Ca^{2+} in myofibrils.
• Skeletal muscle contraction is regulated by a Ca^{2+}-dependent thin filament-based regulatory mechanism.
• Contraction:
  • Nerve impulse stimulates an action potential, which is transmitted throughout the plasma membrane (sarcolemma) and down transverse tubules (yellow).
  • T-tubule action potential stimulates the opening of voltage-gated Ca^{2+} channels in the adjacent sarcoplasmic reticulum (blue), releasing stored Ca^{2+} to raise its sarcoplasm concentration to 10^{-4} M.

Ca^{2+}-Dependent Thin-Filament Regulation of Skeletal Muscle Contraction

• In the absence of Ca^{2+}, tropomyosin blocks the myosin-binding site on actin.
• When Ca^{2+} binds to troponin, tropomyosin shifts, exposing the myosin-binding site.

Myosin Light-Chain Phosphorylation Regulates Smooth Muscle Contraction

• Relaxed: At Ca^{2+} concentrations less than 10^{-6} M, the myosin regulatory light chain is dephosphorylated by MLC phosphatase, and the myosin is folded, blocking head interaction with actin.

Myosin Light-Chain Phosphorylation Regulates Smooth Muscle Contraction

• Activated:
  • Ca^{2+} level rises to >10^{-6} M.
  • Ca^{2+} binds to calmodulin (CaM), which undergoes a conformational change (CaM*).
  • The CaM*-Ca^{2+} complex binds to and activates myosin light-chain kinase (MLC kinase).
  • MLCK phosphorylates the myosin regulatory LC on each head.
  • LC phosphorylation activates the head ATPase activity and unfolds the tail to assemble into bipolar filaments.
  • Active myosin II produces force on actin filaments for contraction.

Cargo Movement by Myosin V

• Myosin V:
  • Transports organelles and secretory vesicles along actin filaments nucleated by formins (purple) into the bud before cell division.
  • Binds the ends of cytoplasmic microtubules (green) to orient the nucleus in preparation for mitosis.

Cytoplasmic Streaming

• Cytoplasmic streaming in cylindrical giant algae.
• Movement driven by myosin along actin filaments.

17.7 Cell Migration: Mechanism, Signaling, and Chemotaxis

• Cell migration involves coordinated activities in different cell regions.
• Rho GTPase family proteins regulate the formation of different actin filament organizations and myosin II activity to direct cell motility.

Steps in Cell Locomotion

1. Extension: Lamellipodium protrusion driven by actin polymerization.
2. Adhesion: Formation of new focal adhesions.
3. Translocation: Cell body movement via actin-myosin contraction.
4. De-adhesion and endocytic recycling: Detachment of old adhesions.

Actin-Based Structures Involved in Cell Locomotion

• Leading edge: Lamellipodia and filopodia.
• Stress fibers: Contractile bundles of actin and myosin.
• Focal adhesions: Sites of attachment to the extracellular matrix.

Regulation of the Rho Family of Small GTPases

• Activation:
  • Cell-surface signaling pathways activate a Rho GEF (guanine nucleotide exchange factor).
  • Rho-GEF activates Rho by stimulating the exchange of GTP for GDP.
  • Rho-GTP activation uncovers a membrane-binding domain.
  • Membrane-bound activated Rho-GTP activates effector proteins that cause changes in the actin cytoskeleton.

Dominant-Active Rho Family Members

• Dominant-active Rho induces stress fiber formation.
• Dominant-active Rac induces lamellipodia formation.
• Dominant-active Cdc42 induces filopodia formation.

Signal-Induced Changes in the Actin Cytoskeleton

• Growth factor, LPA influence different pathways involving Cdc42, Rac, and Rho.
• These pathways affect actin polymerization and myosin activity, leading to filopodia, lamellipodia, and stress fiber formation.

Cell Migration Coordination

• Front:
  • Rac activation leading to Arp2/3 activation.
  • Cdc42 activation at the front.
  • Actin filament assembly and treadmilling in the leading edge.
• Back:
  • Rho activation leading to myosin II activation.
  • Contraction of myosin II filaments in both stress fibers and the cell cortex.