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Dynein and Spindle Orientation

  • Dynein and Dynactin
    • Dynein, in conjunction with dynactin, is crucial for the orientation and positioning of the spindle apparatus during cell division.
    • The interaction of dynein and dynactin facilitates movement towards the poles of the spindle.
  • Cortical Anchoring
    • The presence of a cortical anchor is essential for the action of membrane-associated forces.
    • Inactive cortical anchor: Does not allow forces to act effectively towards spindle movement, causing a stall in movement direction.
    • Active cortical anchor: Enables dynein to exert pulling forces on the spindle, facilitating directional movement.
  • Microtubule Dynamics
    • Before starting actin filament formation, microtubules must be completed to allow proper cellular structure and movement.

Anaphase Dynamics

  • Phases of Anaphase
    • Anaphase A:
    • Characterized by microtubule shortening as chromosomes migrate towards the poles of the spindle.
    • Driven primarily by disassembly of microtubules.
    • Anaphase B:
    • Involves pole separation where motors are utilized for moving poles further apart.
    • Requires energetic processes with motor proteins at work.

Polar Microtubule Functions

  • Role of Polar Microtubules
    • Polar microtubules help localize RhoGTP, which is critical for forming a contractile actin/myosin ring at the metaphase plate.
    • The contractile ring forms as far from the newly formed nuclei as possible, indicating spatial organization in cell division.

Actin Microfilaments Overview

Actin Components and Dynamics

  1. Cortical Nature of Actin
    • Understanding the localization and distribution of actin within the cell cortex.
  2. Properties of Actin
    • Comprehension of the relationship between globular (G-actin) and filamentous (F-actin) forms of actin.
  3. Nuclei and Filament Elongation
    • Explanation of why the presence of a nucleus leads to an increase in actin filament elongation speed.
  4. Treadmilling Concept
    • Discussion of the consequences of differences in critical concentrations (Cc) at the (+) and (-) ends of actin and how this leads to treadmilling phenomena.

Actin Binding Proteins and Regulation

  1. Abundance of G-Actin
    • G-actin is highly abundant; its polymerization is regulated by proteins such as thymosin, profilin, and cofilin.
  2. Formins and Arp2/3
    • Formins play a role in the regulation of unbranched filament assembly, while Arp2/3 facilitates branched filament development.
    • GTP binding proteins also influence these processes.
  3. Role of Arp2/3
    • Arp2/3 is involved in processes like endocytosis and phagocytosis.
  4. Actin Binding Proteins for Bundling and Branching
    • Description of actin-binding proteins that assist in the formation of actin bundles and branched structures.
  5. Actin's Role in Membrane Support
    • Exploration of the mechanisms whereby actin provides structural support and integrity to cellular membranes.

Myosin and the Sarcomere

  1. Myosin Proteins
    • Differentiation of myosin heavy chains, light chains, and the functions of myosin classes (I, II, and V).
  2. Myosin Mechanism
    • Explanation of the sequence of events that occurs as myosin moves towards the (+) end of actin filaments.
  3. Labeling Sarcomeres
    • Ability to label various parts of a sarcomere and the associated proteins that provide support.
  4. Sarcoplasmic Reticulum and T-Tubules
    • Relationship between the sarcoplasmic reticulum and T-tubules concerning muscle contraction regulation.
  5. Calcium Regulation
    • Discussion on how calcium regulates muscle contraction, detailing the events taking place in the sarcomere during contraction.

Actin-Based Structures

  • Types of Actin Structures
    • Microvilli, cell cortex, adherens belts, lamellipodia, and filopodia are highlighted as key actin structures in epithelial and migrating cells.

Actin Polarity and Assembly

Actin Porperties

  1. Actin Polarity
    • Polarity of actin filaments is characterized by a pointed (-) end and a barbed (+) end.
    • Polymerization occurs preferentially at the (+) end.
  2. Total Actin Concentration
    • Monomer and filament concentration influence polymerization dynamics, with critical concentrations for assembly and disassembly significantly impacting cellular activity.
  3. Steady State Dynamics
    • While “steady state” might be reached in vitro, true steady states in cells are rare due to the influence of regulatory proteins.

Regulation of Actin Polymerization

  1. Critical Concentration and Actin Binding Proteins
    • Regulation of actin polymerization by thymosin (sequesters actin), profilin (encourages polymerization), and cofilin (enhances depolymerization).
  2. Capping Proteins
    • CapZ and tropomodulin resolve assembly and disassembly at actin filament ends, crucial in muscle cells.
  3. Disrupting Drugs
    • Cytochalasin induces depolymerization while phalloidin stabilizes actin filaments.

Assembly and Branching Mechanisms

  1. Formins
    • Formins nucleate unbranched filament assembly and their activity is controlled by Rho-GTP.
  2. Arp2/3 and NPFs
    • Nucleation promoting factors (NPFs) like WASp and WAVE activate Arp2/3 for filament branching.
  3. Listeria ActA
    • Acts as an NPF promoting directional movement through polymerization during infection.

Role of Actin in Endocytosis and Phagocytosis

  • Actin facilitates membrane movement, working to either pull membranes inward during endocytosis or push them outward in phagocytosis.

Actin-Binding Proteins and Cellular Structures

  • Functionality of Actin Networks
    • Essential proteins that anchor actin networks to cell membranes in red blood cells and epithelial cells for maintaining cellular structure, shape, and facilitating movement.

Myosin: Actin's Motor Protein

  1. Myosin II
    • Characterized by heavy and light chains, with the head functioning as an ATPase and the tail binding to cargo.
  2. Functions of Myosin Classes
    • Myosin supports functions across contraction, transport, and membrane associations.
  3. Sliding Filament Assay
    • A method to analyze myosin-powered movement. Length of the neck determines the rate of movement in myosin II.

Conformational Changes in Myosin

  • Rigorous State to Movement
    • In the absence of ATP, myosin is in a 'rigor' state, and upon ATP binding, it undergoes conformational changes facilitating muscle contraction.

Skeletal Muscle Structure

  1. Sarcomere Composition
    • Myosin II filaments create the A band which remains unchanged during muscle contraction, while Z disks come closer and I bands decrease in size.
  2. Regulatory Proteins
    • Structures that stabilize sarcomeres and play integral roles in the contractile process.
  3. Sarcoplasmic Reticulum Function
    • Stores and regulates Ca2+ levels, crucial for muscle contractions by releasing calcium ions rapidly upon stimulation.
  4. Calcium and Contraction Cycle
    • The release of Ca2+ from the sarcoplasmic reticulum ignites the muscle contraction cycle, emphasizing the importance of calcium ions in muscle physiology.