cell lecture 8 (m2)

Cytoskeleton Overview

• The cytoskeleton is a network of interconnected filaments and tubules extending through the cytosol.

• Provides structure, shape, and facilitates cell movement and division.

• The cytoskeleton is dynamic and changes according to cell needs.

• Types of Filaments:

  • Microtubules

  • Microfilaments

  • Intermediate filaments

Cytoskeletal Components

Microtubules

• Large (25 nm diameter), hollow tubes made of tubulin proteins.

• Serve as structural elements for cilia and flagella.

Microfilaments

• Small (7 nm wide), twisted chains composed of actin proteins.

• Comprise components of muscle fibrils.

Intermediate Filaments

• Intermediate size (8-12 nm), bundles of protofilaments.

• Composed of different protein classes (6 total).

Prokaryotic vs Eukaryotic Cytoskeleton

• Bacteria and Archaea possess polymer systems similar to eukaryotic cytoskeletal elements:

  • MreB: Actin-like protein involved in DNA segregation.

  • FtsZ: Tubulin-like protein regulating division.

  • Crescentin: A regulator of cell shape.

Microtubules: tubulin in eukaryotes and FtsZ in prokaryotes

Microfilaments: actin in eukaryotes and MreB in prokaryotes

Intermediate filaments: 6 groups in eukaryotes (keratin, vimentin, lamin, etc.) and crescentin in prokaryotes

Techniques to Study the Cytoskeleton

Microscopy Techniques:

1. Fluorescence Microscopy:

   • Fluorescent compounds bind to cytoskeletal proteins, causing them to glow.

   • Example: Fibroblast stained with fluorescent antibodies against actin shows bundles of actin filaments.

2. Live Cell Fluorescence Microscopy:

   • Introduces fluorescent versions of cytoskeletal proteins into live cells.

   • Observes protein function in real-time.

3. Computer-Enhanced Digital Video Microscopy:

   • Uses digital cameras and computer processing to enhance images.

4. Electron Microscopy:

   • Resolves individual filaments with various sample preparation techniques.

Drugs Affecting the Cytoskeleton

• Microtubule Inhibitors:

  • Colchicine, Nocadazole: Inhibit microtubule assembly by binding tubulin.

  • Vinblastine, Vincristine: Prevent polymerization.

  • Taxol: Stabilizes microtubules to prevent disassembly.

• Microfilament Inhibitors:

  • Cytochalasin D: Prevents addition of new actin monomers.

  • Latrunculin A: Sequesters actin monomers.

  • Phalloidin: Stabilizes microfilaments and prevents depolymerization.

Microtubule Structure

• Composition: Microtubules are hollow cylindrical structures made of αβ-tubulin heterodimers.

• Dimensions: They have a 25 nm diameter and are composed of 13 protofilaments arranged in a tube.

• Subunit Arrangement: Each protofilament consists of alternating α-tubulin and β-tubulin dimers.

• Polarity: Microtubules have two distinct ends:

  • Plus end (+): Grows quickly and undergoes polymerization.

  • Minus end (−): More stable and anchored in structures like the microtubule-organizing center (MTOC).

Microtubule Dynamics (Polymerization & Depolymerization)

• Dynamic Instability: Microtubules constantly switch between growth (polymerization) and shrinkage (depolymerization).

• GTP & GDP Control Growth:

  • Tubulin dimers bind GTP, which promotes polymerization.

  • When GTP is hydrolyzed to GDP, the microtubule becomes unstable and depolymerizes.

  • This process allows cells to rapidly reorganize microtubules for different functions.

Functional Domains of Tubulin

• Tubulin has three major domains:

  1. N-terminal GTP-binding domain → Controls polymerization.

  2. Central dimerization domain → Holds α- and β-tubulin together.

  3. C-terminal domain → Interacts with MAPs (Microtubule-Associated Proteins).

Microtubule Arrangements

• Microtubules can form different structures:

  • Singlets: Most common, found in the cytoplasm.

  • Doublets: Found in cilia and flagella.

  • Triplets: Found in centrioles and basal bodies.

Cytoplasmic Microtubules (Dynamic Microtubules)

• These microtubules constantly assemble and disassemble and are involved in:

  • Mitosis & meiosis → Form spindle fibers that separate chromosomes.

  • Cell shape & polarity → Support the structure of cells like neurons.

  • Intracellular transport → Act as tracks for motor proteins (e.g., kinesin and dynein) to move vesicles and organelles.

Axonemal Microtubules (Stable Microtubules)

• These do not disassemble and are involved in:

  • Cilia and flagella → Responsible for movement (e.g., in the respiratory tract and sperm cells).

  • Basal bodies → Anchor cilia and flagella at the cell membrane.

  • Axoneme structure:

    • "9+2" arrangement → 9 doublet microtubules surround 2 central microtubules.

    • Dynein arms → Allow movement of cilia and flagella.

    • Duplets or triplets

Microtubules in Epithelial Function

• Cilia help move mucus and debris out of the lungs.

• Found in respiratory epithelial cells and many developmental structures.

• Basal bodies organize microtubule growth in cilia.

Regulating Microtubules

Microtubule Nucleation and Assembly

• Microtubule Organizing Centers (MTOCs):

  • Act as the site for microtubule nucleation.

  • Centrosomes are the primary MTOCs in animal cells.

  • Basal bodies function as MTOCs in cilia and flagella.

• γ-Tubulin and γ-TuRCs (γ-Tubulin Ring Complexes):

  • γ-tubulin is critical for microtubule nucleation.

  • γ-TuRCs serve as templates for microtubule assembly, ensuring proper growth orientation.

• Centriole Role in MTOCs:

  • Centrioles, composed of nine triplets of microtubules, organize mitotic spindles.

  • Cells lacking centrioles have poorly organized spindles.

Microtubule Polymerization and Dynamics

• Tubulin Monomers and GTP:

  • Microtubules grow when tubulin dimers (α- and β-tubulin) polymerize with bound GTP.

  • GTP hydrolysis leads to destabilization and potential catastrophe.

• Dynamic Instability:

  • Microtubules undergo rapid growth and shrinkage.

  • A stable GTP cap promotes elongation.

  • Catastrophe occurs when GTP is hydrolyzed to GDP, leading to disassembly.

  • Rescue is the return to polymerization after a shrinkage phase.

• Treadmilling:

  • When growth at the plus end is balanced by disassembly at the minus end.

  • Occurs when tubulin concentration is between critical levels.

Microtubule-Associated Proteins (MAPs)

MAPs regulate microtubule stability and function. They are classified into stabilizing and destabilizing proteins.

A. Stabilizing Proteins

• Tau:

  • Forms tight bundles in neurons.

  • Its malfunction leads to neurofibrillary tangles in Alzheimer’s disease.

• MAP2:

  • Organizes microtubules in dendrites.

• +TIP Proteins (Plus-End Tubulin Interacting Proteins):

  • Bind to microtubule plus ends, protecting against catastrophe.

B. Destabilizing Proteins

• Catastrophins (e.g., MCAK):

  • Promote depolymerization at microtubule ends.

• Katanin:

  • Severs microtubules.

• Stathmin/Op18:

  • Binds to tubulin dimers, preventing polymerization.

Microtubule Drugs

Drugs can either stabilize or destabilize microtubules, affecting cell division and cytoskeletal integrity.

• Vinblastine, Vincristine:

  • Bind tubulin dimers, preventing elongation.

  • Used in cancer therapy to inhibit mitosis.

• Taxol:

  • Stabilizes microtubules, preventing disassembly.

  • Also used in cancer treatment to inhibit cell proliferation.

Special Techniques: Speckle Microscopy

• Measures microtubule dynamics by injecting fluorescent tubulin.

• Allows visualization of assembly and movement within cells.

Actin Microfilaments

Structure of Actin Microfilaments

• Composition:

  • Actin microfilaments are made up of G-actin (globular actin) monomers that polymerize to form F-actin (filamentous actin).

  • F-actin consists of two filaments twisted into a helical structure.

• Size:

  • They are 7 nm in diameter, making them the thinnest cytoskeletal element.

• Polarity:

  • They have a plus (+) end and a minus (-) end, with faster elongation at the plus-end due to ATP-actin addition.

Actin Polymerization and Dynamics

• G-actin binds ATP before polymerizing into filaments.

• ATP-actin is added preferentially at the plus-end, leading to filament growth.

• ATP hydrolysis occurs slowly, and the filament mainly consists of ADP-actin in older regions.

• Treadmilling:

  • Actin filaments undergo continuous polymerization at the plus-end and depolymerization at the minus-end.

  • Similar to microtubules' dynamic instability, this allows actin to reorganize rapidly.

Drugs Affecting Actin Polymerization

• Cytochalasins:

  • Fungal metabolites that bind actin filaments and prevent further monomer addition.

• Latrunculin A:

  • A toxin that binds G-actin monomers and prevents polymerization.

• Phalloidin:

  • Stabilizes actin filaments by binding between actin subunits, preventing depolymerization.

Functions of Actin Microfilaments

• Cell Shape and Structural Integrity:

  • Actin filaments maintain cell shape and provide mechanical support.

• Cell Motility:

  • Involved in muscle contraction, cell migration, and cytokinesis (cleavage furrow formation during mitosis).

• Specialized Cell Structures:

  • Microvilli in intestinal epithelium use bundled actin filaments to maintain their shape.

  • Filopodia and lamellipodia are actin-rich protrusions involved in cell movement.

• Intracellular Transport:

  • Actin filaments facilitate vesicle transport within the cell.

Actin in Cell Structure and Membrane Support

• Microvilli Formation:

  • Found in intestinal epithelial cells.

  • Organized, polarized actin bundles are cross-linked by actin-binding proteins.

  • Anchored in the terminal web, which provides rigidity.

• Membrane Interaction:

  • Actin filaments connect with anchoring proteins (e.g., spectrin, ankyrin, Band 4.1) to support plasma membranes.

  • Important in red blood cells and epithelial cells for maintaining structural integrity.

Actin in Cell Crawling and Attachment

• Cell Crawling:

  • Actin filaments are dynamically reorganized at the cell's leading edge.

  • Lamellipodia and filopodia extend from the cell to help with movement.

• Cell Attachment:

  • Stress fibers, composed of bundled actin filaments, help cells adhere to surfaces.

  • These fibers are linked to adhesion sites to provide tension and stability.

Actin in Plant Cells – Cyclosis

• Cyclosis (Cytoplasmic Streaming):

  • In plant cells, actin filaments drive the directed movement of organelles like chloroplasts.

  • This enhances intracellular transport and distribution of nutrients.

Actin Can Organize in Several Ways

• Actin filaments in the cytoskeleton can be arranged in different structural organizations to support cellular shape and function.

• Highly adherent cells (cells that strongly attach to surfaces) contain stress fibers, which are bundles of actin filaments that provide tension.

• Cell cortex: Actin forms a crosslinked gel-like network beneath the plasma membrane, maintaining cell shape during movement.

• Crawling cells use lamellipodia and filopodia:

  • Lamellipodia → a branched network of short actin filaments at the leading edge, allowing the cell to push forward.

  • Filopodia → parallel bundles of actin filaments with plus-ends oriented toward the leading edge for directional movement.

Actin-Binding Proteins

• Actin-binding proteins regulate where actin filaments assemble and disassemble.

• They control:

  • Nucleation: Starting the formation of new filaments.

  • Elongation: Extending existing filaments.

  • Severing: Cutting actin filaments into smaller pieces.

  • Crosslinking: Forming actin networks by linking filaments together.

Key Actin-Binding Proteins:

• Thymosin β4: Prevents actin polymerization by binding free actin monomers.

• Profilin: Competes with thymosin β4 to promote polymerization.

Actin Polymerization & Monomer Regulation

• Actin polymerization is influenced by ATP-bound G-actin (globular actin), which polymerizes into F-actin(filamentous actin).

• The rate of actin filament growth is controlled by monomer binding proteins.

• Thymosin β4 binds actin monomers, making them unavailable for polymerization.

• Profilin competes with thymosin β4, binding actin monomers and promoting filament elongation.

Actin Filament Growth & Length Regulation

• Actin filaments need precise regulation of length for proper function.

• Formins: Promote filament elongation by moving with the barbed (+) end as the filament grows.

• Capping Proteins: Control filament length:

  • CapZ binds the plus-end to prevent uncontrolled elongation.

  • Tropomodulins bind the minus-end to stabilize filaments and prevent disassembly.

Actin Branching & ARP2/3 Complex

• The ARP2/3 complex nucleates new actin branches in lamellipodia.

• Activated by WASP (Wiskott-Aldrich Syndrome Protein) and WAVE/SCAR proteins.

• WASP is autoinhibited but becomes active through PIP₂ signaling, leading to ARP2/3 activation.

• Profillin-actin complexes stimulate growth.

• Capping proteins stop excessive elongation, keeping branched networks organized.

Actin Severing: Gelsolin

• Gelsolin binds F-actin and severs filaments, allowing rapid actin remodeling.

• It caps the plus-end, preventing the addition of new monomers.

• The severed filaments can either:

  • Be crosslinked into new structures.

  • Be disassembled for actin turnover.

• Gelsolin is activated by Ca²⁺ and inhibited by PIP₂, ensuring tight regulation.

Actin Crosslinking: Filamin

• Actin filaments can be loosely crosslinked into networks.

• Filamin is a key crosslinking protein that:

  • Anchors two actin filaments at an angle.

  • Forms gel-like networks rather than parallel bundles.

  • Provides mechanical strength and flexibility to the cytoskeleton.

• Crosslinking networks are deformable, allowing cells to resist stress and reshape under tension.

Actin Bundling in Microvilli

• Microvilli are specialized cellular protrusions that increase surface area (e.g., in intestinal epithelial cells).

• Fimbrin crosslinks parallel actin filaments in microvilli.

• The bundled filaments provide rigid support for stable microvilli structure.

Actin Bundling in Contractile Tissues

• Actin filaments in contractile tissues (e.g., muscle cells) are organized differently.

• α-actinin bundles actin filaments into looser, more dynamic structures.

• In muscle cells:

  • Actin filaments interact with myosin for contraction.

  • α-actinin links filaments together, creating a scaffold for force generation.

Actin Membrane Anchoring

• Actin filaments are anchored to the plasma membrane by proteins that maintain cell shape and structural integrity.

• Key membrane anchoring proteins:

  • Band 3, Band 4.1, Ankyrin, Spectrin:

    • Maintain cell shape during movement and cytokinesis.

    • Especially important in red blood cells (erythrocytes) to prevent membrane damage.

• ERM Proteins (Ezrin, Radixin, Moesin):

  • Attach actin filaments to membrane proteins.

  • Regulate membrane protein movement.

Membrane Anchoring of Actin Networks

Actin filaments are anchored to the plasma membrane by anchoring proteins:

• Band 4.1 facilitates interactions between the cytoskeleton and the plasma membrane, especially in erythrocytes.

• Spectrin & Ankyrin form short chains that crosslink actin with membrane proteins, maintaining cell shape and flexibility.

ERM Proteins and Membrane Dynamics

ERM (Ezrin, Radixin, Moesin) proteins regulate actin-membrane interactions:

• They relocate transmembrane proteins to specific sites on the plasma membrane, enabling clustering and translocation.

• ERM proteins undergo a conformational change upon phosphorylation or PIP₂ binding, switching between inactive and active states.

By linking the actin cytoskeleton to the plasma membrane, ERM proteins contribute to cellular signaling and membrane dynamics.

Regulation of Actin Assembly by PIP₂

• What is PIP₂?
  Phosphatidylinositol-4,5-bisphosphate (PIP₂) is a phospholipid present in the plasma membrane that interacts with several actin-binding proteins.

• How does PIP₂ regulate actin assembly?

  • PIP₂ can bind to proteins such as profilin, WASP, CapZ, and gelsolin, affecting their interaction with actin.

  • It plays a role in recruiting actin-regulating proteins to the membrane.

  • It modulates actin interactions, influencing polymerization and depolymerization.

• Examples:

  • CapZ binding to PIP₂ → causes CapZ removal from actin microfilament (MF) ends, allowing actin polymerization.

  • PIP₂ competes with actin for profilin binding, affecting actin monomer availability.

  • WASP is activated by PIP₂, which promotes actin nucleation (formation of new filaments).

Regulation of Actin Assembly by Rho GTPases

• What are Rho GTPases?
  Rho GTPases are a family of small G proteins (monomeric GTPases) that regulate cytoskeleton dynamics in response to signals like growth factors.

• Key members and their effects:

  • Rho activation → forms stress fibers (contractile actin bundles).

  • Rac activation → promotes lamellipodia formation (broad, sheet-like actin protrusions at the cell's edge).

  • Cdc42 activation → induces filopodia formation (thin, finger-like protrusions).

• How do they regulate actin?

  • Rho GTPases interact with formin proteins and actin regulators to control filament elongation and reorganization.

Molecular Regulation of Rho GTPases

• How are Rho GTPases controlled?

  • Guanine-nucleotide exchange factors (GEFs): Activate Rho GTPases by exchanging GDP for GTP.

  • GTPase-activating proteins (GAPs): Inactivate Rho GTPases by promoting GTP hydrolysis to GDP.

  • Guanine-nucleotide dissociation inhibitors (GDIs): Keep Rho GTPases inactive in the cytosol.

Introduction to Intermediate Filaments (IFs)

• Definition: Intermediate filaments (IFs) are a component of the cytoskeleton that provides mechanical support to cells.

• Characteristics:

  • Not found in plants, but abundant in animal cells.

  • Diverse proteins form filaments 8-12 nm in diameter.

  • Not polarized, meaning they lack a plus and minus end, unlike microtubules and actin filaments.

  • Most stable and least soluble cytoskeletal components.

  • Different IF proteins are unique to different cell types.

Classification of Intermediate Filaments

• Divided into Cytoplasmic and Nuclear IFs:

  • Cytoplasmic IFs:

    • Keratin filaments (epithelial cells).

    • Vimentin and vimentin-related filaments (connective tissue, muscle, and glial cells).

    • Neurofilaments (nerve cells).

  • Nuclear IFs:

    • Nuclear lamins (present in all animal cells, form the nuclear lamina).

Structure and Assembly of Intermediate Filaments

• Basic Unit: Fibrous Dimers

  • Each IF protein has a core rod-like domain (310-318 amino acids long).

  • N- and C-terminal domains differ among IF proteins, giving them cell-type specificity.

• Assembly Process:

  • Dimers form by coiling together.

  • Two dimers associate to form a tetrameric protofilament.

  • Protofilaments align in a staggered manner.

  • 8 protofilaments bundle together to form the final intermediate filament.

Functions of Intermediate Filaments

• Mechanical Support:

  • Bear tension in tissues, preventing excessive stretching.

  • Highly stable and detergent-resistant, unlike microtubules and actin filaments.

  • Not static—they undergo slow turnover and reorganization.

• Role in the Nucleus:

  • Nuclear lamina disassembles during mitosis to allow nuclear breakdown and reformation.

Diseases Related to Intermediate Filaments

• Diseases caused by IF mutations:

  • Keratin mutations → blistering skin diseases (e.g., epidermolysis bullosa).

  • Desmin mutations → cardiac & skeletal myopathies.

  • GFAP (Glial fibrillary acidic protein) mutations → Alexander disease.

  • Neurofilament mutations → amyotrophic lateral sclerosis (ALS).

  • Lamin mutations → cardiomyopathy, lipodystrophy, muscular dystrophies, progeria.

The Nuclear Lamina

• Structure:

  • A 10-40 nm fibrous layer that lines the inner nuclear membrane.

  • Composed of nuclear lamins.

• Functions:

  • Structural support for the nuclear envelope.

  • Regulation of gene transcription by binding and controlling transcription factors.

Laminopathies (Diseases Related to the Nuclear Lamina)

• Mutations in Lamin A cause severe disorders:

  • Muscle wasting diseases.

  • Premature aging disorder (Hutchinson-Gilford Progeria Syndrome, HGPS).

• Fluorescence images:

  • Normal nuclear lamina (A, B) shows smooth, round nuclei.

  • HGPS nuclei (C, D) appear abnormally shaped and fragmented.

Coordination Between Cytoskeletal Components

• Microtubules: Resist bending forces (compression support).

• Microfilaments: Generate tension and contractile forces.

• Intermediate Filaments: Provide elasticity and tensile strength.

Linker Proteins: Spectraplakins (e.g., Plectin)

• Connect all three cytoskeletal components.

• Ensure structural integrity and coordination between cytoskeletal elements.