Cell Biology Chapter 9 The Cytoskeleton

The Cytoskeleton

• Structure:

  • Network of fibrillar proteins organized into filaments and tubules.

  • Composed of different types and exhibits a dynamic structure (grows and breaks).

Functions of the Cytoskeleton

• Major functions include:

  • Scaffold: Maintaining cell shape and enabling shape changes.

  • Muscle contraction.

  • Network of Tracks: Cell movement (inter) and crawling (e.g., axon growth, phagocytosis). direct intracellular transport

    • tracks can assemble and disassemble

  • Organizing the endomembrane system.

• Mitosis Contributions:

  • Chromosome separation through the mitotic spindle.

  • Involvement of centrioles.

• Other functionalities include:

  • Force generating system: Cell motility via cilia and eukaryotic flagella.

    • e.g cilia in the human lung

  • crawling via dynamic rearrangement of the cytoskeleton

Types of Cytoskeletal Elements

• Three main types based on diameter:

  • Actin filaments (6 nm).

  • Intermediate filaments (10 nm).

  • Microtubules (25 nm).

• All types are polymers made from multiple subunits.

Microtubules (thickest)

• Structure:

  • Hollow tubes with a diameter of 25 nm. found in nearly all eukaryotic cells

  • Composed of alpha and beta tubulin heterodimers. that form the protofilaments

  • Both subunits bind GTP/GDP and exhibit a polarized structure: heterodimers

    • b-tubulin: positive (+) end, responsible for rapid polymerization

      • itself is a GTPase which can bind to GAP, and can hydrolyze GTP to GDP-bound tubulin → interaction of heterodimers falls apart = catastrophe of filamentous structure

    • a-tubulin: negative (-) end, anchored at microtubule-organized centers (MTOCs)

    • they form dimers which bind to each other to make structure: 13 protofilaments arranged in a circle . grow towards +

• Function:

  • Intracellular Transport:

    • organelle and vesicle movement (ER to Golgi transport, mRNA localization) using motor proteins

    • transport of peroxisomes, neurotransmitter vesicles (down axons), and endocytic vesicles

      • Kinesisn: move cargo towards plus end (anterograde).

        • vesicles, mitochondria, and peroxisiomes

        • separation of chromosome during mitosos

      • Dyneins: move cargo towards minus end (retrograde)

        • vesicle trafficking, golgi positioning, and cilia/flagella movement

        • Cytoplasmic dyneins:

          • power retrograde transport to neuron

          • 2 heavy chains:

            • stalk binds MTs

            • head: hydrolyzes ATP

          • 2 intermediate & 2 light chains

            • link to cargo

          • Localization:

            • chromosomes (kinetochores) in mitosis

            • Golgi

            • Vesicles undergo retrograde (golgi → ER)

        • axonemal dyneins: generate force for ciliary of flagellar beating

  • Cell shape and structural support

    • radial arrays of mictrotubules define cell architecture

    • in plants: cellulose synthesis

  • Cell division

    • form mitotic spindle for chromosome segregation

    • aid in cytokinesis

  • Motility:

    • enable crawling via dynamic rearrangement

    • axoneme: core structure, 9+2 pattern

• Cytoskeletal interactions in neurons

  • provide axonal transport

  • Anterograde: delivers materials from the cell body to the synaptic terminals

  • Retrograde: receives vesicles from axon terminals back to the cell body

  • Defects in transport cause neurological disorders like ALS

Microtubule Assembly

• Assembly process can occur:

  • In vitro (test tube).

  • In vivo (within a cell).

• Steps involved in formation of microtubule

  • Nucleation (the formation of a small microtubule).

    • slow process

    • Pre-existing MTs (in vitro),

    • gamma-tubulin complexes (in vivo), Rapid

  • Elongation:

    • depends on tubulin heterodimer concentration and temperature.

    • towards + end which has b-tubulin that face the growing end

    • low temp: depolymerization (breakdown) of MTs

    • high temp: increased polymerization

Dynamic Instability of Microtubules

• Microtubules demonstrate:

  • Slow assembly followed by rapid disassembly (catastrophe).

Model of Dynamic Instability

• Factors affecting microtubule dynamics:

  • GTP-bound tubulin stabilizes microtubules (growing).

  • GDP-bound tubulin leads to destabilization (shrinking).

  • Presence of capping proteins may affect polymerization.

Page 11: GTPase Activity and Microtubule Dynamics

• Assembly requires GTP dimers.

• GTPase activity increases, contributing to microtubule dynamics:

  • Hydrolysis of GTP to GDP destabilizes the microtubule.

Page 12: Catastrophe in Microtubule Dynamics

• Loss of the GTP cap triggers catastrophe leading to disassembly:

  • GDP-bound tubulin results in destabilization of microtubules.

Microtubule Organization in Animal Cells

• Nucleation occurs at Microtubule Organizing Centers (MTOCs):

  • Example: centrosomes, facilitating spindle fiber formation during mitosis.

• Types of MTOCs

  • Centrosomes (primary)

    • microtubule nucleation and organization

    • golgi and centrosome positioning

    • spindle formation during mitosis

    • 9+0 formation

    • Polarity

      • minus-end: anchored

      • plus-end

  • Basal Bodies found in cell membrane surface? (cilia/flagella)

    • 9+2 structures

  • Plant cell MTOCs (near nucleus and cortex)

Page 14: MTOC Functions

• Functions of MTOCs include:

  • Organizing mitotic spindle fibers.

  • Facilitating movement of intracellular vesicles.

Page 15: Video Resource on Microtubule Assembly

• Watch: Microtubule Assembly Video

Page 16: GTPase Explained

• GTPase is:

  • A type of enzyme that breaks down GTP.

  • Functions as a molecular switch, involved in cell signaling.

Page 17: Case Study on Cytoskeleton and Fertility

• Title: "More Than Just a Cough: Exploring the Role of the Cytoskeleton in Fertility."

• Author: Carly N. Jordan, Biological Sciences, The George Washington University.

Page 18: Patient Background Summary

• Julia Buckley:

  • Age: 31, Height: 67 in, Weight: 142 lbs.

  • Regular menstrual cycles; previous ectopic pregnancy.

• Robert Buckley:

  • Age: 32, Height: 72 in, Weight: 176 lbs.

  • History: normal sperm count and structure.

Page 19: Video on Cilia and Microtubules in Fertility

• Watch: Cilia’s Role in Fertility.

• Key insights:

  • Sperm head function relies on actin.

  • Sperm tail movement depends on microtubules.

  • Ciliary functions essential in the fallopian tubes.

Page 20: Discussion on Potential Problems

• Possible issues affecting fertility:

  • Sperm head or oocyte malformations.

  • Problems in fertilization or embryo implantation.

Page 21: Focus on Embryo Implantation Issues

• Problematic factors may involve:

  • Sperm, oocyte, fertilization issues, or implantation problems.

Page 22: Further Medical Investigation

• Julia mentions long-term coughing issues without a diagnosed cause despite extensive testing and imaging.

Page 23: Organ with Cilia Associated with Breathing

• The organ containing cilia is:

  • A. Lungs.

Page 24: Ciliary Structure Analysis

• Julia’s fallopian tube sample analysis shows:

  • Normal cilia (9+2 arrangement) vs abnormal (9+0 arrangement) indicating primary ciliary dyskinesia.

• Genetic disorder with a mutation frequency of 1 in 10,000 to 20,000 births.

Page 25: Overview of Microtubule Motors

• Types of motor proteins:

  • Kinesins: Most are + end-directed.

  • Dyneins: - end-directed, inclusive of ciliary dynein.

• All motor proteins hydrolyze ATP for energy.

Page 26: Cytoplasmic Dynein Characteristics

• Composition:

  • 2 heavy chains binding microtubules, with ATP hydrolyzing heads.

• Functions involve:

  • Transportation of chromosomes and vesicles during cell division and intracellular transport.

Page 27: Intermediate Filaments Overview

• Structure:

  • 10 nm fibers with isoforms unique to tissues (e.g., keratin in skin, desmin in muscle).

• Regulated through phosphorylation.

Page 28: Intermediate Filament Assembly

• Stages of assembly:

  • Monomer, dimer, tetramer (no polarity), protofilament formation through end-to-end bonding.

Page 29: Functions of Intermediate Filaments

• Provide structural support and elasticity to skin and muscle, facilitating cell-cell interactions.

• Mutations can lead to skin blistering diseases.

Actin Filaments

• Structure:

  • Comprised of polarized, double-helical filaments made from actin monomers.

  • branched proteins in cytoskeletal elements

    • Barbed (plus) end: rapid polymerization

    • Pointed (minus) end: slower polymerization

  • thin, solid structures forming branched networks

  • Actin monomers bind ATP (globular, G-actin), forming filaments (F-actin) helical strand.

In Vitro Actin Polymerization

• Dynamics of actin polymerization:

  • Continuous polymerization may result in net assembly at the + end and disassembly at the - end, termed 'treadmilling.'

Actin as an ATPase

• Actin’s enzymatic function:

  • Hydrolyzing ATP to ADP + Pi, influencing filament dynamics.

  • ADP-actin → weaker binding affinity compared to ATP-actin, leading to increased filament disassembly

Filament Polarity

• Which cytoskeletal proteins have positive and negative ends?

  • A. Actin filaments and C. Microtubules.

Page 35: Actin-Binding Proteins and Cell Crawling

• Actin polymerization is crucial for neutrophil movement towards bacteria (chemotaxis).

Page 36: Learning Actin Polymerization Mechanisms

• Key players in actin polymerization:

  • Arp2/3 for branching, cofilin for severing, and profilin for inhibiting nucleation.

• F-actin stabilization mechanisms.

Page 37: Parallel Actin Bundles

• Examples include:

  • Filopodia and microvilli, utilizing formins for nucleation and villin for bundling.

Page 38: Actin Motors: Myosins

• Myosin types and functions:

  • ATP-dependent myosins, most are + end directed, with Myosin VI being - end directed.

Page 39: Actinomyosin Contractile Cycle

• Series of events in the cycle:

  • Myosin head binds ATP, releasing from actin filaments.

  • ATP hydrolysis enables re-binding, leading to the power stroke.