Cytoskeleton: Features, Functions and Filaments

Cytoskeleton Features and Functions

• Governs cell shape and polarity.
• Mediates cell and organelle movements, including vesicular transport.
• Provides cell machinery for cell division, like chromosome movement and separation.

Dynamic Properties

• Constantly undergoes assembly-disassembly reactions.
• These reactions are critical for its diverse functions.

Intermediate Filaments (IFs)

• Numerous subunits form long fibrous filaments.
• Intermediate in size relative to muscle thick and thin filaments.
• Major structural element in animal cells and tissues, such as epithelia, muscle, and skin.
• Forms extensive networks of tough durable fibers that enmesh the nucleus.
• Emanates from specialized cell junctions like desmosomes and hemidesmosomes.
• Provides resilient fibers that resist mechanical stress.
• Associates with microtubules in cells.

Types of IFs

• Subunits vary in molecular weight and ability to copolymerize.
• I and II: Keratins
  • Present in epithelial cells.
  • Type I: acidic, small C-terminal globular domains, forming dimers.
  • Type II: basic, large C-terminal globular domains, copolymerizing.
  • Form heterodimers with equimolar ratios of acidic and basic components.
• III: Vimentin
  • Found in cells of mesodermal origin, like fibroblasts and endothelial cells.
  • Desmin is found in muscle cells within smooth and skeletal sarcomeres.
  • Glial Fibrillary Acidic Protein is in Schwann and astrocytes
  • Peripherin is in nerve cells and neurons.
  • Self assembles in homopolymers, but not with keratins.
• IV: Neurofilaments
  • Found in neurons rich in axons.
  • NF 70 kD self-assembles in vitro.
  • Also includes NF 150kD + 210kD.
• V: Nuclear Lamins
  • Present in all eukaryotic cells.
  • Forms nuclear lamina, which stabilizes the nuclear envelope and organizes chromatin.
  • Lamins A, B, and C contain a nuclear localization sequence and assemble into sheets on the inner membrane.
  • Lamin B has specific phosphorylation involved in regulating disassembly during mitosis.

IF Subunit Structure

• Highly elongated fibrous molecules with a common structural motif.
• N-terminal globular head, varies in size.
• 40 nm central rod-like domain with an extending alpha-helical region and long tandem heptad repeats that promote coiled-coil dimer formation between two parallel alpha helices.
• C-terminal globular tail domain, specific to IF isotype.

Higher-Order Assembly

• Two subunits form parallel dimers through a helix.
• These associate to form antiparallel tetramers.
• Tetramers bind on the long axis, forming protofilaments that pack together within the polymer.

Microtubules

• Found in all eukaryotic cells, especially in the brain.
• Function: Vesicle trafficking, cell division (mitotic spindle), maintaining cell shape/polarity, determining the distribution of cytoskeletal filaments, and positioning organelles.
• Organized by Microtubule-Organizing Centers (MTOCs) like centrosomes, spindle poles, and basal bodies.

Tubulin Subunits

• Alpha/beta tubulin: present in all MT structures and encoded by separate genes.
• Gamma tubulin: specific to MTOC centrosomes and spindle poles.
• Monomers self-associate to form an alpha/beta dimer that binds with GTP (building block).

In Vitro Assembly

• Microtubules form by reversible polymerization of tubulin dimers, stimulated by the addition of Mg+/GTP.
• I. Nucleation (lag phase)
  • MTOC serves as the nucleation site in the cell.
  • Tubulin dimers self-associate to form rings.
  • Rings uncoil, forming small protofilaments that laterally associate to form a closed tube with 13 protofilaments making up the wall.
• II. Elongation (growth phase)
  • Tubulin heterodimers assemble into hollow cylinders.
  • Each protofilament is formed by dimeric subunits pointing in the same direction which results in structural polarity.
  • Polarity: One end has alpha subunits (minus end), and the other has beta subunits (plus end).
  • Association and dissociation rate constants are greater at the plus end.

Centrosomes

• Well-defined MTOC in animal cells containing gamma tubulin.
• Contains centrioles - cylindrical structures embedded in the centrosome, arranged at 90 degrees.
• Centrioles organize the centrosome matrix and ensure duplication during cell division.

Dynamic Instability

• Rapid turnover, growing at a constant rate but rapidly shrinking.
• The transition between growing and shrinking is linked to GTP hydrolysis.
• Growth Phase
  • Tubulin assembly is more rapid than GTP hydrolysis, leading to the formation of the GTP-Tubulin Cap.
  • The GTP-Tubulin Cap is stable, with high subunit affinity and growth.
• Shrinking Phase
  • Polymerization slows, and GTP hydrolysis catches up, exposing GDP-tubulin ends.
  • GDP-tubulin has a lower affinity, is more unstable, and depolymerizes.

Microtubule-Associated Proteins (MAPs)

• Bind along the sides of the MT polymer and stabilize it against disassembly.
• Mediate the interaction of MTs with other cellular components.
• Has tubulin-binding motifs and are targets of various kinases.
• Alpha tubulin can be acetylated post-translation.

Microfilaments

• Long, thin filaments and meshworks made of actin subunits.
• Muscle (skeletal/cardiac)
  • Stable, hexagonal crystalline array.
  • Thin filaments (actin) and thick filaments (myosin) form 40% of total muscle protein.
  • Organized in highly ordered sarcomeres within fibers, involved in locomotion.
• Non-muscle cells (epithelial/endothelial, etc.)
  • Highly dynamic.
  • Functions: cell shape, structure, cell polarity, and vesicle trafficking.
  • Less ordered arrays of microfilaments, associated proteins, and myosin molecules.
• Helical array of actin subunits, structurally conserved.

Actin Polymerization

• Reversible assembly-disassembly of actin subunits.
• Nucleation to Elongation to Steady State Equilibrium, globular to filamentous.
• Kinetics are dependent on salt, ATP, and actin concentration.
• Assembly rate differs at the two ends:
  • Barbed end: Fast (plus).
  • Pointed end: Slow (minus).

Cell Specialization

• Cell cortex: structural rigidity and stabilization of the plasma membrane.
• Stress fibers: bundles of actin that are contractile tension-generating elements.
• Cell surface extensions/Protrusions: increase the surface area of absorptive cells, motile activity to probe the environment for cell locomotion.
• Contractile ring: late stage of cell division during cytokinesis.
• Focal adhesion: a dynamic substratum-membrane attachment site.
• The RHO family of monomeric GTPases regulates actin filament organization:
  • Cdc42 → filopodia
  • Rac1 → lamellipodia
  • Rho → stress fibers
  • (These often function as a cascade).

Molecular Motors

• Convert chemical energy into mechanical work.
• Kinesin
  • A microtubule motor that is plus end-directed.
  • A heterotetramer family of related proteins with two heavy chains and two light chains.
  • The heavy chain has three domains: a stalk coiled-coil, a globular N-terminus head with ATPase and tubulin-binding activity, and a C-terminal fan-tail associated with light chains that bind cargo.
  • Mediates movement towards the plus end of MT, starting in the cell center.
  • The rear head is tightly bound to ATP and MT.
  • The front lead is bound to ADP.
  • During a kinesin step, the read head detaches from the tubulin binding site and moves forward, with ATP hydrolysis on the rear head and dissociation of ADP/ATP binding at the leading head.
• Dynein
  • Cytoplasmic, mediates movement of minus end of microtubules.
  • Large oligomeric protein with two heavy chains, two intermediate chains, and varying light chains.
  • Dynactin: Protein that co-purifies with cytoplasmic dynein and is present in similar quantities.
  • Large complex that binds to MTs, dynein itself, and vesicular cargo, involving Arp1.
  • Requires adapter proteins (dynactin) to bind to organelles and vesicular cargos.
• Myosin-Based Movement
  • Major protein in muscle and nonmuscle cells, conventional or un.
  • Composed of a head, tail, and tightly bound light chains, strongly bound to actin.
  • Contains actin-activated ATPases.
• Myosin II, Conventional
  • In skeletal/cardiac muscle cells, 300-400 myosin 2 dimers aggregate laterally and then tail-tail to produce a bipolar thick filament.
  • In smooth muscle/nonmuscle cells, myosin is in equilibrium between extended and folded forms.
  • Folded form: the tail folds back to interact with the head, preventing interaction with F-actin.
  • Extended form: the tail extends away from the head, allowing interaction with F-actin.
  • Regulated by reversible phosphorylation of light chains and Ca2+.
• Unconventional Myosins
  • I, III-XI, specialized functions.
• Microfilaments provide a scaffold for myosin-based motility.

Organelles and Protein Sorting

• Eukaryotic cells are subdivided into functionally distinct membrane-bound compartments.
• Each compartment contains its own set of enzymes and specialized molecules, enabling it to perform distinct functions.
• Biogenesis of organelles and their specialized functions rely on the correct sorting of proteins to the appropriate destination.
• Protein sorting and trafficking require proteins to cross one or more membranes.
• Nucleus: contains DNA and RNA
• Cytoplasm: half the cell volume, intermediary metabolism, protein synthesis, non-membrane mediated biochemical reactions, and function
• Endoplasmic Reticulum: half the total surface area of the cell's membrane, site for synthesis of lipids, membranes, and secreted proteins, as well as CA2+ storage.
• Golgi Apparatus: protein modification and sorting.
• Lysosomes: degradation compartment.
• Endosomes and Plasma Membrane: membrane and macromolecular internalization and recycling.
• Mitochondria: ATP formation from energy sources via oxidative phosphorylation.
• Sorting relies on protein machinery that recognizes and assists gated and trans processes.
• Gated transport: Nucleus, gated transport of large molecules; small cytosolic solutes and proteins can pass freely.
• Transmembrane transport: Mitochondria and ER, protein translocating machinery directs the movement of proteins across the membrane.
• Vesicular Transport: ER, Golgi, secretory, and endocytic pathway, transport vesicles ferry proteins from one compartment to another.
• The topography of membrane proteins and membrane-enclosed proteins is maintained during vesicle transport.

Rules of Protein Sorting

• All proteins are initially synthesized on cytoplasmic ribosomes, regardless of their destination.
• Sorting begins during or after translation.
• Targeting information resides in sorting signals within the polypeptide.
• Adaptor proteins bind the localization sequences and help target new polypeptides to the correct compartment.
• If a protein lacks a sorting signal, it remains in the cytoplasm as a resident protein.
• Target organelles contain machinery that recognizes appropriate sorting signals.
• Organelles grow by expansion of preexisting organelles.

Sorting Signals

• Signal Peptides: a stretch of 15-60 amino acids that dictates targeting, often removed by cleavage of a signal peptidase, direct proteins from the cytosol to the appropriate compartment.
• Signal Patches: a 3D arrangement of amino acids on the surface of a folded protein organized into a signal recognition structure, which directs enzymes and proteins to the correct compartments.

Nuclear Transport

• Nucleo-cytoplasmic exchange allows for the movement of proteins and DNA into and out of the nucleus at relatively high volume and with high rates of exchange.
• Proteins involved in nuclear functions have to be imported, and tRNAs and mRNAs have to be exported.
• Some ribosomal proteins are first imported, assembled with rRNA, and then exported as part of a ribosomal particle.
• Exchange occurs through gated-pores – Nuclear Pore Complexes.
• Mediates bidirectional transport of proteins and nucleic acids post-translationally.
• Allows free diffusion of small solutes or low MW proteins across the nuclear envelope.