11.08.11+-+Cytoskeletal+Based+Movement+&+Mitosis

Cytoskeletal Based Movement & Mitosis

Dynein walks toward minus end || None || Hydrolysis of ATP destabilizes G-Actin-G-Actin bond.
 * || Actin || Microtubules || Intermediate Filaments ||
 * Subunits || G-Actin || a, B Tubulin || Intermediate filament proteins ||
 * Nucleation Factors || Arp 2/3 || g-Tubulin || N/A ||
 * Assembly || Self-assembly || Self-assembly || Self-assembly ||
 * Functions/Locations || Muscle contraction; micro-villi core; base of focal adhesions and zonula adhesions; assembly causes leading edge of motile cells; short distance intracellular transport || Flagella; mitosis (retraction of chromatid); cilia; intracellular transport (large distances e.g. axonal transport) || Desmosomes; hemidesmosomes; cellular structure; holds myofibrils in place (gives banding pattern); called keratin (skin), nestin (stem cells), or neurofilaments (neurons) depending on location ||
 * Polarity || +/- || +/- || None ||
 * Orientation || In motile cells plus end leads; in muscle fibers plus ends at z bands; at adhesions plus end toward the membrane || Except for in epithelium (plus end toward basal surface), the plus end is always toward the action (chromatid in mitosis, tip of cilia, tip of flagella, synapse of axon) || Randomly oriente ||
 * Motor Proteins || Myosin walks on actin toward + end || Kinesin walks toward plus end
 * Disassembly || Actin w/ ATP is more stable than actin w/ ADP.

Cofilin, a protein that disassembles NTP/NDP or NDP/NDP actin bonds disassembles the back end of the actin pushing the leading membrane edge in the direction of cellular movement || Microtubules w/ GTP are more stable than microtubules w/ GDP. Hydrolysis of GTP destabilizes microtubule structure.

If the more stable GTP cap at the end of microtubules is removed by hydrolysis, microtubule disassembles rapidly through process called catastrophe. || Far more stable ||
 * NTP || ATP || GTP || None ||

Actin filaments and microtubules
 * Subunits: monomeric G-actin; heterodimeric ß-tubulin
 * Subunits are pointed in same direction, imparting polarity
 * An implication of polarity is that the two ends differ functionally. One end is termed “plus”, the other “minus”. This terminology is based on very old studies wherein the polymers were assembled in a test tube from purified subunits. These assembly reactions showed that one end added subunits faster. This end was termed the “plus-end”.
 * G-actin and ß-tubulin are conserved across different cells
 * Bind and hydrolyze ATP (actin) or GTP (tubulin). This activity underlies the fascinating dynamics of these polymers.
 * Functions: Intracellular transport, force production & spatial organization

Intermediate filaments
 * Subunits termed intermediate filament proteins.
 * Intermediate filament proteins can vary between different cells, e.g. keratins in skin keratinocytes; nesting in stem cells, neurofilaments in neurons.
 * Polymers lack polarity: Intermediate filament proteins form anti-parallel dimers, which are symmetric, and these dimerize to make symmetric tetramers of IF protein that assemble into the polymer.
 * Do not bind nucleotide and are relatively static
 * Function is to contribute mechanical stability to the cell

Self-assembly: the spontaneous assembly of subunits (typically proteins) to build an ordered structure. Subunits interact through non-covalent bonds

Polymer dynamics: nucleation and elongation

Nucleation factors for actin filaments and microtubules:
 * Bind to one end of the polymer
 * Control where the polymer assembles and its orientation
 * Cap the minus-end (i.e. prevent disassembly from this end).
 * Arp2/3 complex (actin), g -Tubulin complex (microtubules)

Orientation of microtubules in cells (motile fibroblasts, cilia, mitotic cell at metaphase, epithelial cell, neuron)

Orientation of actin filaments in cells (muscle sarcomeres; leading and trailing parts of a motile cell)

Motor proteins > one direction along the polymer
 * Enzymes that hydrolyze ATP and generate movement and/or force in
 * Motor domains and cargo-binding domains (“tails”)
 * Kinesins, dyneins, myosins and their functions

Representative functions of motor proteins
 * Muscle contraction
 * Membrane transport (secretory vesicles, lysosomes, Golgi)
 * Axonal transport
 * Mitosis

Cilia
 * Motile cilia
 * Primary cilia
 * Axoneme (the part of the cilia internal to the membrane; axonemal dyneins).
 * Intraflagellar transport along outer axonemal microtubules (uses a kinesin and cytoplasmic dynein).
 * Ciliopathies

The hydrolysis of ATP (actin) and GTP (tubulin) in actin filaments and microtubules
 * General idea: hydrolysis accelerates the rate that subunits disassemble.
 * Accelerating the rate of disassembly is what makes these polymers so unusual and dynamic.
 * Mechanism of nucleotide-dependent acceleration of disassembly differs between microtubules and actin.

Mechanism of nucleotide-dependent disassembly: Actin > mechanism to that of the protrusion of the leading edge of motile cells.
 * Co-filin cleaves the helical actin filament between ADP-actin subunits
 * Cell locomotion: Protrusion of the leading edge through oriented assembly and disassembly of actin filaments; myosin II – driven retraction of the cell’s rear end.
 * Listeria monocytogenes pathogenesis: “comet tail” of actin nucleated by Arp 2,3 propels the bacteria through the cytoplasm. Similarity of

Mechanism of nucleotide-dependent disassembly: Microtubules
 * Dynamic instability
 * GTP cap
 * Protofilaments
 * Physical model: Seam zipping and unzipping associated with GTP hydrolysis
 * Catastrophe versus simple disassembly
 * Microtubule structure stores the energy from GTP hydrolysis.
 * Dynamic instability helps explain how the prophase mitotic spindle microtubules “find” kinetochores

Mitosis - overview
 * Mitosis is one particular stage of the cell-cycle
 * Stages of mitosis: prophase, metaphase, anaphase, cytokinesis
 * Cyclins and cyclin-dependent phosphorylation
 * Cell cycle and the logic of the mitotic check-point

Mitosis – Components of the mitotic cell and their functions
 * Chromosome: Centromere, kinetochore, condensin complex (“glue”)
 * Mitotic spindle organization: Centrosome, microtubules (astral, polar, & kinetochore microtubules)
 * Spindle dynamics: metaphase, anaphase A, anaphase B

The mitotic (“anaphase onset”) checkpoint
 * Bipolar and monopolar attachments of kinetochores to microtubules
 * The role of tension
 * One unattached kinetochore is sufficient to generate the checkpoint signal to the “anaphase promoting complex” (APC). APC tags proteins with ubiquitin, leading to their proteolysis.
 * The anti-mitotic drug taxol stabilizes microtubules, prevents their dynamics and hence proper attachment of kinetochores to microtubules. Cell cycle arrest via interminable checkpoint signal from unattached kinetochores..

Proteins associated with actin filaments or microtubules: Motor proteins Nucleating Other proteins: bundling, cross-linking, capping, nucleating, severing/depoymerizing, gel-forming, capping