08. MODULE 8.pdf

Definition of Cytoskeleton Internal cell framework. Three types of the cytoskeleton in eukaryotic cells: (1) actin filaments (microfilaments), (2) microtubules, and (3) intermediate filaments. Definition of Cytoskeleton Provides structure, localizes organelles, and supports the plasma memb...

Definition of Cytoskeleton Internal cell framework. Three types of the cytoskeleton in eukaryotic cells: (1) actin filaments (microfilaments), (2) microtubules, and (3) intermediate filaments. Definition of Cytoskeleton Provides structure, localizes organelles, and supports the plasma membrane. Pulls the chromosomes apart at mitosis, splits the dividing cell into two. Facilitates intracellular transport (organelles, mRNAs, etc.). Enables some cells to swim, move or contract. Serves as a signaling transducer. Composition of Cytoskeleton Intermediate filaments provide mechanical strength and resistance to shear stress. Microtubules determine the positions of organelles, facilitate intracellular transport, segregate chromosomes during cell division. Actin filaments determine the cell shape and are necessary for whole-cell locomotion and the pinching of one cell into two. Accessory proteins link the different filaments to other cell components, as well as to each other. Motor proteins move organelles along the filaments or move the filaments themselves. Building Blocks of Cytoskeleton Cytoskeletal polymers are built of protofilaments, strings of subunits. Intermediate filaments: fibrous subunits, whereas actin filaments and microtubules: globular subunits. Subunits are held together by hydrophobic interactions and noncovalent bonds. The protofilaments twist around one another in a helical lattice. The multiple protofilament structure yields filaments resistant to thermal breakage.  Image: A hypothetical example, the stable filament is formed from five protofilaments; the bonds holding the subunits together are in red. The Cytoskeleton Is Dynamic Microtubules: a star-like array from the cell center, form (1) a bipolar mitotic spindle during cell division, (2) cilia and flagella, or (3) bundles - tracks for the transport of materials. Actin filaments form (1) protrusions that cells use to move, (2) a contractile ring during cytokinesis, (3) arrays that allow cells to brace themselves against an underlying substratum and enable muscles to contract. Intermediate filaments (1) line the inner side of the nuclear envelope, (2) hold epithelial cell sheets together, (3) help neuronal cells to extend axons, and (4) form tough appendages such as hair and fingernails. NB: Intermediate filaments are less dynamic than actin filaments and microtubules. Microtubule Assembly Microtubules: stiff, hollow cylinders of 13 parallel protofilaments. Each protofilament is a polymer of heterodimers of α-tubulin and β-tubulin, non-covalently bound. GTP on α-tubulin is never hydrolyzed or exchanged; GTP on β-tubulin can be hydrolyzed or exchanged. Assembly is head-to-tail, giving the microtubule polarity, with α-tubulins exposed at one end (minus end) and β-tubulins at the other end (plus end). Microtubule-associated proteins connect microtubules to each other. Microtubules Are Nucleated by γ-Tubulin γ-tubulin is required for microtubule nucleation from a microtubule-organizing center (MTOC), with the plus end growing outward. Most animal cells have a single MTOC (centrosome) near the nucleus. Embedded in the centrosome is a pair of “cylinders” (centrioles) at a right angle to each other; the centrioles are modified microtubules with accessory proteins. The centrosome duplicates during interphase; two daughter centrosomes at the opposite sides of the nucleus form the poles of the mitotic spindle. Microtubules exhibit branching nucleation (from the side of pre-existing microtubules); research has proven that actin filaments also branch. Actin Assembly The actin subunit is a globular monomer (G-actin) with a binding site for ATP (or ADP). Actin subunits assemble head-to-tail into filaments (F-actin) with a structural polarity. Each actin filament consists of two parallel protofilaments that twist around each other. Actin filaments are more flexible than microtubules. Actin filaments are cross-linked together by accessory proteins, making the structure Actin Polymerization Nucleation: ATP-G-actin monomers (in red) slowly form actin complexes (blue). Elongation: The nuclei are rapidly elongated by the addition of subunits to both ends of the filament. At elongation, there is an end with faster growth/shrinkage (plus or barbed end) and an end of slower growth (minus or pointed end). Steady state: Rates of addition and loss of subunits are equal. Soon after incorporation into a filament, subunits hydrolyze ATP and become ADP-F-actin (white color). Nucleation is catalyzed by actin-related proteins (ARPs) from the minus end, allowing elongation at the plus end; ARPs can also attach to the side of actin filaments, building filaments into a web. Steady State At a steady state when the rate of addition equals the rate of release, the concentration of free subunits is constant (this is the critical concentration, Cc). The Cc at the (+) end (i.e., Cc+) of the filament is lower than the Cc at the (−) end (i.e., Cc−). At [G-actin] < Cc+, the filament does not grow; at [G-actin] between Cc+ and Cc−, growth occurs at the (+) end; and above Cc−, growth occurs at both ends, but faster at the (+) end. The steady state is reached at [G-actin] between the Cc values for the (+) and (−) ends when subunits are added to the (+) end and lost from the (−) end, a phenomenon called treadmilling. T- and D-Forms of Filaments The actin and tubulin subunits are enzymes that catalyze the hydrolysis of ATP or GTP, respectively. After adding a subunit to a filament, nucleotide hydrolysis occurs; therefore, the filament exists in T- or D-form (i.e., ATP/GTP or ADP/GDP). Most of the free energy from hydrolysis is stored in the polymer, making the free energy change upon dissociation of a D-form polymer higher than that of the T-form polymer; therefore, D-form depolymerization occurs more readily than T-form. The equilibrium constant for dissociation KD = koff/kon for the D-form polymer equals the critical concentration Cc(D), and is higher than the equilibrium constant for the T-form polymer; therefore, for certain concentrations of free subunits, D-form polymers shrink, and T- form polymers grow. More on Treadmilling Elongation is faster than hydrolysis at the (+) end, and the terminal subunits there are in T- form. At the (-) end, hydrolysis is faster than elongation, and the terminal subunits there are in D- form. The critical concentration for polymerization on a T-form end is lower than for a D-form end. If subunit concentration is between the two values, the (+) end grows and the (-) end shrinks; this is treadmilling. Dynamic Instability On a filament, one end might grow in a T-form, but then change to a D-form and shrink, even at a constant free subunit concentration. This conversion between a growing and shrinking state is called dynamic instability. The change to shrinkage is called a catastrophe; the change to growth is called a rescue. Dynamic instability predominates in microtubules, whereas treadmilling predominates in actin filaments. Putting It Together The cytoskeleton organizes spatially the cytoplasm through a network of protein filaments. The cytoskeleton has three types of filaments: microtubules, actin filaments, and intermediate filaments, all with helical assemblies of subunits that self-associate using a combination of end-to-end and side-to-side protein contacts. The cytoskeletal filaments undergo constant assembly and disassembly: microtubules and actin filaments add and lose subunits only at their ends, with one end (the plus end) growing faster than the other. Tubulin and actin bind and hydrolyze nucleoside triphosphates (tubulin binds GTP and actin binds ATP). Actin filaments predominantly undergo treadmilling (filament assembly at one end and simultaneous disassembly at the other end). Microtubules predominantly display dynamic instability (a microtubule end undergoes alternating bouts of growth and shrinkage).