Microtubule Assembly and Disassembly By: Cherish Harmon Temperature Dependency • Assembly and stability of microtubules are temperature dependent. • If they are cooled to 4ºC, they depolymerize into αβ-tubulin dimers. • When they are warmed to 37ºC in the presence of GTP, the tubulin dimers polymerize into microtubules. • Change in temperature is used to purify. Temperature Affects Tubulin Polymerization • At αβ-tubulin concentrations above the critical concentration (Cc), the dimers polymerize into microtubules, whereas at concentrations below the Cc, microtubules depolymerize. • The nucleotide bound to the β-tubulin causes the Cc for assembly at the + and – ends of the microtubule to differ. Preferred assembly is at the + end. Tubulin Polymerization Cont. • At αβ-tubulin concentration higher than the Cc for polymerization, dimers add to the + end. • When the αβ-tubulin concentration is higher that the Cc at the + end but lower than the Cc at the – end, microtubules can treadmill by adding subunits to one end and dissociating subunits for the opposite end. Tubulin Polymerization Cont. • Because the intracellular concentration of the assembly-competent tubulin is much higher that the Cc of assembly, polymerization is highly favored in a cell. • The initial rate of tubulin polymerization is accelerated in the presence of nuclei (microtubule-based structures of fragments). Addition of Microtubule Fragments Microtubule Assembly • Three steps: • 1: Protofilaments assemble from αβ- tubulin subunits. • 2: Protofilaments associate to form the wall of the microtubule. • 3: The addition of more subunits to the ends of the protofilaments elongates the microtubule. Stages in Assembly of Microtubules Disassembly of Microtubules • Microtubule shortening: • Under shortening conditions, the microtubule ends are splayed, as if the lateral interactions between protofilaments may have been broken. • When frayed apart and freed from lateral stabilizing interactions, the protofilaments may depolymerize by endwise dissociation of tubulin subunits. • This provides clues about the instability of a microtubule. Observation of Disassembled Microtubules The End!!!
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