lecture 30

lecture 30 - LECTURE 30 06 November 2009 (P. J. Hollenbeck)...

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LECTURE 30 06 November 2009 (P. J. Hollenbeck) BIOL231 Assembly of the Cytoskeleton Read: chap. 17: 580-82; 591-94; DVD 17.2 - 17.4; Problems: exam III ‘07, #9; exam IV ‘07, #2 I. Filament Assembly <Cytoskeletal filaments are constantly assembling and disassembling in the cell. We can study this using purified actin or tubulin in vitro , because the subunits are able to self-assemble , that is, they are able to assemble into filaments without any help from other proteins. If we examine the assembly of actin or MTs we can see that it has 3 phases. These can be discerned quantitatively by looking at the course of assembly over time. We’ll consider the assembly of actin in more detail here, but MT assembly from tubulin subunits is very similar.> (A) Actin assembly: nucleation, elongation, and steady state (1) Nucleation. If we put pure actin monomers in a tube and measure their assembly over time, very little happens at first. This is because the slowest step in assembly is nucleation, the initial formation of something stable and bigger than a single subunit. Actins diffuse in solution, bumping into each other, until two of them fit together in the orientation that allows them to bind. When a third actin bumps into them and fits correctly to form a trimer, then more rapid assembly of additional subunits can occur. (2) Elongation. Subunits can add rapidly to the trimer nucleus, at both ends of the growing filament. There are different methods used to follow assembly quantitatively, but they all give results like those below. (3) Steady state. Eventually the subunit “on” and “off” rates become equal, and there is no net assembly. Do not confuse this with a lack of assembly and disassembly! The filaments are dynamic, but not undergoing net growth. -1-
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(B) Measuring filament growth (1) There is more than one useful way to measure and analyze the growth of filaments. The graph above looked at % assembly versus time. A common way to analyze an assembly experiment is to plot the rate of elongation versus the concentration of free actin subunits. At high [actin], the rate of assembly is high. But as you lower the [free actin], the rate drops until, at some [actin], it is zero. This [actin], below which there is net disassembly, and above which there is net assembly, is called the critical C concentration for assembly, or C . (See the figure below, at left.) (2) Note that this description – start with high [free actin], get rapid assembly, and then watch the assembly rate slow to zero as the [free actin] goes down – is exactly what happens when you put some actin in a tube and let it assembly to steady-state (see first
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This note was uploaded on 12/18/2009 for the course BIOL 101 taught by Professor Wormer during the Fall '08 term at Purdue.

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lecture 30 - LECTURE 30 06 November 2009 (P. J. Hollenbeck)...

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