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shortening, and finally shrinks back to the centrosome (Komarova et al.,
2002b). Some microtubules detach from the centrosome (Abal et al., 2002;
Keating et al., 1997), which usually leads to their rapid shortening from the
released minus end (Komarova et al., 2002b). Enucleated cell fragments
(cytoplasts) lacking centrosomes provide a convenient model for the studies of
such non-centrosomal microtubules (Chausovsky et al., 2000; Rodionov et al.,
1999). These microtubules demonstrate 'treadmilling' dynamics, in which
rapid depolymerization at the free minus end is balanced by the growth at the
plus end (Rodionov et al., 1999; Rodionov and Borisy, 1997). The steady state
is possible if the concentration of the non-polymerized tubulin is suciently
high. Therefore the mass of the microtubule polymer in the centrosome-free
cytoplasts is substantially smaller than in centrosome-containing cytoplasts or
in intact cells (Rodionov et al., 1999) (see also Figure 5.6).
Examining effects of mDia1 on microtubule dynamics, we have shown that
in cells transfected with the active form of mDia1 microtubules grow more
slowly and their growth is less tenacious than in control cells (the average
value of the growth speed was twofold lower, and the variance about twofold
higher). However, the velocity of microtubule shortening during the
oscillation phase at the cell periphery was also strongly reduced, while the
frequencies of the transitions from growth to shrinking and vice versa were
not significantly changed. Thus, the amplitude of the oscillations of the
microtubule plus ends significantly decreased in cells expressing the active
mDia1. These dynamic changes correlated with an apparent increase of
overlap between microtubule ends and focal adhesions at the cell leading edge
(Figure 5.5). The effect of mDia1 on the minus end microtubule dynamics was
also dramatic. Microtubule polymer level in the centrosome-free cytoplasts
produced from the cells expressing constitutively active mDia1 did not
decrease, as in control cytoplasts, but remained almost as high, as in intact
cells (Figure 5.6). This suggests that active mDia1 protects microtubule minus-
ends from rapid disassembly in the absence of the centrosome. In this
connection, it is interesting that both transfected and endogenous mDia1 was
enriched at the centrosomes of intact cells (Figure 5.4). Effect of mDia1 on the
microtubule minus-end required cooperation with another Rho target,
ROCK, which is also localized to the centrosome (Chevrier et al., 2002).
What could the mechanism of mDia1-mediated microtubule regulation be,
and what role might it play in the coordination of microtubule and actin
functions in the processes of cell polarization and directional migration? The
answer to the first question depends on revealing possible partners of mDia1
among proteins that directly control microtubule dynamics. As mentioned
above, there are several groups of such proteins, including proteins that
sequester non-polymerized tubulin dimers, 'classical' MAPs that bind to the
microtubules along their entire length, and microtubule tip-binding proteins
(Heald and Nogales, 2002). The last group of proteins is perhaps most
