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quantitation and mechanistic evaluation of comet formation and movement.
Interestingly, transient expression of the activated form of the small GTPase
Arf6 also results in comet formation (Schafer et al., 2000).
While imaging cultured cells expressing Dyn2-GFP, we observed small,
comet-like structures that had bright Dyn2 puncta associated with their vesicle
'heads' and a more faintly stained 'tail'. In the case of macropinosomes, Dyn2
labelling was confined to the side of the macropinosome opposite the direction
of movement, the same as for actin and cortactin (Kaksonen et al., 2000; Orth
et al., 2002). These structures appeared identical to the actin comets induced
by PIP5KIa and Arf6. Based on these data, we sought to determine the role of
Dyn2 in actin comet formation and movement. Cells expressing PIP5KIa and
Dyn2-GFP revealed a prominent co-localization of Dyn2, cortactin and actin
in comet structures (Figure 12.1B) (Orth et al., 2002). Interestingly, comet
formation and motility were normal in wildtype Dyn2-GFP expressing cells
but significantly altered in cells expressing Dyn2 mutants. Cells expressing a
GTPase-deficient Dyn2 (Dyn2K44A-GFP) contained fewer comets, while
those that did form were shorter than normal, and progressed with a
considerably decreased velocity. In contrast, comets in cells expressing
Dyn2DPRD-GFP did not incorporate the mutant Dyn2 protein, suggesting
that the PRD has a role in Dyn2 recruitment to the comet. Furthermore, these
Dyn2DPRD comets were significantly longer and slower than those in control
cells. These novel findings were supported by a similar study from Lee and
DeCamilli, which showed that Dyn2 is a component of comet structures
induced by Listeria and PIP5KIa, that the PRD has a role in targeting Dyn2
to the comets and that Dyn2 regulates comet formation and velocity (Lee and
De Camilli, 2002). Consistent with these studies, recent in vitro data showed
that Dyn2 mediates actin polymerization through cortactin, and affects F-
actin organization extended from PIP 2 -containing liposomes, although how
Dyn2 exerts this effect was not defined (Schafer et al., 2002). Together, these
studies suggest that Dyn2 functions at the interface of membranes and the
actin cytoskeleton to regulate membrane tracking and F-actin polymeriza-
tion and organization.
Dynamin and cytokinesis
Cytokinesis involves coordinated interactions between the microtubule and
actin cytoskeletons and cellular membranes to form and constrict the cleavage
furrow, completing daughter cell separation (for reviews see Glotzer, 2001;
Straight and Field, 2000; Robinson and Spudich, 2000). More recently it has
also been noted that proteins involved in vesicle tra cking, such as members
of the Rab family of small GTPases, vesicle coat proteins and syntaxins, are
also present at the cleavage furrow and contribute to the completion of
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