Poster: Cytoskeleton: Structure & Function

Abs # 1106: Ultrastructural and morphometric analysis of cytochalasin-induced rewidening of preprophase bands in high pressure frozen cells

Presenter:	Mineyuki, Yoshinobu , mineyuk@hiroshima-u.ac.jp
Authors	Mineyuki, Yoshinobu  (A)   Takashi, Murata  (B)   Ichirou, Karahara  (C)   Andrew, Staehelin  (D)
Affiliations:	(A): Graduate School of Science, Hiroshima University (B): Graduate School of Arts and Science, The University of Tokyo (C): Faculty of Science, Toyama University (D): Molecular Cellular and Developmental Biology, University of Colorado at Boulder

The preprohase band (PPB) of microtubules (MTs) marks the future site of cell division. It is assembled during G2, narrows during prophase, and disappears prior to prometaphase. The actin-disrupting drug cytochalasin D (CD) disrupts PPB narrowing and also rewidens mature PPBs. In this study we have investigated the mechanism of the PPB rewidening process by comparing the organization of the cortical cytoskeleton in control and CD treated, cryofixed onion and tobacco cells. In control onion epidermal cells visualized by dual-axis electron tomography (~7 nm 3-D resolution), the PPB zone contains only a few, long microfilaments, whereas outside the PPB more numerous, randomly-oriented microfilaments are seen. In CD-treated cells, the preferential loss of randomely-oriented filaments make this distinction less clear. Furthermore, the few remaining microfilaments in the broad PPB region become closely aligned with the MTs. Quantitative analysis of the images of aphidicolin-synchronized tobacco root tip cells demonstrated that in CD-treated cells the PPBs were twice as wide as in control cells. Within these widened PPBs, the number of MTs was unchanged, but the clusters of parallel MTs were more dispersed. No change in either the spacing between MTs inside clusters or between the MTs and the plasma membrane were observed. Together, these results suggest that the narrowing of the PPB during its assembly is driven by a loss of actin filaments within the PPB zone, and the assembly of more actin filaments outside the PPB. CD-caused disassembly of the actin filaments outside the PPB allows the PPB to rewiden. Supported by grants NIH-GM59787 to LAS, by JSPS grants 09044225 to YM and by Japan-U.S. Cooperative Program.