Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells¹
Evidence for Interaction with Microtubules

David A. Collings^*, Tetsuhiro Asada, Nina S. Allen, and Hiroh Shibaoka

Department of Biology, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560, Japan (D.A.C., T.A., H.S.); and Department of Botany, Box 7612, North Carolina State University, Raleigh, North Carolina 27695-7612 (D.A.C., N.S.A.)

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K⁺ and Ca²⁺ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.

Plant Physiol. (1998) 118: 917-928
Copyright Clearance Center:   0032-0889/98/118//12
© 1998 American Society of Plant Physiologists

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