Role of the Centrosome in Organizing the Interphase Microtubule Array : Properties of Cytoplasts Containing or Lacking Centrosomes ERIC KARSENTI , SUMIRE KOBAYASHI

To study the role of the centrosome in microtubule organization in interphase cells, we developed a method for obtaining cytoplasts (cells lacking a nucleus) that did or did not contain centrosomes . After drug-induced microtubule depolymerization, cytoplasts with centrosomes made from sparsely plated cells reconstituted a microtubule array typical of normal cells . Under these conditions cytoplasts without centrosomes formed only a few scattered microtubules . This difference in degree of polymerization suggests that centrosomes affect not only the distribution but the amount of microtubules in cells . To our surprise, the extent of microtubules assembled increased with the cell density of the original culture . At confluent density, cytoplasts without centrosomes had many microtubules, equivalent to cytoplasts with centrosomes . The additional microtubules were arranged peripherally and differed from the centrosomal microtubules in their sensitivity to nocodazole . These and other results suggest that the centrosome stabilizes microtubules in the cell, perhaps by capping one end . Microtubules with greater sensitivity to nocodazole arise by virtue of change in the growth state of the cell and may represent free or uncapped polymers . These experiments suggest that the spatial arrangement of microtubules may change by shifting the total tubulin concentration or the critical concentration for assembly . Microtubules have many important functions in cells, including a role in cell motility, intracellular transport, determination of cell shape, and segregation of chromosomes in mitosis (10, 26) . For all of these functions microtubules must have the proper location and orientation in the cell . The mechanisms that determine the location and orientation of microtubules are not well understood. In the last several years, however, a better picture of the overall arrangement ofmicrotubules in cells has been developed by immunofluorescence microscopy using antibody to tubulin as well as by electron and light microscopy . These methods have demonstrated that microtubules originate near the nucleus and fan out toward the cell periphery in what has been termed the cytoplasmic microtubule complex (4, 11, 12, 28) . Since the location of the microtubules in the cell is to some extent determined by their sites of origin, considerable attention has been directed toward study ofthese sites. By electron microscopy the centrosome region of interphase animal cells seems to be the site where microtubule density is higher than in any other portion of the cell (25) . However, THE JOURNAL OF CELL BIOLOGY " VOLUME 98 MAY 1984 1763-1776 0 The Rockefeller University Press 0021-9525/84/05/1763/14 $1 .00 outside this region the overall arrangement ofmicrotubules is not readily apparent from thin sections. The visualization of microtubules is better achieved by immunofluorescence of whole cells . These images suggest that many of the microtubules of the cell originate near the nucleus, perhaps in the vicinity of the centrosome . However, the exact origin of the microtubules cannot be distinguished on the light microscope level (40) . Except in cells displaying very sparse microtubule arrays, such as neutrophils (30), it is difficult, even by electron microscopy of whole-mount preparations, to distinguish the points oforigin ofmost microtubules . This is due to problems of resolution, superposition, and tracking . Therefore, although it is possible to say that some microtubules originate at the centrosome and that most microtubules originate near the nucleus, it is not possible to say that all or even most microtubules arise from the centrosome . The strongest argument supporting the idea that microtubules are nucleated from sites associated with centrioles has come from regrowth experiments where the cytoplasmic microtubule complex is first depolymerized with drugs like 1763 on M arch 0, 2017 D ow nladed fom Published May 1, 1984