Resul ts Site of Initiation for Microtubule Reorganization in Severed Arms

Many different cell types possess microtubule patterns which appear to be polarized and oriented, in part, by cytoplasmic factors not directly associated with a centrosome. Recently, we demonstrated that cytoplasmic extensions ("arms") of teleost melanophores will reorganize their microtubule population outward from their centers after surgical isolation (McNiven, M. A., M. Wang, and K. R. Porter. 1984. Cell. 37:753-765). In the study reported here, we examine microtubule dynamics within the centrosomefree fragments and find that, after severing, microtubule reorganization is initiated at the proximal (cut) end of an arm and migrates distally with the aggregated pigment mass until it becomes permanently positioned at the middle of the arm. Computer-aided image analysis demonstrates that this middle position is located at the arm centroid, implicating the action of a cytoplasmic gel in this process. Morphological studies of arms devoid of pigment reveal that microtubules do not emanate from a single site or structure within the centroid area, but from a more generalized region. Taken together, these findings suggest that factors distributed throughout cytoplasm participate in microtubule assembly and organization. T HE mechanisms by which eukaryotic cells regulate the number, polarity, orientation, and spatial distribution of their microtubules are poorly understood. It is believed that specific central structures, such as centrosomes, spindle poles, or basal bodies provide an essential environment for the initiation of tubulin assembly and organized microtubule growth (for reviews see Raft, 1979; Tucker, 1979; Brown et al., 1982; Mclntosh, 1983). Although these nucleating structures undoubtedly play an important role, it has become apparent that more peripherally placed cytoplasmic constituents are also involved in both the assembly and disposition of microtubules. An example of this peripheral influence can be found in the scleroblasts located on the fibrillary plate of teleost scales. When stained with antibodies to tubulin and viewed in situ, the microtubules are seen to extend out radially from the cells' organizing centers, and then curve abruptly to become aligned with the collagenous fibers of the underlying scale. It is impressive that microtubules in adjacent cells are similar in number, all oriented in the same manner, and all share a nearly identical pattern (Byers et al., 1980). It seems highly unlikely that the centrosome alone can induce changes in the direction of microtubule growth from a distance of many micrometers without the aid of other organizing components situated in peripheral cytoplasm. In addition, there are cell types in which cytoplasmic microtubules are not associated with an obvious nucleating structure, as in the epidermal cells of the developing leg tarsomere of the blowfly. In these cells there are two different microtubule populations. The first exhibits conventional radial growth from a centrosomal complex of dense bodies, while the second consists of a cortical layer of microtubules that is independent of the centrosomal microtubule population (Tucker, 1979). Perhaps the most graphic demonstration of a cell's ability to organize microtubules independent of a centrosome can be found in elongating axons. Despite the fact that axoplasm may extend outward over great distances from the centrosomal complex of the perikaryon, microtubules are ordered in their arrangement and uniformly polarized. A substantial number of studies have examined the length, number, and continuity of axonal microtubules to determine whether individual microtubules can span the entire nerve fiber. The findings indicate that microtubules are not continuous structures but show numerous starts and stops over the length of the axon (Nadelhaft, 1974; Bray and Bunge, 1981; Sasaki et al., 1983). In fact, the peripheral axon may possess more microtubules than are found within the cell body itself (Zenker and Hohberg, 1973). These observations suggest that an axon is capable of initiating microtubule assembly at points along its length, quite independently of a single centrosome. An alternative view, which should not be discounted, is that microtubules first nucleate and grow from a centrosome within the cell body, later become fragmented, and then are translocated outward into the distal regions of the axon. This model is supported by the fact that tubulin is a major component of slow axonal transport (Hoffman and Lasek, 1975; Black and Lasek, 1980). Recently, we set out to test whether peripheral cytoplasm within cells can assemble uniformly polarized and wellordered microtubule bundles in the absence of a centrosome. For several reasons, the teleost melanophore is a favorable cell model for such a study. These neuronally derived cells exhibit rapid intracellular pigment movements in response to caffeine and epinephrine and, most importantly, they have a stellate morphology with many dendritelike branches or © The Rockefeller University Press, 0021-9525/88/05/1593/13 $2.00 The Journal of Cell Biology, Volume 106, May 1988 1593-1605 1593 on Jne 6, 2017 D ow nladed fom Published May 1, 1988