Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

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Metaphase PtK~ ceils, lysed into polymerization-competent microtubule protein, maintain a spindle which will gain or lose birefringence depending on the concentration of disassembled tubulin subunits used in the lysis medium. Concentrations of tubulin subunits greater than the equilibrium monomer value promote a rate and extent of birefringence increase that is proportional to the subunit concentration. Increase in spindle birefringence can be correlated with an increase in tubule number, though the relationship is not strictly linear. Increase in spindle tubule number is due to an in vivo-like initiation of tubules at the mitotic centers, as well as tubulin addition onto pre-existing spindle fragments. Colcemid-treated prometaphase cells lysed into polymerization-competent tubulin develop large asters in the region of the centrioles and short tubules at kinetochores, making it unlikely that all microtubule formation in lysed cell preparations is dependent on tubulin addition to short tubule fragments. Asters can also form in colcemidtreated prometaphase cells lysed in tubulin that is incapable of spontaneous tubule initiation, suggesting that the centriolar region serves a tubule-initiator function in our lysed cell preparations. The ability of the centriole to initiate microtubule assembly is a time-dependent process--a ripening effect takes place between prophase and late prometaphase. Ripening is expressed by an increase in the number and length of tubules found associated with the centriolar region.Formation of the mitotic spindle is dependent on the assembly of microtubules (MT) from tubulin subunits. This assembly is generally controlled with respect to time in the cell cycle, location within the cell. and orientation of the microtubules. Most, if not all, of the proteins required for the formation of the mitotic spindle are present before the onset of mitosis (9,12,29,34,38). Weisenberg (43) has shown that in eggs of the surf clam Spisula tubulin concentration in unactivated eggs is comparable to that during metaphase in activated eggs. These observations suggest that tubule assembly for spindle formation is not initiated by an immediate increase in the concentration of tubulin; a change must occur in the cytoplasm that shifts the tubulin-assembly reaction to favor polymer formation. There are studies which strongly suggest that the formed spindle is in equilibrium with an unassembled state of its subunits (16,35,36,37). Many factors have been identified that affect this equilibrium, but little is known at present of the ways in which the cell regulates spindle formation.Understanding the mechanism(s) by which the cell shifts its equilibrium toward MT formation will not necessarily explain the spatial control of

Abstract. Metaphase and anaphase spindles in cultured newt and PtK~ cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation. THE UV microbeam offers a means by which structures containing microtubules (MTs) 1 can be experimentally manipulated by local disruption. This possibility exists because MTs are sensitive to irradiation of between 260-300 nM (20,52). The technique has been used mostly for studying spindle structure and function (for example, see references 2, 12, 18-23, 26, 27), particularly by Forer and his colleagues (for example, see references 4, 7-9, 40, 41); on occasion, it has been used to probe other MT-based motility systems (for example, see references 25, 28; see also Using our first UV microbeam apparatus and working with diatoms, our previous observations on spindle MT dynamics (26,27) were significantly different from those reported by Forer in crane fly spermatocytes (7,8; see Discussion). Specifically, Forer describes areas of reduced birefringence (ARBs) created by the irradiation, as moving polewards at metaphase and anaphase at about the rate of anaphase chromosome movement. This observation was widely interpreted as indicating the existence of a polewards flux of MT subunits in spindle fibers with the likelihood that the MTs were being assembled at the kinetochores during metaphase and disassembled at the poles. In our experiments on diatom central spindles, the two cut ends of MTs in the ARB behaved quite differently, with the polewards end of severed MTs disassembling polewards (increasing the size of...

Mitotic cells Iysed into solutions of polymerizable microtubule protein contain a spindle which is similar to the living spindle in two respects: it will lose and gain birefringence when cooled and warmed, and it will move anaphase chromosomes to the opposite ends of the cell. Early anaphase cells lysed into buffers containing high molecular weight polyethylene glycol and nucleotide triphosphates will continue chromosome motion and spindle elongation in the absence of exogenous spindle subunits. These results suggest that while spindle growth requires microtubule polymerization, anaphase motions do not.Considerable effort has gone into the study of cell-free preparations of the mitotic apparatus (MA). Much has been learned about the structure of the isolated MA (1-4), and something is known of its chemistry (5-12), but two interesting questions have remained unanswered: what is the character of the equilibrium between the spindle and its subunits, and what is the nature of the motors that move the chromosomes?Previous studies have isolated the MA by "stabilizing" it, i.e., making it reversibly less labile by lysing mitotic cells into buffers which are poor solvents for protein (9). In such buffers the MA will keep its birefringence and overall form for hours, but it will not shrink and regrow as a function of temperature the way the spindle does in situ (13). Since the work of Hoffman-Berling on chromosome motion in glycerol-extracted cells (14), no one has, to our knowledge, been able to study the physiology of anaphase-like processes in vitro.In this paper we describe an experimental system for studying spindle functions in vitro and present some of our early results. We have taken advantage of the recent discovery of techniques for the reassembly of isolated microtubule protein (tubulin) (15, 16) to design a procedure based upon an equilibrium between assembled and disassembled subunits. Independent investigations based upon the same experimental design have been initiated by two groups at Woods Hole, Massachusetts. Preliminary reports of their findings have recently appeared in abstract form (17, 18). We lyse mitotic Pt K1 cells with a nonionic detergent into buffers containing various concentrations of polymerizable tubulin, and we monitor the magnitude and longevity of spindle birefringence and spindle size with the light microscope. Our results show that the spindle is stable for more than 1 hr after lysis in solutions containing sufficient concentrations of tubulin. The Abbreviations: PIPES, Piperazine-N-N'-bis[2-ethane Sulfonic Acid]; EGTA, ethylene bis(oxyethylene-nitrilo) tetraacetic acid; GTP, guanosine triphosphate; GEP, GTP + EGTA + PIPES; ATP, adenosine triphosphate; Tx, Triton X-100; MA, mitotic apparatus. stabilized spindles will lose birefringence when cooled and regain at least some of it when rewarmed. Cells deprived of spindle birefringence by cold treatment and then lysed into tubulin solutions will regrow birefringence after lysis when the preparation is warmed to 370. Spindles pre...