Tubulin hooks as probes for microtubule polarity: an analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle.

The structural polarity of cellular microtubules can be visualized in situ by lysing cells in special buffers containing tubulin. Under these conditions, the tubulin polymerizes to form curved sheets which attach to the walls of the endogenous microtubules. When such decorated microtubules are cut in cross section and viewed in the electron microscope, they appear to bear hooks curving clockwise or counter-clockwise. The direction of hook curvature is defined by the orientation of the decorated microtubule and thus serves as a probe for microtubule polarity. In this paper we describe a way to analyze the relative frequencies of hooks of different curvatures so as to measure the fidelity of the relation between hook curvature and microtubule polarity. The assumptions of the method are tested and found to be valid to a reasonable accuracy. The correlation between hook curvature and microtubule orientation is shown to be at least 0.98 for the spindles of PtK cells and Haemanthus endosperm at all stages of division and at all places in the spindle. The correlation is shown to be valid for each hook that forms, so the polarity of those microtubules that bear multiple hooks is specified with even better certainty than 0.98. This property of hook decoration is used to reinvestigate the possibility that some of the microtubules of the kinetochore fiber might be oriented with their plus ends distal to the kinetochore (opposite to the direction previously shown to predominate). Close analysis fails to identify such oppositely oriented microtubules. The scoring of tubules bearing multiple hooks also shows that individual interzone fibers at anaphase are constructed from clusters of antiparallel microtubules. The method for estimating the correlation between hook decoration and microtubule polarity is shown to be applicable to many structures and circumstances, but we find that the hook decoration assay for microtubule polarity is not uniformly accurate. We suggest that future studies using hook decorations should employ the method of data analysis presented here to assess the accuracy of the results obtained.

Polarity of midbody and phragmoplast microtubules.

A newly discovered method (Heidemann and McIntosh, 1980, Nature [Lond.] 286:517) for displaying the molecular polarity of microtubules (MTs) has been slightly modified and applied to the midbodies of cultured mammalian cells and the phragmoplasts of Haemanthus endosperm. The method involves the decoration of preexisting MTs in lysed cells with curved ribbons of tubulin protofilaments; the direction of curvature of these C-shaped appendages as seen in cross section reflects the intrinsic polarity of the MTs. In travsverse sections of midbodies from HeLa and PtK cells, we find that essentially all the MTs in a given region of the structures have the same direction of hook curvature, and hence the same polarity. The midbody MTs that lie on one side of the spindle equator show the opposite polarity from those on the other side, indicating that the midbody is constructed from two families of antiparallel MTs. Midbody MTs are arranged with their fast-growing ends overlapping at the spindle equator, consistent with the hypothesis that the midbody is formed by the interdigitation of aster MTs. The polarities of the MTs from the phragmoplast of endosperm cells are the same as those found in the mammalian midbody. Our results eliminate one model for mitosis, but are consistent with others. The systematic and reproducible polarities observed favor the concept that MT polarity is an important factor in the formation and/or the function of these two mitotic structures.

Mechanism of centrosome positioning during the wound response in BSC-1 cells.

Locomoting cells are characterized by a pronounced external and internal anterior-posterior polarity. One of the events associated with cell polarization at the onset of locomotion is a shift of the centrosome, or MTOC, ahead of the nucleus. This position is believed to be of strategic importance for directional cell movement and cell polarity. We have used BSC-1 cells at the edge of an in vitro wound to clarify the causal relationship between MTOC position and the initiation of cell polarization. We find that pronounced cell polarization (the extension of a lamellipod) can take place in the absence of MTOC repositioning or microtubules. Conversely, MTOCs will reposition even after lamellar extension and cell polarization have occurred. Repositioning requires microtubules that extend to the cell periphery and is independent of selective detyrosination of microtubules extending towards the cell front. Significantly, MTOCs maintain, or at least attempt to maintain, a position at the cell's centroid. This is most clearly demonstrated in wounded monolayers of enucleated cells where the MTOC closely follows the centroid position. We suggest that the primary response to the would is the biased extension of a lamellipod, which can occur in the absence of microtubules and MTOC repositioning. Lamellipod extension leads to a shift of the cell's centroid towards the wound. The MTOC, in an attempt to maintain a position near the cell center, will follow. This will automatically put the MTOC ahead of the nucleus in the vast majority of cells. The nucleus as a reference for MTOC position may not be as meaningful as previously thought.

Structural polarity of kinetochore microtubules in PtK1 cells.

The polarity of kinetochore microtubules (MTs) has been studied in lysed PtK1 cells by polymerizing hook-shaped sheets of neurotubulin onto walls of preexisting cellular MTs in a fashion that reveals their structural polarity. Three different approaches are presented here: (a) we have screened the polarity of all MTs in a given spindle cross section taken from the region between the kinetochores and the poles, (b) we have determined the polarity of kinetochore MTs are more stable to cold-treated spindles; this approach takes advantage of the fact that kinetochore MTs are more stable to cold treatment than other spindle MTs; and (c) we have tracked bundles of kinetochore MTs from the vicinity of the pole to the outer layer of the kinetochore in cold-treated cells. In an anaphase cell, 90-95% of all MTs in an area between the kinetochores and the poles are of uniform polarity with their plus ends (i.e., fast growing ends) distal to the pole. In cold-treated cells, all bundles of kinetochore MTs show the same polarity; the plus ends of the MTs are located at the kinetochores. We therefore conclude that kinetochore MTs in both metaphase and anaphase cells have the same polarity as the aster MTs in each half-spindle. These results can be interpreted in two ways: (a) virtually all MTs are initiated at the spindle poles and some of the are "captured" by matured kinetochores using an as yet unknown mechanism to bind the plus ends of existing MTs; (b) the growth of kinetochore MTs is initiated at the kinetochore in such a way that the fast growing MT end is proximal to the kinetochore. Our data are inconsistent with previous kinetochore MT polarity determinations based on growth rate measurements in vitro. These studies used drug-treated cells from which chromosomes were isolated to serve as seeds for initiation of neurotubule polymerization. It is possible that under these conditions kinetochores will initiate MTs with a polarity opposite to the one described here.

Evidence for an involvement of actin in the positioning and motility of centrosomes.

Cultured human polymorphonuclear leukocytes exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) spread on the substratum and undergo centrosome splitting. The two centrioles may separate by a distance of several micrometers, each being surrounded by an aster of microtubules. Here we show that the centriole/aster complexes are in constant, rapid motion through the cytoplasm, carrying with them some of the cytoplasmic granules while pushing aside others, or deforming and displacing the nucleus. An analysis of this unique motility phenomenon was undertaken. We show that intact microtubules are required for TPA-induced centrosome splitting and aster motility, but not for cell spreading. More importantly, disruption of the actin network inhibits both centrosome splitting and cell spreading, and even reverses splitting (induces convergence and fusion of asters) in polymorphonuclear leukocytes pretreated with TPA alone. These observations indicate the existence of a dynamic relationship between microtubules and actin networks and provide evidence for a role of actin in determining the position of the centrosome by way of interaction with the microtubules radiating from it.

Polarity of spindle microtubules in Haemanthus endosperm.

Structural polarities of mitotic spindle microtubules in the plant Haemanthus katherinae have been studied by lysing endosperm cells in solutions of neurotubulin under conditions that will decorate cellular microtubules with curved sheets of tubulin protofilaments. Microtubule polarity was observed at several positions in each cell by cutting serial thin sections perpendicular to the spindle axis. The majority of the microtubules present in a metaphase or anaphase half-spindle are oriented with their fast-growing or "plus" ends distal to the polar area. Near the polar ends of the spindle and up to about halfway between the kinetichores and the poles, the number of microtubules with opposite polarity is low: 8-20% in metaphase and 2-15% in anaphase cells. Direct examination of 10 kinetochore fibers shows that the majority of these microtubules, too, are oriented with their plus ends distal to the poles, as had been previously shown in animal cells. Sections from the region near the spindle equator reveal an increased fraction of microtubules with opposite polarity. Graphs of polarity vs. position along the spindle axis display a smooth transition from microtubules of one orientation near the first pole, through a region containing equal numbers of the two orientations, to a zone near the second pole where the opposite polarity predominates. We conclude that the spindle of endosperm cells is constructed from two sets of microtubules with opposite polarity that interdigitate near the spindle equator. The length of the zone of interdigitation shortens from metaphase through telophase, consistent with a model that states that during anaphase spindle elongation in Haemanthus, the interdigitating sets of microtubules are moved apart. We found no major changes in the distribution of microtubule polarity in the spindle interzone from anaphase to telophase when cells are engaged in phragmoplast formation. Therefore, the initiation and organization of new microtubules, thought to take place during phragmoplast assembly, must occur without significant alteration of the microtubule polarity distribution.

Polarity of kinetochore microtubules in Chinese hamster ovary cells after recovery from a colcemid block.

The polarity of kinetochore microtubules was determined in a system for which kinetochore-initiated microtubule assembly has been demonstrated. Chinese hamster ovary cells were treated with 0.3 micrograms/ml colcemid for 8 h and then released from the block. Prior to recovery, microtubules were completely absent from the cells. The recovery was monitored using light and electron microscopy to establish that the cells progress through anaphase and that the kinetochore fibers are fully functional. Since early stages of recovery are characterized by short microtubule segments that terminate in the kinetochore fibrous corona rather than on the outer disk, microtubule polarity was determined at later stages of recovery when longer kinetochore bundles had formed, allowing us to establish unambiguously the spatial relationship between microtubules, kinetochores, and chromosomes. The cells were lysed in a detergent mixture containing bovine brain tubulin under conditions that allowed the formation of polarity-revealing hooks. 20 kinetochore bundles were assayed for microtubule polarity in either thick or thin serial sections. We found that 95% of the decorated kinetochore microtubules had the same polarity and that, according to the hook curvature, the plus ends of the microtubules were at the kinetochores. Hence, the polarity of kinetochore microtubules in Chinese hamster ovary cells recovering from a colcemid block is the same as in normal untreated cells. This result suggests that microtubule polarity is likely to be important for spindle function since kinetochore microtubules show the same polarity, regardless of the pattern of spindle formation.

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements.

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable.

Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.

Viscoelastic properties of vimentin compared with other filamentous biopolymer networks.

The cytoplasm of vertebrate cells contains three distinct filamentous biopolymers, the microtubules, microfilaments, and intermediate filaments. The basic structural elements of these three filaments are linear polymers of the proteins tubulin, actin, and vimentin or another related intermediate filament protein, respectively. The viscoelastic properties of cytoplasmic filaments are likely to be relevant to their biologic function, because their extreme length and rodlike structure dominate the rheologic behavior of cytoplasm, and changes in their structure may cause gel-sol transitions observed when cells are activated or begin to move. This paper describes parallel measurements of the viscoelasticity of tubulin, actin, and vimentin polymers. The rheologic differences among the three types of cytoplasmic polymers suggest possible specialized roles for the different classes of filaments in vivo. Actin forms networks of highest rigidity that fluidize at high strains, consistent with a role in cell motility in which stable protrusions can deform rapidly in response to controlled filament rupture. Vimentin networks, which have not previously been studied by rheologic methods, exhibit some unusual viscoelastic properties not shared by actin or tubulin. They are less rigid (have lower shear moduli) at low strain but harden at high strains and resist breakage, suggesting they maintain cell integrity. The differences between F-actin and vimentin are optimal for the formation of a composite material with a range of properties that cannot be achieved by either polymer alone. Microtubules are unlikely to contribute significantly to interphase cell rheology alone, but may help stabilize the other networks.

A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells.

Treatment of epithelial African green monkey kidney (BSC-1) cells with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid and reversible redistribution of actin and vinculin that is detectable after only 2 min of treatment. Within 20-40 min, stress fibers disappear, while at the same time large actin-containing ribbons resembling ruffles develop both at the cell periphery and in more central regions. Vinculin is associated with these actin ribbons or bands in a punctate or patchy staining pattern. Adhesion to the substratum is changed from predominantly focal contacts associated with stress fiber ends in untreated cells to broad zones of close contact after TPA treatment. High voltage electron microscopic observations disclose the ribbons to consist of highly cross-linked actin filament networks. Thus, association of vinculin with filament networks, rather than (the ends of) filament bundles, is demonstrated. The integrity of microtubules and vimentin filaments is not affected by TPA treatment, but their distribution is altered to conform with the highly distorted cell shape. The response to TPA is neither prevented nor modified by nocodazole-induced depolymerization or taxol-induced stabilization of microtubules. An intact intermediate filament network seems not required either since colcemid-induced collapse of vimentin filaments towards the nucleus does not affect the cell's response to TPA. Rapid redistribution of actin and vinculin also takes place in enucleated cells and in the presence of cycloheximide, but is prevented by dinitrophenol or oligomycin. TPA-induced cytoskeletal alterations are independent of fibronectin expression and not mimicked, modified, or prevented by calmodulin inhibitors or experimentally elevated levels of calcium and cyclic AMP. Thus the morphological response to TPA involves rapid redistribution of actin and vinculin independent of transcription and translation, fluctuations in the levels of calcium or cyclic AMP, or changes in the organization of microtubules, intermediate filaments, and fibronectin.

Centrosome-dependent exit of cytokinesis in animal cells.

As an organelle coupling nuclear and cytoplasmic divisions, the centrosome is essential to mitotic fidelity, and its inheritance could be critical to understanding cell transformation. Investigating the behavior of the centrosome in living mitotic cells, we documented a transient and remarkable postanaphase repositioning of this organelle, which apparently controls the release of central microtubules from the midbody and the completion of cell division. We also observed that the absence of the centrosome leads to defects in cytokinesis. Together with recent results in yeasts, our data point to a conserved centrosome-dependent pathway that integrates spatial controls into the decision of completing cell division, which requires the repositioning of the centrosome organelle.

Unusual centrosome cycle in Dictyostelium: correlation of dynamic behavior and structural changes.

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a gamma-tubulin-green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyostelium centrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G1/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of gamma-tubulin-green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.

Release of enzymes of intermediary metabolism from permeabilized cells: further evidence in support of a structural organization of the cytoplasmic matrix.

Cultured BSC-1 cells were exposed to the mild ionic detergent, Brij 58, and the time course of the release of three enzymes of intermediary metabolism (lactate dehydrogenase, aldolase, and creatine phosphokinase) was determined spectrophotometrically. Their release correlates well with the overall decrease in structural complexity of the cytoplasmic matrix. However, each of the three enzymes tested has its own characteristic time-dependent release profile, a result suggesting enzyme-specific variability in their association with the cytomatrix. Cells lysed for 5 min in Brij 58 and then transferred to detergent-free glycolysis medium were able to produce lactate from glucose, an observation consistent with the idea that all enzymes of the glycolytic pathway remained in the cytomatrix. These observations are consistent with a structural rather than viscous organization of the cytoplasm and suggest that cytoplasmic components other than cytoskeletal filaments are able to form parafilamentous, metastable complexes.

Isolation of nucleation-competent centrosomes from Dictyostelium discoideum.

The centrosome of Dictyostelium discoideum is a box-shaped, layered core structure surrounded by a corona which is made up of dense nodules embedded in amorphous material. It is also known as nucleus-associated body. Because of its tight association with the nucleus the centrosome has resisted so far all attempts for isolation in sufficient purity and quantity for biochemical analysis. Here we report on the large-scale isolation of D. discoideum centrosomes after treatment of nucleus-centrosome complexes with a buffer containing sodium pyrophosphate. Following heparin treatment and a filtration step, centrosomes were further purified by density gradient centrifugation. Immunofluorescence analysis of the isolated centrosomes revealed the presence of the D. discoideum 350-kDa antigen, a centrosomal marker protein, gamma-tubulin, and the D. discoideum homologues of pericentrin, Spc110p, and Cdc31p. The structural integrity of the isolated centrosomes was demonstrated by confocal laser microscopy and electron microscopy. Microtubule nucleation assays with purified pig brain tubulin showed that the isolation procedure did not only preserve the structure but also the functionality of the isolated centrosomes. D. discoideum centrosomes should now become an attractive new model system in addition to, and for comparison with, centriolar centrosomes and yeast spindle pole bodies.

Polarity of marginal-band microtubules in vertebrate erythrocytes.

Erythrocytes from three different vertebrate species (bullfrog, mudpuppy, and stinkpot) were isolated, and the polarity of the marginal-band microtubules was determined using a modification of the method described by Heidemann and McIntosh [12]. A considerable part of each marginal band was examined in serial sections to see whether any changes in the polarity pattern occurred along the length of the band. On the basis of analyses of over 45 marginal bands, the following observations were made: (i) different polarity patterns were present within a given species; (ii) the polarity pattern seen in one cross-section of the marginal band remained the same along its entire length; no evidence for a limited zone of overlap was obtained; (iii) centrioles were found in erythrocytes from all three species. The data on the polarity patterns and the presence of centrioles in erythrocytes are compatible with a model recently proposed for the organization of marginal-band microtubules in blood clams that invokes the activity of a microtubule organizing center [20]. We suggest that a modification of the process of marginal-band formation described in that model can account for our observations on the polarity of marginal-band microtubules.

Centrosomes, microtubules and cell migration.

Directed cell movement is an immensely complex process that depends on the co-operative interaction of numerous cellular components. Work over the past three decades has suggested that microtubules play an important role in the establishment and maintenance of the direction of cell migration. This chapter summarizes recent work from our laboratory designed to determine the roles of the microtubules and centrosome position relative to the direction of cell migration in a variety of cell types, and discusses these observations in the context of work from other laboratories. The results suggest that microtubules are required for stabilization of the direction of migration in many, but not all, cell types. For the centrosome to act as a stabilizer of cell migration requires that it is repositioned behind the leading edge. However, the process of repositioning does not precede the extension of a leading edge and the establishment of a new direction of cell migration. Rather, the centrosome follows the repositioning of the leading edge in response to other stimuli and, in doing so, stabilizes cell movement.