Positioning and capture of cell surface-associated microtubules in epithelial tendon cells that differentiate in primary embryonic Drosophila cell cultures

Using primary embryonic Drosophila cell cultures, we have investigated the assembly of transcellular microtubule bundles in epidermal tendon cells. Muscles attach to the tendon cells of previously undescribed epidermal balls that form shortly after culture initiation. Basal capture of microtubule ends in cultured tendon cells is confined to discrete sites that occupy a relatively small proportion of the basal cell surface. These capturing sites are associated with hemiadherens junctions that link the ends of muscle cells to tendon cell bases. In vivo, muscle attachment and microtubule capture occur across the entire cell base. The cultured tendon cells reveal that the basal ends of their microtubules can be precisely targeted to small, pre-existing, structurally well-defined cortical capturing sites. However, a search and capture targeting procedure, such as that undertaken by kinetochore microtubules, cannot fully account for the precision of microtubule capture and positioning in tendon cells. We propose that cross-linkage of microtubules is also required to zip them into apicobasally oriented alignment, progressing from captured basal plus ends to apical minus ends. This involves repositioning of apical minus ends before they become anchored to an apical set of hemiadherens junctions. The proposal is consistent with our finding that hemiadherens junctions assemble at tendon cell bases before they do so at cell apices in both cultures and embryos. It is argued that control of microtubule positioning in the challenging spatial situations found in vitro involves the same procedures as those that operate in vivo.     

Desmoplakin: an unexpected regulator of microtubule organization in the epidermis

Despite their importance in cell shape and polarity generation, the organization of microtubules in differentiated cells and tissues remains relatively unexplored in mammals. We generated transgenic mice in which the epidermis expresses a fluorescently labeled microtubule-binding protein and show that in epidermis and in cultured keratinocytes, microtubules stereotypically reorganize as they differentiate. In basal cells, microtubules form a cytoplasmic network emanating from an apical centrosome. In suprabasal cells, microtubules concentrate at cell-cell junctions. The centrosome retains its ability to nucleate microtubules in differentiated cells, but no longer anchors them. During epidermal differentiation, ninein, which is a centrosomal protein required for microtubule anchoring (Dammermann, A., and A. Merdes. 2002. J. Cell Biol. 159:255-266; Delgehyr, N., J. Sillibourne, and M. Bornens. 2005. J. Cell Sci. 118:1565-1575; Mogensen, M.M., A. Malik, M. Piel, V. Bouckson-Castaing, and M. Bornens. 2000. J. Cell Sci. 113:3013-3023), is lost from the centrosome and is recruited to desmosomes by desmoplakin (DP). Loss of DP prevents accumulation of cortical microtubules in vivo and in vitro. Our work uncovers a differentiation-specific rearrangement of the microtubule cytoskeleton in epidermis, and defines an essential role for DP in the process.     

The unusual microtubule polarity in teleost retinal pigment epithelial cells

In cells of the teleost retinal pigment epithelium (RPE), melanin granules disperse into the RPE cell's long apical projections in response to light onset, and aggregate toward the base of the RPE cell in response to dark onset. The RPE cells possess numerous microtubules, which in the apical projections are aligned longitudinally. Nocodazole studies have shown that pigment granule aggregation is microtubule-dependent (Troutt, L. L., and B. Burnside, 1988b Exp. Eye Res. In press.). To investigate further the mechanism of microtubule participation in RPE pigment granule aggregation, we have used the tubulin hook method to assess the polarity of microtubules in the apical projections of teleost RPE cells. We report here that virtually all microtubules in the RPE apical projections are uniformly oriented with plus ends toward the cell body and minus ends toward the projection tips. This orientation is opposite that found for microtubules of dermal melanophores, neurons, and most other cell types.     

Changes in microtubule polarity orientation during the development of hippocampal neurons in culture

Microtubules in the dendrites of cultured hippocampal neurons are of nonuniform polarity orientation. About half of the microtubules have their plus ends oriented distal to the cell body, and the other half have their minus ends distal; in contrast, microtubules in the axon are of uniform polarity orientation, all having their plus ends distal (Baas, P.W., J.S. Deitch, M. M. Black, and G. A. Banker. 1988. Proc. Natl. Acad. Sci. USA. 85:8335-8339). Here we describe the developmental changes that give rise to the distinct microtubule patterns of axons and dendrites. Cultured hippocampal neurons initially extend several short processes, any one of which can apparently become the axon (Dotti, C. G., and G. A. Banker. 1987. Nature [Lond.]. 330:477-479). A few days after the axon has begun its rapid growth, the remaining processes differentiate into dendrites (Dotti, C. G., C. A. Sullivan, and G. A. Banker. 1988. J. Neurosci. 8:1454-1468). The polarity orientation of the microtubules in all of the initial processes is uniform, with plus ends distal to the cell body, even through most of these processes will become dendrites. This uniform microtubule polarity orientation is maintained in the axon at all stages of its growth. The polarity orientation of the microtubules in the other processes remains uniform until they begin to grow and acquire the morphological characteristics of dendrites. It is during this period that microtubules with minus ends distal to the cell body first appear in these processes. The proportion of minus end-distal microtubules gradually increases until, by 7 d in culture, about equal numbers of dendritic microtubules are oriented in each direction. Thus, the establishment of regional differences in microtubule polarity orientation occurs after the initial polarization of the neuron and is temporally correlated with the differentiation of the dendrites.     

XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover

We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs.     

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.     

Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells

MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E.H.K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M.H. Bre, G. Griffiths, and E. Karsenti. 1990. 110:1123-1136). In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced double-immunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4 microns/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a t1/2 of less than 30 min, whereas in confluent monolayers, a large population of microtubules had a t1/2 of greater than 2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.     

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.     

Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy

We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell.     

Posttranslational modification of distinct microtubule subpopulations during cell polarization and differentiation in the mouse preimplantation embryo

During the course of preimplantation development, the cells of the mouse embryo undergo both a major subcellular reorganization (at the time of compaction) and, subsequently, a process of differentiation as the phenotypes of trophectoderm and inner cell mass cell types diverge. We have used antibodies specific for tyrosinated (Kilmartin, J. V., B. Wright, and C. Milstein. 1982. J. Cell Biol. 93:576-582) and acetylated (Piperno, G., and M. T. Fuller. 1985. J. Cell Biol. 101:2085-2094) alpha-tubulin in immunofluorescence studies and found that subsets of microtubules can be distinguished within and between cells during the course of these events. Whereas all microtubules contained tyrosinated alpha-tubulin, acetylated alpha-tubulin was detected only in a subpopulation, located predominantly in the cell cortices. Striking differences developed between the distribution of the two populations during the course of development. Firstly, whereas the microtubule population as a whole tends to redistribute towards the apical domain of cells as they polarize during compaction (Houliston, E., S. J. Pickering, and B. Maro. 1987. J. Cell Biol. 104:1299-1308), the microtubules recognized by the antiacetylated alpha-tubulin antibody became enriched in the basal part of the cell cortex. After asymmetric division of polarized cells to generate two distinct cell types (termed inside and outside cells) we found that, despite the relative abundance of microtubules in outside cells, acetylated microtubules accumulated preferentially in inside cells. Treatment with nocodazole demonstrated that within each cell type acetylated microtubules were the more stable ones; however, the difference in composition of the microtubule network between cell types was not accompanied by a greater stability of the microtubule network in inside cells.     

Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Uniform apicobasal polarity of microtubules and apical location of gamma-tubulin in polarized intestinal epithelium in situ

Polarized differentiation of the intestinal epithelium has been previously shown to depend on an intact microtubular system that is essential for vectorial delivery of apical membrane proteins to the apical cell surfaces. Uniform alignment and polarization of microtubules have been suggested to provide the ultrastructural basis for vectorial transport between the Golgi apparatus and the apical cell surface. In the present study we applied the hook decoration technique to analyse the polarity of microtubules in the rat jejunal epithelium. By immunocytochemistry we studied the subcellular location of gamma-tubulin, an essential component of microtubule-organizing centers. Microtubules were found to be mainly aligned parallel to the apicobasal axis of the cells and to extend from the subterminal space underneath the apical terminal web down to the cellular basis. We found that 98% out of 1122 decorated microtubules displayed uniform apicobasal polarity with the growing ends (plus ends) pointing basally and the non-growing ends (minus ends) pointing towards the cellular apex. No differences were observed with respect to microtubular polarity between the apical, perinuclear and infranuclear cellular portions. Immunostaining specific for gamma-tubulin was restricted to the apical subterminal space underneath the rootlets of microvilli. These findings indicate that the apical subterminal space of enterocytes serves as the predominant if not exclusive microtubule-organizing compartment from which uniformly polarized microtubules grow out with their plus ends towards the cellular basis.     

Peripheral mechanisms take central stage in microtubule dynamics: Commentary on "Signaling function of α-catenin in microtubule regulation" by Shtutman et al.

Although the centrosome is traditionally viewed as cell’s principle microtubule organizing center (MTOC), regulation of microtubule dynamics at the cell cortex plays an equally important role in the formation of the steady-state microtubule network. Several recent studies, including one published in this issue, reveal that complex signaling mechanisms associated with adherence junctions influence both microtubule nucleation at the centrosome, and the stability of non-centrosomal microtubules.

In the mid 1980s Marc Kirschner and Timothy Mitchison proposed an elegant “search-and-capture” hypothesis that seemed to explain how cells manage to convert a simple radial array of microtubules produced by the centrosome into the complex and precisely regulated asymmetric network found in a typical polarized cell. The key to this mechanism was the selective stabilization of inherently dynamic microtubule plus ends at the certain parts of cell cortex.4 Subsequently, it was shown that microtubule plus ends can in fact be captured and stabilized at diverse cortical loci including focal adhesions and adherence junctions. These observations provided direct support to the search-and-capture hypothesis. However, in recent years it became clear that role of cell cortex in the regulation of microtubule dynamics goes beyond simple stabilization of the plus ends. For example, there is evidence that integrin β1 is involved in the regulation of microtubule nucleation at the centrosome.6 Further, in polarized epithelia, cell cortex serves as the dominant MTOC, effectively replacing the centrosome.5 Thus, cell-cortex mechanisms affect microtubule dynamics both at their plus- and minus ends. The challenge now is to identify molecular pathways underlying this regulation.

A study in this issue of Cell Cycle (Shtutman et al.) suggests that α-catenin, a major component of adherence junctions is responsible for promoting microtubule nucleation and/or stability in a centrosome-independent fashion. Shtutman and coworkers used centrosome-free cytoplasts. The number of microtubules in these cytoplasts is low in the absence of cell-cell contacts but increases to near-normal levels in confluent cultures3 or upon overexpression of cadherins1 suggesting that adherence junctions somehow regulate microtubule dynamics. Shtutman and coworkers now demonstrate a similar increase in microtubule density can be induced by overexpression of a membrane-targeted α catenin. This is an exciting finding because α-catenin is also directly involved in the regulation of actin dynamics2 and thus this molecule emerges as a central player in the global regulation of the cytoskeleton in response to extracellular interactions. Interestingly, expression of non-membrane-targeted α-catenin only mildly increased the density of microtubule network in centrosome-free cytoplasts suggesting that α-catenin needs to be engaged in an activation event at the cell cortex, perhaps within the adherence junction.

Although formation of cell-cell junctions clearly increases the density of microtubule network, microtubule nucleation appears to occur throughout the cytoplasm and not preferentially at adherence junctions in these cells.1 Thus, local interactions at adherence junctions ultimately result in the propagation of a certain factor(s) that influences global microtubule dynamics. The exact nature of this factor or even the general layout of the pathway that alters microtubule dynamics in response to cortical interactions remain unknown. However, the demonstration that α-catenin is one of the molecular players required for this pathway is an important towards the understanding the link between extracellular interactions and microtubule dynamics.

Further Reading

Chausovsky A, Bershadsky AD, Borisy GG. Cadherin-mediated regulation of microtubule dynamics. Nat Cell Biol 2000; 2:797- 804. Gates J, Peifer M. Can 1000 reviews be wrong? Actin, alpha-Catenin, and adherens junctions. Cell 2005; 123:769-72. Karsenti E, Kobayashi S, Mitchison T, Kirschner M. Role of the centrosome in organizing the interphase microtubule array: properties of cytoplasts containing or lacking centrosomes. J Cell Biol 1984; 98:1763-76. Kirschner M, Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell 1986; 45:329-42. Reilein A, Yamada S, Nelson WJ. Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells. J Cell Biol 2005; 171:845-55. Reverte CG, Benware A, Jones CW, LaFlamme SE. Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis. J Cell Biol 2006; 174:491-7.     

Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.     

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.     

Identification of a Microtubule-associated Motor Protein Essential for Dendritic Differentiation

The quintessential feature of the dendritic microtubule array is its nonuniform pattern of polarity orientation. During the development of the dendrite, a population of plus end–distal microtubules first appears, and these microtubules are subsequently joined by a population of oppositely oriented microtubules. Studies from our laboratory indicate that the latter microtubules are intercalated within the microtubule array by their specific transport from the cell body of the neuron during a critical stage in development (Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol. 130:93– 104). In addition, we have established that the mitotic motor protein termed CHO1/MKLP1 has the appropriate properties to transport microtubules in this manner (Sharp, D.J., R. Kuriyama, and P.W. Baas. 1996. J. Neurosci. 16:4370–4375). In the present study we have sought to determine whether CHO1/MKLP1 continues to be expressed in terminally postmitotic neurons and whether it is required for the establishment of the dendritic microtubule array. In situ hybridization analyses reveal that CHO1/MKLP1 is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Immunofluorescence analyses indicate that the motor is absent from axons but is enriched in developing dendrites, where it appears as discrete patches associated with the microtubule array. Treatment of the neurons with antisense oligonucleotides to CHO1/MKLP1 suppresses dendritic differentiation, presumably by inhibiting the establishment of their nonuniform microtubule polarity pattern. We conclude that CHO1/MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite.     

A novel role for aquaporin-5 in enhancing microtubule organization and stability

Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function.     

Polarity orientation of axonal microtubules

The polarity orientation of cellular microtubules is widely regarded to be important in understanding the control of microtubule assembly and microtubule-based motility in vivo. We have used a modification of the method of Heidemann and McIntosh (Nature (Lond.). 286:517-519) to determine the polarity orientation of axonal microtubules in postganglionic sympathetic fibers of the cat. In fibers from three cats we were able to visualize the polarity of 68% of the axonal microtubules; of these, 96% showed the same polarity orientation. Our interpretation is that the rapidly growing end of all axonal microtubules is distal to the cell body. We support Kirschner's hypothesis on microtubule organizing centers. (J. Cell Biol. 86:330- 334), although this interpretation raises questions about the continuity of axonal microtubules. Our results are inconsistent with a number of models for axonal transport based on force production on the surface of microtubules in which the direction of force is determined by the polarity of microtubules.     

Distinct cytoskeleton populations and extensive crosstalk control Ciona notochord tubulogenesis

Cell elongation is a fundamental process that allows cells and tissues to adopt new shapes and functions. During notochord tubulogenesis in the ascidian Ciona intestinalis, a dramatic elongation of individual cells takes place that lengthens the notochord and, consequently, the entire embryo. We find a novel dynamic actin- and non-muscle myosin II-containing constriction midway along the anteroposterior aspect of each notochord cell during this process. Both actin polymerization and myosin II activity are required for the constriction and cell elongation. Discontinuous localization of myosin II in the constriction indicates that the actomyosin network produces local contractions along the circumference. This reveals basal constriction by the actomyosin network as a novel mechanism for cell elongation. Following elongation, the notochord cells undergo a mesenchymal-epithelial transition and form two apical domains at opposite ends. Extracellular lumens then form at the apical surfaces. We show that cortical actin and Ciona ezrin/radixin/moesin (ERM) are essential for lumen formation and that a polarized network of microtubules, which contributes to lumen development, forms in an actin-dependent manner at the apical cortex. Later in notochord tubulogenesis, when notochord cells initiate a bi-directional crawling movement on the notochordal sheath, the microtubule network rotates 90° and becomes organized as parallel bundles extending towards the leading edges of tractive lamellipodia. This process is required for the correct organization of actin-based protrusions and subsequent lumen coalescence. In summary, we establish the contribution of the actomyosin and microtubule networks to notochord tubulogenesis and reveal extensive crosstalk and regulation between these two cytoskeleton components.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.