Cell Motility and the Cytoskeleton 35:85-93 (1996) Views and Reviews Making a Connection: The “Other” Microtubule End Michael P. Koonce Division of Molecular Medicine, Wadsworth Center, and Department of Biomedical Sciences, State University of New York, Albany, New York INTRODUCTION A recent congruence of structural, biochemical, and molecular genetic work has identified a likely candidate for the long sought centrosomal component that nucleates and anchors the minus ends of microtubules in many eukaryotic cells [Moritz et al., 1995; Zheng et al., 19951. This important work will no doubt lead to a better understanding of where the microtubule cytoskeleton comes from and its regulation throughout the cell cycle. However, determining the morphogenetic events behind establishing and maintaining the spatial patterns of microtubules in interphase and dividing cells are separate and equally important issues. There is no doubt that the dynamic nature of microtubules plays a major role in providing a constant supply of polymer from the centrosome. But, to what extent do interactions at the “other” microtubule end contribute to the overall pattern of this cytoskeletal component? The seminal paper by Kirschner and Mitchison that, in 1986, ushered in a new era of thinking about microtubules and the cytoskeleton elaborated the idea of “capping structures near the cell periphery” that bind and selectively stablize the microtubule plus ends. If appropriately regulated, this could lead to a morphogenetic differentiation of the microtubule network and overall cell shape. While the kinetochore region of condensed chromosomes has served as a paradigm for such structures, a number of molecules and events have now been identified that could participate in maintaining other arrays of microtubules. This review examines what is known about structural interactions at or near the plus ends of microtubules, and how these would contribute to the spatial organization of a cellular array. The primary focus here is on interphase microtubules [for recent reviews of morphogenetic events in mitotic spindle assembly, see McIntosh, 1994; Inoue and Salmon, 1995; Hyman and Karsenti, 19961. 0 1996 Wiley-Liss, Inc. The Spatial Microtubule Pattern is Actively Maintained In a generic eukaryotic cell, the interphase microtubule network primarily functions to organize the cytoplasmic membrane systems [Cole and LippincottSchwartz, 19951. It helps direct endo- and exocytotic trafficking of vesicles between the endoplasmic reticulum (ER), Golgi, plasma membrane, and lysosomes. It further organizes the Golgi membranes as well as the position of the endoplasmic reticulum and other membrane-bound organelles. The microtubule polarity (minus ends anchored at the centrosome, plus ends distal) provides direction for motor molecule activity, and the dynamic radial pattern provides access to all regions of the cell [e.g., Holy and Liebler, 19941. Most microtubules probably function as a support for organelle motility. Further, it is generally assumed that in a radial pattern, each microtubule acts independent of its neighbor. In specialized cell processes where structural support is required (e.g., axons, dendrites, and kinetochore fibers), the microtubules are bundled together for stability and rigidity, and their removal with nocodazole profoundly affects the processes structure. There are at least three components to the architecture of a given microtubule array. First is the association of the microtubule at its organizing center. A y-tubulincontaining ring complex has been found in Drosophilu and Xenopus that serves as a nucleating template to anchor the minus ends in the centrosome [Moritz et al., 1995; Zheng et al., 19951. Given that nearly all eukaryotic cells maintain some form of a microtubule-organizing center and that y-tubulin stains positive in many of Received May 22, 1996; accepted June 10, 1996 Address reprint requests to Dr. Michael Koonce, Wadsworth Center, Empire State Plaza, PO Box 509, Albany, NY 12201-0509. 86 Koonce these structures, it is tempting to speculate that these ring complexes will be conserved. This will no doubt be addressed experimentally within the next few years. Although some cell fragments can form aster-like arrays in the absence of a conventional centrosome, these are likely due to the aggregation of microtubule ends and associated components to form a short term, centrosomelike structure [McNiven and Porter, 1988; Maniotis and Schliwa, 19911. In vitro work suggests that this could be assembled in large part by a dynein-like motility [Verde et al., 19911. The second architecture component is the dynamic character of the polymer. This is in part due to the microtubule’s inherent structural instability and in part to modifications imposed by associated proteins and post-translational modifications. Dynamics do play an important role in vivo since forced polymerization and stabilization with tax01 results in disorganized arrays and bundles. These structures do not appear over time to resolve themselves into an aster. Third, in order for the microtubule system to be functional, there must also be distal interactions of the polymer with other cellular structures, be that a second or third microtubule in a bundle, or a vesicle, membrane, or other protein. As discussed below, these distal interactions are critical for determining and maintaining the overall form of the microtubule network. In many active cells, the centrally focused, radial microtubule array is maintained as movement or shape change occurs [Schliwa and Honer, 19931. In some cells, rearrangement of the microtubule network can precede cell movement, suggesting that there are active forces that sense and govern its spatial pattern. It is unlikely that microtubule dynamics alone could account for these rapid rearrangements. Instead, distal interactions of the polymer with organelles or the cell cortex are probably necessary to anchor or generate a force along the polymer to connect microtubules to the cytoplasm. As shape changes occur, these connections help maintain the radial pattern and cell polarity. Indeed, there are several examples in the literature which demonstrate that forces acting along the sides or from the ends of microtubules can be strong enough to move a microtubule array through the visoelastic cytoplasmic matrix [Lutz et al., 1988; Hyman, 1989; Palmer et al., 1992; Waters et al., 19931. Recent work from our laboratory [Koonce and Sams6, 19961 suggests that by overexpressing the head domain of a cytoplasmic dynein in Dictyostelium, the microtubule network appears uncoupled from its cytoplasmic anchorage. In this work, many interphase cells no longer have a radial array of microtubules. Instead the microtubules appear loosely bundled around the nucleus (Fig. 1). While initial microtubule regrowth from centrosomes following nocodazole treatment appears similar to wild-type cells, these radial patterns are not main- tained in cells expressing the truncated dynein. These data further argue that there are distal connections to the microtubules that are necessary to keep the pattern radial. Where Would a Distal Anchor Lie? Distal microtubule anchors could act in at least three sites to control overall pattern and dynamics: (1) at the very tip of the polymer, (2) anchored in the cell cortex and interacting with the side of the polymer at or near its end, or (3) existing as the sum of all protein and organelle interactions along the length of the polymer. Tip anchors. Two of the best characterized tip anchors are found at the ends of axonemal microtubules in cilia and flagella, and at the kinetochore in dividing cells [for reviews of kinetochore-microtubule interactions, see Murray and Mitchison, 1994; InouC and Salmon, 1995; Hyman and Karsenti, 19961. Capping structures that appear to plug the ends of the doublet and central pair microtubules of axonemes and link these structures to the plasma membrane have been observed in a variety of organisms (reviewed in Dentler, 1990). One of these structures has been characterized in some detail as a large multisubunit complex of 1,500-2,OOO kDa [Wang et al., 19931. A 97-kDa component of this complex shares antibody reactivity with a polypeptide(s) located at the kinetochores of some mitotic chromosomes, leading to the interesting suggestion that these two structures share some conserved function [Miller et al., 19901. Axonemal caps may function to regulate assembly or stability of the core microtubule structure, or they may play a role in coordinating the motility of cilia or flagella. A cytoplasmic equivalent complex that plugs individual microtubules and links them to the membrane has not (yet) been described. However, interphase extracts of Xenopus eggs contain ER-derived vesicles that favor connection to microtubule tips in vivo [WatermanStorer et al., 19951. If these vesicles were part of extensive membrane systems in situ then assembly/disassembly reactions of the microtubule polymer could be sufficient to generate a tip force or a transient cytoplasmic anchor [e.g., InouC and Salmon, 1995; Lombillo et al., 1995a,b]. Additionally, the cortical bud site in S. cerevisiue may be in part an analogous structure [Lew and Reed, 19951. During cell division, the bud site interacts with astral microtubules and plays an anchorage role in determining the spindle position [Palmer et al., 19921. Cell cortex anchor. There are several examples of specialized cortical areas that interact with microtubules in meaningful ways. The term cortex here is meant to describe the nebulous region between the plasma membrane and the organelle-filled portion of the cytoplasm. In many cells this region is filled with an actin-rich fil- Microtubule Connections 87 Fig. 1. Dictyostelium amoeba visualized with a tubulin antibody (A,C) and corresponding phase contrast (B,D). A,B show a wild type AX-2 Dictyostelium cell. Note the radial microtubule pattern typical of most AX-2 cells and of most eukaryotes. C,D show one example typical of transformed AX-2 cells that overexpress a 380-kDa head domain fragment of the dynein heavy chain. Here the microtubule array has lost its radial character and appears whorled around the nucleus. We favor the idea that this morphology results from microtubules having lost their distal cellular connections so that as cells move, the array becomes compressed around the nucleus. See text and Koonce and SamsB  for more details. Scale bar = 10 pm. arnent meshwork which may act as a physical barrier to microtubules. During oogenesis and early development, interactions of astral microtubules with the cell cortex are important for positioning and anchoring of some spindles [Strome, 1993; Ault and Rieder, 1994; Karsenti et al., 19961. In Xenopus, a massive parallel layer of microtubules transiently forms near the interface between the cortex and interior shortly after fertilization. This layer directs a cortical rotation necessary to establish the dorsaVventra1 axis of the embryo [Elinson and Rowning, 1988; see also Larabell et al., 19961. When helper T-cells encounter their appropriate target [Geiger et al., 19821 and in epithelial cells activated to move by wounding of a monolayer [Kupfer et al., 1982; Gotlieb et al., 1983; Euteneuer and Schliwa, 19921, the microtubule array appears stimulated by a cortical-related event to polarize the cell in the direction in which movement will occur. Recent work with T-cells has correlated a signaling cascade identified in yeast with the regulation of the cytoskeletal rearrangements involved with activation [Stowers et al., 19951. Many cortical interactions have not been directly demonstrated but can be well-inferred by subsequent events. For example, during neuronal cell differentiation, microtubule bundles fill and support the growing axons and dendrites. The initial recruitment events that bundle microtubules behind the nascent growth cone are not well understood but likely involve stabilization directed in part by a localized cortical activity. Conidia germination in some fungi (Aspergillus, Neurospora) produces an elongating tube (hypha) into which nuclei migrate and assume evenly spaced positions [Morris et 88 Koonce Fig. 2. Network extension in Reticulomyxa. Two examples of cell processes growing from the cell body. The video-processed phase contrast images show microtubule bundles extending to the tips of these reticulopodia (arrowheads). Despite a rapid rate of growth, microtubules are nearly always found in association with the ends of growing and branching tubes. Scale bar = 5 km. al., 19951. Microtubules are essential for this motility and bundles extend into a phase-dense matrix at the hypha end. Although the polarity of the microtubules between the last nuclear-associated MTOC and the hypha end is not known, the event that specifies the site of conidia outgrowth likely involves an initial and subsequently maintained microtubule-cortex interaction. In a similar but extreme case, colinear bundles of microtubules and microfilaments support network extensions from the cell body of the freshwater amoeba, Reticulomyxa [Koonce et al., 19861. In these structures, which can extend at rates in excess of 100 km/min, dense bundles of microtubules maintain connections at or near the growing tip. These often terminate in a phase-dense plaque (Fig. 2). The microtubules provide structural support and serve to transport growth components to the ends. To what extent this phase-dense plaque resembles the structures seen in hyphal tubes or analogous components in neurons remains to be determined. Nevertheless, these structures appear to coordinate assembly (and/ or sliding) and bundling of microtubules with process growth. Summation anchor. Integration of all the motordriven movements along the length of a microtubule would generate a large viscous drag. This alone would provide a substantial “sea anchor” for the polymer in the face of local cytoplasmic movements. If some of these motor-driven movements were connected to additional cytoplasmic elements, further tension could be placed on the polymer. Work by Hiramoto [ 19861 using a focused UV light to locally inactivate the microtubule poison colcemid resulted in a polarized growth of microtubules from the centrosome. The resulting partial aster moved in the direction of polymer growth, suggesting that a substantial pulling force exists on the microtubule system. In a complete microtubule array this force would be radially balanced. While no net translocation would occur, the whole system should be under tension. The site of force production here is not known, but either a kinesin-like activity at the MTOC or a dynein-like activity Microtubule Connections in the cytoplasm could produce the same effect. In another example, kinesin is believed to be at least partially responsible for extending the intermediate filament network in some cells [Gyoeva and Gelfand, 19911. At some point a balance must be reached between the extensile force of a kinesin molecule and the elastic force of the intermediate filament network. If the cortex anchors the microtubule ends, then the two cytoskeletal systems could be linked under tension into a tensegrity-like structure. A number of structural MAPShave been characterized that bundle microtubules (MAP-2, tau, etc.), others bind the polymer side and presumably regulate dynamics [reviewed in Mandelkow and Mandelkow, 19951. Still other MAPS are believed to connect microtubules to cytoplasmic components, either providing structural links or localize proteins, enzyme complexes, RNAs, etc. One type of cytoplasmic linker protein (CLIP- 170) has been characterized in some detail to couple microtubules and endosome vesicles. Interestingly, this binding seems to favor a subset of microtubules, and at peripheral positions near their plus ends [Rickard and Kreis, 19901. This activity may serve a prerequisite step for binding motor molecules or may serve as a cytoplasmic anchorage. A recent review describes several motifs in this protein that are shared with other microtubule-related polypeptides, suggesting they may constitute a family of related activities [Rickard and Kreis, 19961. Molecular Connections As the preceding sections illustrate, several mechanisms exist to “connect” the microtubules to the cytoplasm. In doing so, they either maintain polarity important for directional activities (polarized transport or secretion) or could be used to establish an asymmetry important for division or differentiation. The three categories are not mutually exclusive: complexes bound to the microtubule tip may also exert a force near the polymer end (as in the case for a kinetochore). In some instances, clear cortical connections between the plasma membrane and microtubule can be seen all along the polymer length (as in cilia and flagella). The structural linkages are also likely to be modulated by a regulatory system that can sense the microtubule requirements of a given cell activity. Several components and mutations have recently been described that appear important in maintaining the overall microtubule pattern and act at a site distal from the centrosome. These are summarized in Table I. Some of these may form part of structural complexes, others are motor protein components that may also provide a structural role. Two of the more general themes emerging from these data are that cytoplasmic dyneins play important roles in anchoring the peripheral ends of mi- 89 crotubules and that there is an intimate relationship between the microtubule and actin filament-based cytoskeletal systems. Cytoplasmic dynein-like activity has long been implicated to provide a traction force along the microtubule polymer. If anchored in the cortex, it has the proper polarity of movement to interact with and pull on the peripheral microtubule ends. If uniformly positioned throughout the cortex, transient activity would reinforce the radial interphase array. Alternatively, if dynein activity was coupled to cortex-generated signals, it could be manipulated to engage a subset of microtubules and to reorient the microtubule organizing center. Although dynein antibodies do not dramatically localize this protein to the cortex of many cells, small functional amounts at the cell periphery would be difficult to distinguish above the greater, soluble, cytoplasmic pool. Some areas of focused microtubule-cortex interaction do show dynein localization [Xiang et al., 1995b; Yeh et al., 19951, lending support for a general cortical anchor role. Mutations and manipulations of the dynein heavy chain gene and related components are also consistent with a corticalbased function. Dynein’s role here does not necessarily require processive force generation. It could provide an anchor function by acting as an ATP-sensitive coupler that in turn is pulled on by microtubule-based motor proteins located elsewhere on the polymer. Another intriguing aspect of microtubule organization lies in its relationship with the actin filament network. The actin filament machinery provides the basic locomotive force for cell movement and shape. While not necessary for movement, microtubules maintain the cytoplasmic organization necessary to direct growth components and provide structural support [Schliwa and Honer, 19931. Thus, microtubules are important for persistent, directed locomotion and differentiation. It seems reasonable to expect that the two filament systems share a common machinery that coordinates their interaction. It has been known for some time that physical connections exist between the two filament systems [e.g., Schliwa and van Blerkom, 19811. More recent demonstrations that individual vesicles can move on either substrate [Langford, 19951 and that perturbations of regulatory components can affect both microtubule and microfilament organizations [Chen et al., 1994; Ursic et al., 1994; Guo and Kemphues, 1995; Stowers et al., 19951 emphasize the biochemical connections between these two assemblies. Furthermore, these observations highlight motor-molecule activity as one avenue for crosstalk. Vesicles containing myosin, dynein, and kinesin activities on their surface potentially serve as dynamic force-producing crosslinkers to connect the two filament systems. In addition, disruption of the actinrelated component of dynactin, the molecular complex Koonce 90 TABLE I. Connections to the Distal Ends of Microtubules (MTs) ~~ STRUCTURAL Cytoplasmic dynein and related oroteins Dynein heavy chain (DHC) Minus end directed organelle motor. In yeast and other fungi, possible cortical-attached MT anchor for cytoplasmic and mitotic astral MTs [Eshel et al., 1993; Li et al., 1993; Plamann et al., 1994; Xiang et al., 19941. Essential in Drosophila [Gepner et al., 19961 and Dictyosteliurn [Koonce et al., 19941, and participates in bipolar spindle formation in some mammalian cells [Vaisberg et al. , 19931. Dynein light chain 8-kda subunit, nudG locus in Aspergillus [Beckwith and Moms, 19951. Mutations abolish dynein localization at growing hyphal tip and produce a phenotype similar to mutations in the heavy chain. Dynactin Multi subunit complex, potential receptor/anchor for cytoplasmic dynein [Schroer, 1994; Schroer et al., 1996; Vallee and Sheetz, 19961. p 150G'"ed Links dynactin to DHC via interaction with dynein intermediate chain [Vaughan and Vallee, 19951. Also has regulated MT-binding activity. Shares some domain homology with CLIP-170 [Rickard and Kreis, 19961. Actin-related protein Strikingly similar phenotype as dynein heavy chain mutations in yeast and filamentous fungi [Clark and Meyer, 1994; Muhua et al., 1994; Plamann et al., 1994; Schroer, 1994; Schroer et al., 19961. JNM- 1 44-kDa budding yeast protein [McMillan and Tatchell, 19941. Mutants show a nuclear migration phenotype very similar to DHC mutations. Potential anchor component for astral MTs in bud site. NUMlp Cortex-associated 3 13-kDa budding yeast protein believed to interact with astral MTs to align the nuclear spindle [Farkasovsky and Kuntzel, 19951. BIK 1 60-kDa budding yeast polypeptide that colocalizes with MTs [Berlin et al., 19901. Mutants show aberrant localization of spindle during mitosis and abnormal cytoplasmic MT morphologies. Shares some domain homology with CLIP-170 [Rickard and Kreis, 19961. CLIP- I70 Cytoplasmic-linker protein from humans [Rickard and Kreis, 19901; 170-kDa MT-binding protein that favors a subset of plus ends. It is believed to mediate linkages between endocytotic vesicles and MTs. May also facilitate docking of motor protein for vesicle movement. Shares domain homology with pl5OGIuedand BIKl, suggesting that there may be a conserved family of such linkers [Rickard and Kreis, 19961. MT-membrane attachments This is a vast field. However, one demonstration of note is the association of the RER with MTs in vitro, mediated by globular domains enriched with active MT motors [Allan and Vale, 19941. MT-capping proteins Axoneme complex Membrane-associated MT plugs. Large, multisubunit complex [Miller et al., 1990; Wang et al., 19931. TACs Membrane-MT tip attachement complexes in interphase-arrested Xenopus egg extracts [Waterman-Storer et al., 19951. Motor protein-mediated Some (but not all) MT-based motor proteins can couple latex microspheres to the ends of shrinking MTs [Lombillo et al., 1995a,b]. Though this was demonstrated with components assembled in vitro, analogous mechanisms may be employed in the TACs mentioned above, and in kinetochores during cell division. MDMl 5 1-kDa budding yeast intermediate filament-like protein [McConnell and Yaffe, 19921. Deletions affect mitochondria distribution and mitotic spindle position. Could act as a structural scaffold to orient microtubule-based motilities. Axonemal kinesin Kinesin-related protein forms a motile "raft" that appears to connect plasma membrane components to the sides of the axonemal MTs [Kozminski et al., 19951. REGULATORY CDC-42 Small ras-like GTPase involved in signal transduction [Stowers et al., 19951. Deletion affects both actin-membrane interactions and ability of cells to orient MT array. Homologs found in several organisms. PAR- 1 126-kDa C. elegans serinelthreonine protein kinase [Guo and Kemphues, 19951. Polarized cortical location in early embryos. Mutations affect p-granule partitioning and mitotic spindle orientation. Homologs found in several organisms. nudF 49-kDa Aspergillus polypeptide related to the @-subunitof heterotrimeric G-proteins [Xiang et al., 1995al. Deletion causes a phenotype indistinguishable from mutations in the dynein heavy chain. This component may help regulate dynein's role in MT-based nuclear migration. TCP-1lBin 2pIBin 3pIANC2 Related family of polypeptides that share homology with chaperonins [Chen et al., 1994; Ursic et al., 1994; Vinh et al., 19941. When mutated in yeast, they affect both MT and actin filament structure and associated processes. Microtubule Connections believed to anchor dynein on vesicles in the cytoplasm [Schroer, 1994; Vallee and Sheetz, 19961, can produce the same phenotype as dynein heavy chain mutations. Given its sequence relationship to actin, this component may provide a link between dynein and the microfilament system and thus provide the firmest association between the microtubules and the cortical meshwork of actin filaments [see also Schroer et al., 19961. Review and View Linking the “other” microtubule end to the cytoplasm is one mechanism by which a cell controls the spatial pattern of its microtubule array. Distal connections can act to modify microtubule dynamics and produce a viscous drag that contributes to the elastic nature of the cytoplasm. In addition, there are numerous examples that demonstrate an intimate association of the microtubules with the cell cortex. If these linkages were coupled with cortical regulatory machinery, a mechanism would be created that enables cells to transduce extracellular signals into intracellular architectural changes that specify cell polarity and function. The componentdevents outlined in Table I likely represent the initial players of complex interactions and regulation; genetic and biochemical analyses will no doubt uncover many more [e.g., Verdeetal., 1995; Brunoetal., 19961. It is interesting to compare these interactions with microtubule-kinetochore connections formed during division. The kinetochore is a small structure that assembles onto the primary constrictions of condensing chromosomes and functions, in part, to capture microtubule plus ends and produce force. Its activity is temporally regulated, probably by multiple mechanisms. The cortical surface of a cell could be viewed as a “diffuse kinetochore,” that is, a regulated machine that can engage the distal ends of microtubules and produce force. Unlike the chromosomal kinetochore, components could be continuously mantained over the entire cell cortex. If these components were normally turned “off,” they would not interfere with microtubule dynamics or vesicle trafficking. In addition, they may not normally be detected unless a cell is stimulated to move or differentiate. Regulation, either through a global activation, or local interactions dictated by intra- or extracellular cues, could activate cortex components to engage underlying microtubules and produce a force/anchorage. This exertion could stabilize the local polymers, and thus provide a structural anchor, and target a transport direction for exo/ endocytosis of membrane-bound components. Cytoplasmic dynein, the dynactin complex, and a ciliary capping protein localize to some mitotic kinetochores and may provide a cortical anchor in interphase cells [Miller et al., 1990; Vallee and Sheetz, 1996; Schroer et al., 19961. Thus, there is some evidence to suggest that a common 91 machinery can be shared between kinetochores and the cell cortex. As additional components are characterized, it may be interesting to see whether this comparison holds up. ACKNOWLEDGMENTS We thank Drs. Alexey Khodjakov, Jeff Ault, and Conly Rieder for stimulating discussions on this topic. The data for Figure 2 were obtained while M.P. Koonce was a graduate student in Dr. Manfred Schliwa’s laboratory. The work in our lab is supported in part by the NIH (GM51532) and in part by institutional support from the Wadsworth Center. NOTE ADDED IN PROOF Two additional microtubule-binding proteins have come to our attention: Gephyrin, a 93 kDa polypeptide appears to link glycine receptor function with microtubules located at synapses [Prior et al., Neuron 8: 11611170, 19921, and adenomatous polyposis coli (APC), a 3 10 kDa tumor supressor protein appears to bind a subset of microtubule ends and may link these to cadherin junctions involved with active cell migration in epithelial cells [Nathke et al., J. Cell Biol. 134:165-179, 19961. REFERENCES Allan, V.J., and Vale, R.D. (1994): Movement of membrane tubules along microtubules in vitro: Evidence for specialized sites of motor attachment. J . Cell Sci. 107:1885-1897. Ault, J.G., and Rieder, C.L. (1994): Centrosome and kinetochore movement during mitosis. Curr. Opin. Cell Biology. 6:41-49. Beckwith, S.M., and Moms, N.R. (1995): Cytoplasmic dynein is missing at the tip in nu&, a nuclear migration mutant in Aspergillus nidulans. Mol. Biol. Cell. 6s:5a. Berlin, V., Styles, C.A., and Fink, G.R. (1990): BIKl, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J . Cell Biol. 111:2573-2586. Bruno, K.S., Tinsley, J.H., Minke, P.F., and Plamann, M. (1996): Genetic interactions among cytoplasmic dynein, dynactin, and nuclear distribution mutants of Neurospora crassa. Proc. Natl. Acad. Sci. USA 93:4775-4780. Chen, X . , Sullivan, D.S., and Huffaker, T.C. (1994): Two yeast genes with similarity to TCP-1 are required for microtubule and actin function in vivo. Proc. Natl. Acad. Sci. USA 91:91119115. Cole, N . B . , and Lippincott-Schwartz, J. (1995): Organization of organelles and membrane traffic by microtubules. Curr. Opin. Cell Biol. 755-64. Clark, S.W., and Meyer, D.I. (1994): ACT3: A putative centractin homologue in S. cerevisiae is required for proper orientation of the mitotic spindle. J. Cell Biol. 127:129-138. Dentler, W.L. (1990): Linkages between microtubules and membranes in cilia and flagella. In: Bloodgood, R.A., (ed), “Cil- 92 Koonce iary and Flagellar Membranes.” New York Plenum Press, pp Koonce, M.P., Euteneuer, U., and Schliwa, M. (1986): Reticulomyxu, a new model system for the study of intracellular trans31-64. port. J. Cell Sci. Suppl. 5:145-159. Elinson, R.P., and Rowning, B. (1988): A transient array of parallel microtubules in frog eggs: Potential tracks for a cytoplasmic Koonce, M.P., Pope, T., and Knecht, D.A. (1994): Disruption of the cytoplasmic dynein heavy chain gene in Dictyostelium. Mol. rotation that specifies the dorso-ventral axis. Dev. Biol. 128: Biol. Cell 5s:287a. 185-197. Eshel, D., Urrestarazu, L.A., Vissers, S., Jauniaux, J.-C., van Vliet- Kupfer, A., Louvard, D.D., and Singer, S.J. (1982): Polarization of the Golgi apparatus and the microtubule-organizing center in Reedijk, J.C., Planta, R.J., and Gibbons, I.R. (1993): Cytocultured fibroblasts at the edge of an experimental wound. plasmic dynein is required for normal nuclear segregation in Proc. Natl. Acad. Sci. USA 79:2603-2607. yeast. Proc. Natl. Acad. Sci. USA 90:11172-11176. Euteneuer, U., and Schliwa, M. (1992): Mechanism of centrosome Langford, G.M. (1995): Actin- and microtubule-dependent organelle motors: Interrelationships between the two motility systems. positioning during wound response in BSC-1 cells. J. Cell Curr. Opin. Cell Biol. 7:82-88. Biol. 116:1157-1166. Farkasovsky, M., and Kuntzel, H. (1995): Yeast Numlp associates Larabell, C.A., Rowning, B .A., Wells, J., Wu, M., and Gerhart, J.C. (1996): Confocal microscopy analysis of living Xenopus eggs with the mother cell cortex during S/G2 phase and affects miand the mechanism of cortical rotation. Development 122: crotubular functions. J. Cell Biol. 131:1003-1014. 1281-1289. Geiger, B., Rosen, D., and Berke, G. (1982): Spatial relationships of microtubule-organizing centers and the contact area of cyto- Lew, D.J., and Reed, S.I. (1995): Cell cycle control of morphogenesis in budding yeast. Curr. Opin. Genet. Dev. 5:17-23. toxic T-lymphocytes and target cells. 1. Cell Biol. 95:137-143. Gepner, J., Li, M-G., Lundmann, S., Kortas, C., Boylan, K., Iya- Li, Y.-Y., Yeh, E., Hays, T., and Bloom, K. (1993): Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc. Natl. durai, S.J.P., McGrail, M., and Hays, T.S. (1996): CytoplasAcad. Sci. USA 90:10096-10100. mic dynein function is essential in Drosophilu melunogaster. Lombillo, V., Nislow, C., Yen, T., Gelfand, V.I., and McIntosh, Genetics. 142:865-878. J.R. (1995a): Antibodies to the kinesin motor domain and Gotlieb, A.I., Subrahmanyan, L., and Kalnins, V.I. (1983): MicroCENP-E inhibit depolymerization-dependent motion of chrotubule-organizing centers and cell migration: Effect of inhibimosomes in vitro. J. Cell Biol. 128:107-115. tion of migration and microtubule disruption in endothelial Lombillo, V.A., Stewart, R.J., and McIntosh, J.R. (1995b): Minuscells. J. Cell Biol. 96:1266-1272. end-directed motion of kinesin-coated microspheres driven by Guo, S., and Kemphues, K.J. (1995): par-1, a gene required for microtubule depolymerization. Nature 373: 161-164. establishing polarity in C. eleguns embryos, encodes a putative Lutz, D.A., Hamaguchi, Y., and Inoue, S. (1988): Micromanipulaser/thr kinase that is asymmetrically distributed. Cell 81:611620. tion studies of the asymmetric positioning of the maturation spindle in Chuetopterus sp. oocytes. I. Anchorage of the spinGyoeva, F.K., and Gelfand, V.I. (1991): Coalignment of vimentin dle to the cortex and migration of a displaced spindle. Cell intermediate filaments with microtubules depends on kinesin. Motil. Cytoskel. 11:83-96. Nature. 353:44-448. Hiramoto, Y., Hamaguchi, Y., Hamaguchi, M.S., and Nakano, Y. Mandelkow, E., and Mandelkow, E.-M. (1995): Microtubules and microtubule-associated proteins. Curr. Opin. Cell Biol. 7:72-81. (1986): Roles of microtubules in pronuclear migration and spindle elongation in sand dollar eggs. In: Ishikowa, H., Ha- Maniotis, A., and Schliwa, M. (1991): Microsurgical removal of centano, s.,and Sato, H. (eds.), “Cell Motility: Mechanism and trosomes blocks cell reproduction and centriole generation in BSC 1 cells. Cell 67:495-504. Regulation.” New York: Alan R. Liss, pp 349-356. Holy, T.E., and Leibler, S. (1994): Dynamic instability of microtu- McConnell, S.J., and Yaffe, M.P. (1992): Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures bules as an efficient way to search in space. Proc. Natl. Acad. Sci. USA 915682-5685. defined by the MDMl protein. J. Cell Biol. 385-395. Hyman, A.A. (1989): Centrosome movement in the early divisions of McIntosh, J.R. (1994): The role of microtubules in chromosome Cuenorhubditis eleguns: A cortical site determining cenmovement. In: Hyams, J.H., and Lloyd, C.W. (eds.), “Mitrosome position. J. Cell Biol. 109:1185-1193. crotubules.” New York: Wiley-Liss, pp. 413-434. Hyman, A.A., and Karsenti, E. (1996): Morphogenetic properties of McMillan, J.N., and Tatchell, K. (1994): The JNMl gene in the yeast microtubules and mitotic spindle assembly. Cell 84:401-410. Succharomyces cerevisiue is required for nuclear migration and spindle orientation during the mitotic cell cycle. J. Cell Biol. Inouk, S., and Salmon, E.D. (1995): Force generation by microtubule 125: 143-158. assemblyidisassembly in mitosis and related movements. Mol. McNiven, M.A., and Porter, K.R. (1988): Organization of microtuBiol. Cell 6:1619-1640. bules in centrosome free cytoplasm. J. Cell Biol. 106:1593Karsenti, E., Boleti, H., and Vernos, I. (1996): The role of microtu1605. bule dependent motors in centrosome movements and spindle Miller, J.M., Wang, W., Balczon, R., and Dentler, W.L. (1990): pole organization during mitosis. Sem. Cell Dev. Biol. 7:367378. Ciliary microtubule capping structures contain a mammalian kinetochore antigen. J. Cell Biol. 110:703-714. Kirschner, M.W., and Mitchison, T. (1986): Beyond self assembly: From microtubules to morphogenesis. Cell 45:329-342. Moritz, M., Braunfeld, M.B., Sedat, J.W., Alberts, B., and Agard, D.A. (1995): Microtubule nucleation by y-tubulin-containing Kozminski, K.G., Beech, P.L., and Rosenbaum, J.L. (1995): The Chlumydomonus kinesin-like protein FLAlO is involved in morings in the centrosome. Nature 378:638-640. tility associated with the flagellar membrane. J. Cell Biol. 131: Moms, N.R., Xiang, X.,and Beckwith, S.M. (1995): Nuclear mi1517-1 527. gration advances in fungi. Trends Cell Biol. 5:278-282. Koonce, M.P., and Sams6, M. (1996): Overexpression of cytoplasmic Muhua, L., Karpova, T.S., and Cooper, J.A. (1994): A yeast actindynein’s globular head causes a collapse of the interphase mirelated protein homologous to that found in vertebrate dynactin crotubule network in Dictyostelium. Mol. Biol. Cell. 7, 7:935complex is important for spindle orientation and nuclear mi948. gration. Cell 78:669-679. Microtubule Connections Murray, A.W., and Mitchison, T.J. (1994): Mitosis. Kinetochores pass the 1.Q. test. Curr. Biol. 4:38-41. Palmer, R.E., Sullivan, D.S., Huffaker, T., andKoshland, D. (1992): Role of astral microtubules and actin in spindle orientation and migration in the budding yeast Saccharomyces cerevisiae. J. Cell Biol. 119583-593. Plamann, M., Minke, P.F., Tinsley, J.H., and Bruno, K.S. (1994): Cytoplasmic dynein and actin-related protein Arpl are required for normal nuclear distribution in filamentous fungi. J. Cell Biol. 127:139-149. Rickard, J.E., and Kreis, T.E. (1990): Identification of a novel nucleotide-sensitive microtubule-bindingprotein in HeLa cells. J. Cell Biol. 110:1623-1633. Rickard, J.E., and Kreis, T.E. (1996): CLIPS for organelle-microtubule interactions. Trend. Cell Biol. 6:178-183. Schliwa, M., and Honer, B. (1993): Microtubules, centrosomes, and intermediate filaments in directed cell movement. Trend Cell Biol. 3:377-380. Schliwa, M., and van Blerkom, J. (1981): Structural interaction of cytoskeletal components. J. Cell Biol. 90:222-235. Schroer, T.A. (1994): New insights into the interaction of cytoplasmic dynein with the actin-related protein, ARP-1. J. Cell Biol. 127:1-4. Schroer, T.A., Bingham, J.B., and Gill, S.R. (1996): Actin-related protein 1 and cytoplasmic dynein-based motility-what's the connection? Trend Cell Biol. 6:212-215. Stowers, L., Yelon, D., Berg, L.J., and Chant, J. (1995): Regulation of the polarization of T cells toward antigen-presentingcells by ras-related GTPase CDC42. Proc. Natl. Acad. Sci. USA 9 2 5027-503 1. Strome, S. (1993): Determination of cleavage planes. Cell 72:3-6. Ursic, D., Sedbrook, J.C., Himmel, K.L., and Culbertson, M.R. (1994): The essential yeast Tcpl protein affects actin and microtubules. Mol. Biol. Cell. 5:1065-1080. Vallee, R.B., and Sheetz, M.P. (1996): Targeting of motor proteins. Science, 27 1:1539-1544. Vaisberg, E.A., Koonce, M.P., and McIntosh, J.R. (1993): Cytoplasmic dynein plays a role in mammalian mitotic spindle formation. J. Cell Biol. 1235349-858. Vaughan, K.T., and Vallee, R.B. (1995): Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150p'ued. J. Cell Biol. 131:1507-1516. 93 Verde, F., Berrez, J.M., Antony, C., and Karsenti, E. (1991): Taxolinduced microtubule asters in mitotic extracts of Xenopus eggs: Requirement for phosphorylated factors and cytoplasmic dynein. J. Cell Biol. 112:1177-1187. Verde, F., Mata, J., and Nurse, P. (1995): Fission yeast cell morphogenesis: Identification of new genes and analysis of their role during the cell cycle. J. Cell Biol. 131:1529-1538. Vinh, D.B-N., and Drubin, D.G. (1994): A yeast TCP-I-like protein is required for actin function in viva Proc. Natl. Acad. Sci. USA 91:9116-9120. Wang, W., Suprenant, K., and Dentler, W.L. (1993): Reversible association of a 97 kDa protein complex found at the tips of ciliary microtubules with in vitro assembled microtubules. J. Biol. Chem. 268:24796-24807. Waters, J.C., Cole, R.W., and Rieder, C.L. (1993): The force-producing mechanism for centrosome separation during spindte formation in vertebrates is intrinsic to each aster. J. Cell Biol. 1221361-372. Waterman-Storer, C.M., Gregory, J., Parsons, S.F., and Salmon, E.D. (I 995): Membrane/microtubuletip attachment complexes (TACS) allow the assembly dynamics of plus ends to push and pull membranes into tubulovesicular networks in interphase Xenopus egg extracts. J. Cell Biol. 130:1161-1169. Xiang, X., Beckwith, S.M., and Moms, N.R. (1994): Cytoplasmic dynein is involved in nuclear migration in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 91:2100-2104. Xiang, X., Osmani, A.H., Osmani, S.A., Xin, M., and Morris, N.R. (1995a): NudF, a nuclear migration gene in Aspergillus nidulam, is similar to the human LIS- 1 gene required for neuronal migration. Mol. Biol. Cell. 6297-310. Xiang, X., Roghi, C., and Moms, N.R. (1995b): Characterization and colocalization of the cytoplasmic dynein heavy chain in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 91:21002104. Yeh, E., Skibbens, R.V., Cheng, J.W., Salmon, E.D., and Bloom, K. (1995): Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae. J. Cell Biol. 130:6587-700. Zheng, Y., Wong, M.L., Alberts, B., and Mitchison, T. (1995): Nucleation of microtubule assembly by a y-tubulin-containing ring complex. Nature 378:578-583.