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Nuclear positioning: the means is at the ends
54
Nuclear positioning: the means is at the ends N Ronald Morris
The nucleus, like other smaller organelles in the cell, is dynamic and can move about in the cytoplasm. In some cells, nuclear movements are concerned with mitosis or meiosis; in others, they are concerned with orienting nuclear divisions; and in still others, they deal with distributing nuclei through the cytoplasm. Recent interest in nuclear positioning has shown that nuclear movements are often mediated by the interactions of dynein and other proteins at the plus ends of astral microtubules with the cell cortex. How the microtubule minus ends interact with the nucleus also affects nuclear movements. Addresses Department of Pharmacology, UMDNJ ± Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:54±59 0955-0674/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(02)00004-2 Abbreviations APC adenomatous polyposis coli MT microtubule MTOC microtubule-organizing center RNAi RNA-mediated interference SPB spindle pole body
Microtubules and nuclear positioning
Nuclear movements have been studied in a wide variety of eukaryotic cells, tissues, and organisms, and include such apparently diverse phenomena as the entry of mitotic nuclei into the bud in Saccharomyces cerevisiae [1±7,8], meiotic nuclear oscillations in Schizosaccharomyces pombe [9,10], pronuclear migration, rotation, and nuclear migration concurrent with cell migration in Caenorhabditis elegans [11±13,14,15], and nuclear migrations in Drosophila melanogaster [16±18], ®lamentous fungi [19,20,21,22,23,24,25] (Figure 1) and plants [26]. In some cases, like the cyclical nuclear movements seen in paraventricular brain neurons during development, in bipolar epithelial cells, and in the ommatidia of the Drosophila eye [18], the meaning of these nuclear movements can only be guessed at. Much of what we know about the mechanisms of nuclear movement comes from genetic studies in model organisms, speci®cally budding and ®ssion yeasts, ®lamentous fungi, Drosophila and Caenorhabditis. In animals and fungi, dynein, dynactin and other proteins found at the plus ends of microtubules (MTs) play a special role in this interaction. In higher plants, which lack dynein and its associated proteins [27], Current Opinion in Cell Biology 2003, 15:54±59
nuclear positioning is mediated by the preprophase MT band and actin [26]. This review will deal primarily with nuclear positioning in fungi and animal cells.
Nuclear positioning in fungi
The analysis of nuclear positioning in fungi has been extensive, in part because these organisms have excellent genetic systems and in part because the rigid morphologies of their cell walls provide landmarks against which the position of the nucleus can be charted. Mutations that affect nuclear movements in these organisms have mainly been identi®ed by direct visual inspection. The yeasts S. cerevisiae and S. pombe [1±7,8,9,10] and two ®lamentous fungi, Aspergillus nidulans and Neurospora crassa [19,20,21,22,23], provide the bulk of what we know about nuclear migration in the fungi. Signi®cant observations have also recently been made in Podospora anserina, and Ashbya gossypii [24,25]. Mitosis in S. cerevisiae provides a rough guide to nuclear movements in these fungi. In S. cerevisiae and other fungi, the nuclear microtubuleorganizing center (MTOC) is an organelle known as the spindle pole body (SPB) embedded in the nuclear envelope (Figure 2a). Astral MTs generated from the SPB continuously scan the mother cell cortex and the bud neck, cortex and tip. By interacting with these cortical sites they orient the mitotic spindle toward the bud, move the mitotic nucleus into the bud neck, and pull the daughter nucleus into the bud. Four different MTdependent motors, dynein and the Kar3p, Kip2p and Kip3p kinesins, are required for different steps in this process. Mutations in these motors also affect MT dynamics. A microtubule capture apparatus, in which the key component is Kar9p, is assembled at the bud tip with the help of the formin bni1, bud6, myosin and actin [1±3]. Kar9p captures and stabilizes MTs via a MT plus-end-associated protein, Bim1p/Yeb1p, a mammalian EB1 homologue [4,5]. Following this capture and stabilization MT shrinkage pulls the mitotic nucleus to the bud neck and a subsequent sliding interaction between MT-bound dynein and the bud cortex pulls the daughter nucleus into the bud [6]. The sliding interaction is mediated by Num1p, a cortical protein that interacts with Pac11p, the yeast dynein intermediate chain [7,8]. This general pattern of dynamic interactions between astral MTs and the cell cortex is also seen during mating in S. cerevisiae [28]. Nuclei move toward the tips of the mating cells before cell tip fusion, and following tip fusion move toward each other to achieve karyogamy. These movements are conspicuously coupled to MT plus-end growth and shrinkage [28]. A similar process occurs during meiotic prophase in the ®ssion yeast S. pombe, in which www.current-opinion.com
Nuclear positioning: the means is at the ends Morris 55
Figure 1
One of the most striking nuclear migration phenotypes is caused by mutations that affect dynein, dynactin, NUDF and other nuclear distribution proteins in the fungus Aspergillus nidulans. In the left panel, nuclei are distributed along the length of a young wild-type mycelium. A nuclear distribution mutant is shown on the right. Although this spore has produced two germ tubes (only one is clearly visible in the picture) and the nuclei have divided, they have not migrated out from the original spore portion of the mycelium. Nuclei are stained with DAPI.
the nucleus oscillates between the cell ends pulled by anteriorly oriented astral MTs from the SPB, which grow and shrink to pull the nucleus ®rst in one direction then the other [9,10]. These oscillations require dynein. Nuclear positioning at the center of interphase S. pombe cells has similarly been attributed to MT growth and shrinkage, but in this case has been ascribed to MTs pushing against the ends of the cells to move the nucleus [29]. Although it is not completely certain to what extent the motor activities of dynein and kinesins versus their effects on MT plus-end dynamics are responsible for nuclear positioning, it is clear from the experimental data, and from the fact that tubulin mutations affecting MT dynamics also affect nuclear movement [30,31], that growth and shrinkage of MTs can move nuclei. In multinucleate ®lamentous fungi such as Aspergillus and Neurospora, the nuclei are distributed more or less evenly along the hyphae. As in yeasts, the nuclei appear to be pulled along via astral MTs acting on their SPBs. Mutations in subunits of dynein [19,20,21,25], dynactin [19,21], the num1p-related protein apsA [24], kinesin [22], and in three additional proteins required for dynein function, NUDF (Pac1p in S. cerevisiae), NUDE and NUDC [20,32], cause migration to be abnormal (Figure 1). Mutations in these proteins also alter MT stability and plus-end dynamics [20,22]. Interestingly, green ¯uorescent protein (GFP) fusions of these same proteins are found at the plus ends of astral MTs, where one might expect to ®nd them if they were in¯uencing plus-end dynamics [19,20,21,23]. How these proteins affect MT plus-end dynamics is not understood.
affect nuclear movements in animal cells [11,12,15±17]. As in the fungi, GFP fusions have revealed that dynein and a set of dynein-interactive proteins are carried on the plus ends of astral MTs. The ®rst such protein to be observed was the mammalian cytoplasmic linker protein CLIP-170 [33,34,35]. CLIP-170 targets dynein and dynactin to the MT ends and links dynein to MTs [33,34,35]. Subsequently, many other proteins have been observed at the plus ends of MTs in mammalian systems including dynein [33,34], dynactin [34,35,36], Lis1 (the mammalian homologue of NUDF) [33], CLIP-115 (a relative of CLIP-170) [37,38], and CLASP proteins (which interact with CLIP-170 and CLIP-115) [38]. The CLASP proteins are of particular interest because they stabilize MTs and are speci®cally and uniquely found only on MTs oriented toward the leading edge of migrating cells. The human colon tumor suppressor adenomatous polyposis coli (APC) [39,40] and an associated protein EB1 [41], are also found at MT tips. Dynein [42] and APC [43] are associated with the cell cortex as well as with astral MTs and may act as astral MT attachment sites. How dynein, a plus-end-directed motor, and these other proteins come to be associated with MT plus ends are still matters for speculation. One such speculation has been that a plus-end-directed motor Ð a kinesin Ð brings these proteins to the MT tip. The fact that APC interacts with KAP3, a KIF3A/B kinesin-associated protein, has provided some support for this idea [43]. Because a dominant-negative KAP3 mutant interferes with APC plus-end localization, it has been suggested that the plus-end-directed KIP3A/B kinesin motor might target APC to MT ends.
Nuclear positioning in animal cells
Cortical capture proteins
Nuclear positioning in higher eukaryotes has some similarities to the fungal systems and some conspicuous differences. Mutations in dynein and several of the same proteins that affect nuclear migration in the fungi also www.current-opinion.com
By analogy with similar processes in yeast, it has been supposed that cortical APC, although not a Kar9p homologue, captures EB1 [44] and that cortical dynein interacts with MTs [42]. IQGAP1, a protein associated with Current Opinion in Cell Biology 2003, 15:54±59
56 Cell structure and dynamics
Figure 2
(a)
(b)
Current Opinion in Cell Biology
This highly schematized figure illustrates a significant difference in the organization of the nuclear MTOC and astral MTs between fungi and animal cells. (a) In fungi, the minus ends of astral MTs are rigidly anchored in the spindle pole body (blue), an organelle that is embedded in the nuclear envelope. The plus ends of the MTs, which are oriented toward the cell cortex, are dynamic, and proteins (green) concentrated at the plus ends interact with receptors (red) on the cell cortex. In S. cerevisiae, two such interactions have been described, MT Yeb1p with cortical Kar9p and MT dynein with cortical Num1p. (b) In animal cells, the minus ends of the astral MTs originate from a centrosomal MTOC. The plus ends of these astral MTs near the cell cortex are dynamic. Proteins (green) at the plus ends interact with receptors (red) on the cell cortex. These interactions, described in the text, are less well defined than in yeast. MTs from this MTOC may also interact with nuclear receptors, to couple the centrosome to the nucleus and position the centrosome (see [11]). Whether plus-end proteins are involved in this interaction, as hypothesized here, and, if so, whether they are the same plus-end proteins that interact with the cell cortex, remains to be determined.
cortical actin at the leading edge of migrating cells, has also been suggested as a cortical capture protein. IQGAP1 interacts with both CLIP-170 and with the small Rhofamily GTPases, Rac1 and Cdc42, which are important at the cortex for cell polarization and directed cell movement [45]. PLAC-24, a protein discovered by virtue of its interaction with the dynein intermediate chain, has also been localized to actin rich MT-interacting sites of cell±cell contact in epithelial cells [46]. It has been suggested that PLAC-24 is part of an evolutionarily conserved cortical MT binding complex. Current Opinion in Cell Biology 2003, 15:54±59
The centrosomal microtubule-organizing center
In animals, unlike fungi, the centrosomal MTOC at the minus ends of the astral MTs is not passively embedded in the nuclear envelope, but is a dynamic organelle attached to the nucleus by a dynein-dependant linkage [11] (Figure 2b). This is illustrated by the effect of RNAmediated interference (RNAi)-induced dynein de®ciency in C. elegans one-cell-stage embryos. In the presence of dynein heavy chain RNAi the centrosomes of the male pronucleus fail to separate to opposite sides of the www.current-opinion.com
Nuclear positioning: the means is at the ends Morris 57
nucleus, the pronuclei don't migrate, mitotic spindle orientation is abnormal, and most importantly the centrosomes detach from the pronucleus [11,15]. Similar defects in centrosomal positioning and nuclear attachment have been found in dynein de®cient Drosophila embryos [47]. The fact that dynein is found on the nuclear periphery in the C. elegans embryos suggests that MTOC positioning is caused by an interaction with dynein on the nuclear envelope [11]. In some cell types that migrate in tissue culture the centrosome re-orients toward the direction of cell locomotion [48,49±51,52]. For example, in a scratched astrocyte culture the cells moving into the cell-depleted gap produce a long anterior extension and the centrosomal MTOC, the Golgi apparatus and MTs all re-orient toward this extension [48]. In this system integrin-mediated recognition of lost cell±cell contacts recruit Cdc42 to the leading edge to activate a signal pathway that initiates an MTOC re-orientation [48]. The centrosome re-orientation requires dynein and dynactin [48,49]. However, in migrating PtK cells the centrosome lags behind the nucleus [50]. Thus the notion that the centrosome is positioned in migrating cells by astral MTs interacting with receptors at the leading edge of the cortex is probably an oversimpli®cation of a more varied and complex process. The data from PtK cells would seem to be equally compatible with an interaction between astral MTs and dynein associated with the nuclear envelope.
Nuclear migration and cell migration
Nuclear migration plays a role in some cell migrations. In C. elegans, for example, during embryonic development P cell nuclei migrate ventrally through an extended process. This is followed by retraction of the dorsal cytoplasm to leave the nucleus and cell body in a new position [13,14]. A similar process may occur during neuronal migration in humans. This possibility is based in part on the fact that lissencephaly, a disease in which neuronal migration to the cerebral cortex is retarded, is caused by haplode®ciency of Lis1, a homologue of the fungal nuclear migration gene NUDF, which is required for dynein function (see [20] for references). In migrating neonatal cerebellar neurons the centrosome re-orients in the direction of migration, and Lis1 co-localizes with dynein and NUDC around the centrosome and at neurite tips [52], suggesting that dynein and Lis1 mediate karyokinesis during cell migration. Nuclear migration in D. melanogaster oocytes also depends on Lis1 and dynein [16,17]. Co-localization and extensive physical interactions between Lis1, NUDC, NUDE, dynein, dynactin and CLIP-170, support the idea that Lis1 affects dynein function in animals [32,33,52,53±55,56]. A screen for C. elegans mutations that interfere with P cell and other nuclear migrations has produced two genes, UNC-83 and UNC-84, whose gene products interact with each other and bind to lamin in the nuclear envelope www.current-opinion.com
[13,14]. UNC-84 resembles the S. pombe sad1p SPB protein and a human inner nuclear membrane protein, suggesting that the role of UNC-84 in nuclear migration may be evolutionarily conserved [57]. Another dramatic nuclear migration that also involves a perinuclear protein occurs during photoreceptor cell development in the developing eye of D. melanogaster. Iterative up and down nuclear migrations involved in the morphogenesis of the photoreceptor fail to occur in the absence of a protein, Klarsicht [17], initially discovered as affecting lipid droplet movements in Drosophila embryos. Interestingly, there is a mammalian Klarsicht-related protein, Syne-1, which is speci®cally associated with nuclei clustered beneath the neuromuscular junction in muscle cells [58]. These nuclei migrate through a multinucleate myotube to accumulate at the neuromuscular junction, leading to the speculation that Syne-1 plays a role in this migration. Whether Klarsicht/Syne-1 is an integral nuclear membrane protein, and whether UNC-83/ UNC-84 interacts with MTs, anchors dynein to the nuclear envelope or acts by some other mechanism remains to be determined.
Conclusions
The systems that orient and move nuclei are moderately well conserved from fungi to animal cells. In both cases dynein, dynactin and other proteins on the plus ends of astral MTs appear to interact with proteins on the cell cortex to generate forces that act on the nuclear MTOC. Interactions between centrosomal MTs and the nuclear envelope may also position the centrosome in animal cells. Although the outlines of the mechanisms responsible for nuclear positioning are relatively well understood in S. cerevisiae, and partially understood in fungi, exactly how nuclei are moved and to what extent nuclear migration is coupled to cell migration is `early days' in animal systems. Much needs to be learned about the details of component interactions and the spatial and temporal regulation of nuclear migration in all systems.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Yin H, Pruyne D, Huffaker TC, Bretscher A: Myosin V orientates the mitotic spindle in yeast. Nature 2000, 406:1013-1015.
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Beach DL, Thibodeaux J, Maddox P, Yeh E, Bloom K: The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast. Curr Biol 2000, 10:1497-1506.
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Miller RK, Cheng SC, Rose MD: Bim1p/Yeb1p mediates the Kar9p-dependent cortical attachment of cytoplasmic microtubules. Mol Biol Cell 2000, 11:2949-2959.
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Korinek WS, Copeland MJ, Chaudhuri A, Chant J: Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science 2000, 287:2257-2259. Current Opinion in Cell Biology 2003, 15:54±59
58 Cell structure and dynamics
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Adames NR, Cooper JA: Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J Cell Biol 2000, 149:863-874.
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Heil-Chapdelaine RA, Oberle JR, Cooper JA: The cortical protein Num1p is essential for dynein-dependent interactions of microtubules with the cortex. J Cell Biol 2000, 151:1337-1344.
8.
Farkasovsky M, Kuntzel H: Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast. J Cell Biol 2001, 152:251-262. In addition to the interaction described in the title of this paper, the authors also show, as in Adames and Cooper (2000) [6] and Farkasovsky and Kuntzel (2000) [7], that the bud tip location of GFP±Num1p does not require Kar9p, Dyn1p or cytoplasmic MTs, but does require the formin Bni1p. 9.
Yamamoto A, Tsutsumi C, Kojima H, Oiwa K, Hiraoka Y: Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in ®ssion yeast. Mol Biol Cell 2001, 12:3933-3946. Nuclei of Schizosaccharomyces pombe undergo vigorous oscillations between the poles of the cell at meiotic prophase. GFP-tagged spindle pole body MTs lead these nuclear oscillations and interact distally with the cell cortex in wild-type cells, but not in dynein mutants. The authors propose a model in which nuclear oscillations are driven by cortical/ microtubule interactions and the dynamics of microtubule disassembly at the cortex. 10. Miki F, Okazaki K, Shimanuki M, Yamamoto A, Hiraoka Y, Niwa O: The 14-kDa dynein light chain-family protein Dlc1 is required for regular oscillatory nuclear movement and ef®cient recombination during meiotic prophase in Fission Yeast. Mol Biol Cell 2002, 13:930-946. Dlc1 belongs to the Tctex family of dynein light chains.
21. Zhang J, Han G, Xiang X: Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans. Mol Microbiol 2002, 44:381-392. The authors use mutations and GFP-labeled proteins to show that localization of the dynein intermediate and heavy chains to MT plus ends in A. nidulans requires both dynein subunits. They also demonstrate that heavy chain localization to MT plus ends requires NUDF, the A. nidulans homolog of mammalian Lis1. 22. Requena N, Alberti-Segui C, Winzenburg E, Horn C, Schliwa M, Philippsen P, Liese R, Fischer R: Genetic evidence for a microtubule-destabilizing effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution in Aspergillus nidulans. Mol Microbiol 2001, 42:121-132. 23. Lee IH, Kumar S, Plamann M: Null mutants of the Neurospora actin-related protein 1 pointed-end complex show distinct phenotypes. Mol Biol Cell 2001, 12:2195-2206. This paper demonstrates that the abnormal hyphal morphology of ropy mutants is not a necessary consequence of the nuclear migration defect seen in most of these mutants. Although mutations in the RO2 (p65), RO7 (Arp11), and RO12 (p25) subunits of the dynactin pointed end complex have abnormal curled hyphae, as do all the other Neurospora crassa ropy mutants, nuclear distribution is normal in RO12. 24. Graia F, Berteaux-Lecellier V, Zickler D, Picard M: ami1, an orthologue of the Aspergillus nidulans apsA gene, is involved in nuclear migration events throughout the life cycle of Podospora anserina. Genetics 2000, 155:633-646.
11. Gonczy P, Pichler S, Kirkham M, Hyman AA: Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J Cell Biol 1999, 147:135-150.
25. Alberti-Segui C, Dietrich F, Altmann-Johl R, Hoepfner D, Philippsen P: Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the ®lamentous ascomycete Ashbya gossypii. J Cell Sci 2001, 114:975-986. In contrast to other fungi in which dynein ablation causes nuclei to be clustered at the spore end of the hyphae, dynein ablation in Ashbyacauses nuclei to be clustered at the hyphal tips.
12. Yoder JH, Han M: Cytoplasmic dynein light intermediate chain is required for discrete aspects of mitosis in Caenorhabditis elegans. Mol Biol Cell 2001, 12:2921-2933.
26. Chytilova E, Macas J, Sliwinska E, Rafelski SM, Lambert GM, Galbraith DW: Nuclear dynamics in Arabidopsis thaliana. Mol Biol Cell 2000, 11:2733-2741.
13. Starr DA, Hermann GJ, Malone CJ, Fixsen W, Priess JR, Horvitz HR, Han M: unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration. Development 2001, 128:5039-5050. 14. Lee KK, Starr D, Cohen M, Liu J, Han M, Wilson KL, Gruenbaum Y: Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans. Mol Biol Cell 2002, 13:892-901. This interesting paper establishes a physical basis for the nuclear envelope localization of UNC-84. 15. Gonczy P, Echeverri C, Oegema K, Coulson A, Jones SJ, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E et al.: Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 2000, 408:331-336. 16. Swan A, Nguyen T, Suter B: Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning. Nat Cell Biol 1999, 1:444-449. 17. Lei Y, Warrior R: The Drosophila lissencephaly1 (DLis1) gene is required for nuclear migration. Dev Biol 2000, 226:57-72. 18. Mosley-Bishop KL, Li Q, Patterson L, Fischer JA: Molecular analysis of the Klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye. Curr Biol 1999, 9:1211-1220. 19. Xiang X, Han G, Winkelmann DA, Zuo W, Morris NR: Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1. Curr Biol 2000, 10:603-606. 20. Han G, Liu B, Zhang J, Zuo W, Morris NR, Xiang X: The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics. Curr Biol 2001, 11:719-724. In addition to reporting that Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics, this paper also contains useful references to earlier papers demonstrating that NUDF is required for dynein function. Current Opinion in Cell Biology 2003, 15:54±59
27. Lawrence CJ, Morris NR, Meagher RB, Dawe RK: Dyneins have run their course in plant lineage. Traf®c 2001, 2:362-363. 28. Maddox P, Chin E, Mallavarapu A, Yeh E, Salmon ED, Bloom K: Microtubule dynamics from mating through the ®rst zygotic division in the budding yeast Saccharomyces cerevisiae. J Cell Biol 1999, 144:977-987. 29. Tran PT, Marsh L, Doye V, Inoue S, Chang F: A mechanism for nuclear positioning in ®ssion yeast based on microtubule pushing. J Cell Biol 2001, 153:397-411. Using GFP±tubulin to visualize microtubules (MTs), the authors demonstrate that cytoplasmic MTs emanating from the nucleus dynamically sense the positions of the ends of the cell and maintain the nucleus in the center of the cell by pushing against the ends. 30. Caron JM, Vega LR, Fleming J, Bishop R, Solomon F: Single site alpha-tubulin mutation affects astral microtubules and nuclear positioning during anaphase in Saccharomyces cerevisiae: possible role for palmitoylation of alpha-tubulin. Mol Biol Cell 2001, 12:2672-2687. 31. Dougherty CA, Sage CR, Davis A, Farrell KW: Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis. Biochemistry 2001, 40:15725-15732. This tubulin mutation, like the one described in Tran et al. (2001) [29] affects nuclear positioning. The import of these two papers for dyneinmediated nuclear positioning is that because microtubule dynamics affect nuclear positioning, the effect of dynein on positioning could just as well be mediated via an effect of dynein on microtubule dynamics as by dynein motor activity per se. 32. E®mov VP, Morris NR: The Lis1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein. J Cell Biol 2000, 150:681-688. 33. Coquelle FM, Caspi M, Cordelieres FP, Dompierre JP, Dujardin DL, Koifman C, Martin P, Hoogenraad CC, Akhmanova A, Galjart N et al.: Lis1, CLIP-170's key to the dynein/dynactin pathway. Mol Cell Biol 2002, 22:3089-3102. www.current-opinion.com
Nuclear positioning: the means is at the ends Morris 59
This paper provides evidence that Lis1 acts as an adaptor between CLIP170 and cytoplasmic dynein by demonstrating that they colocalize and interact physically. 34. Vaughan KT, Tynan SH, Faulkner NE, Echeverri CJ, Vallee RB: Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends. J Cell Sci 1999, 112:1437-1447. 35. Valetti C, Wetzel DM, Schrader M, Hasbani MJ, Gill SR, Kreis TE, Schroer TA: Role of dynactin in endocytic traf®c: effects of dynamitin overexpression and colocalization with CLIP-170A. Mol Biol Cell 1999, 10:4107-4120. 36. Vaughan PS, Miura P, Henderson M, Byrne B, Vaughan KT: A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport. J Cell Biol 2002, 158:305-319. This exciting report shows that recruitment of dynactin P150glued to microtubule plus ends is modulated by phosphorylation. Of particular interest is a demonstration that interactions between microtubule plus ends and Golgi membranes precede centripetal, dynein-mediated membrane transport. 37. Hoogenraad CC, Akhmanova A, Grosveld F, De Zeeuw CI, Galjart N: Functional analysis of CLIP-115 and its binding to microtubules. J Cell Sci 2000, 113:2285-2297. 38. Akhmanova A, Hoogenraad CC, Drabek K, Stepanova T, Dortland B, Verkerk T, Vermeulen W, Burgering BM, De Zeeuw CI, Grosveld F, Galjart N: Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile ®broblasts. Cell 2001, 104:923-935. The CLASP proteins are found on distal segments of microtubules oriented toward the leading edge of migrating cells. The authors provide evidence that CLASPs regulate MT dynamics in response to positional clues. 39. Mogensen MM, Tucker JB, Mackie JB, Prescott AR, Nathke IS: The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells. J Cell Biol 2002, 157:1041-1048. It had previously been shown (see Mimori-Kiyosue et al. [2000] [41]) that EB1, a protein associated with the adenomatous polyposis coli (APC) protein, is found at the plus ends of microtubules. This paper rounds out the picture by showing that APC co-locates with EB1 at microtubule plus ends in polarized epithelial cells in which microtubule polarity has been de®nitively established by hook decoration. The authors show that in mice heterozygous for APC, there are fewer microtubule bundles, suggesting that the presence of APC stabilizes microtubules. 40. Zumbrunn J, Kinoshita K, Hyman AA, Nathke IS: Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation. Curr Biol 2001, 11:44-49.
45. Fukata M, Watanabe T, Noritake J, Nakagawa M, Yamaga M, Kuroda S, Matsuura Y, Iwamatsu A, Perez F, Kaibuchi K: Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell 2002, 109:873-885. This beautiful paper demonstrates that IQGAP1, an effector of the Rac1 and Cdc42 GTPases, interact with the microtubule plus-end protein CLIP-170 to target the plus ends of cytoplasmic microtubules to the polarized cell cortex. 46. Karki S, Ligon LA, DeSantis J, Tokito M, Holzbaur EL: PLAC-24 is a cytoplasmic dynein-binding protein that is recruited to sites of cell±cell contact. Mol Biol Cell 2002, 13:1722-1734. 47. Robinson JT, Wojcik EJ, Sanders MA, McGrail M, Hays TS: Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila. J Cell Biol 1999, 146:597-608. 48. Etienne-Manneville S, Hall A: Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell 2001, 106:489-498. Cells migrating into a wound re-orient their microtubule organizing center and microtubules by a dynein-requiring process initiated by the interaction of integrins with the extracellular matrix. 49. Palazzo AF, Cook TA, Alberts AS, Gundersen GG: mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat Cell Biol 2001, 3:723-729. 50. Yvon AM, Walker JW, Danowski B, Fagerstrom C, Khodjakov A, Wadsworth P: Centrosome reorientation in wound-edge cells is cell type speci®c. Mol Biol Cell 2002, 13:1871-1880. 51. Chevrier V, Piel M, Collomb N, Saoudi Y, Frank R, Paintrand M, Narumiya S, Bornens M, Job D: The Rho-associated protein kinase p160ROCK is required for centrosome positioning. J Cell Biol 2002, 157:807-817. 52. Aumais JP, Tunstead JR, McNeil RS, Schaar BT, McConnell SK, Lin SH, Clark GD, Yu-Lee LY: NUDC associates with Lis1 and the dynein motor at the leading pole of neurons. J Neurosci 2001, 21:RC187. NUDC co-localizes with Lis1 and the cytoplasmic dynein intermediate chain in a polarized manner at the microtubule organizing center in migrating cerebellar granule cells. It also co-immunoprecipitates with CDIC and cytoplasmic dynein. These data suggest that a functional interaction of NUDC with Lis1 and dynein underlies neuronal migration. 53. Kitagawa M, Umezu M, Aoki J, Koizumi H, Arai H, Inoue K: Direct association of Lis1, the lissencephaly gene product, with a mammalian homologue of a fungal nuclear distribution protein, rNUDE. FEBS Lett 2000, 479:57-62. 54. Tai CY, Dujardin DL, Faulkner NE, Vallee RB: Role of dynein, dynactin, and CLIP-170 interactions in Lis1 kinetochore function. J Cell Biol 2002, 156:959-968.
41. Mimori-Kiyosue Y, Shiina N, Tsukita S: The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr Biol 2000, 10:865-868.
55. Niethammer M, Smith DS, Ayala R, Peng J, Ko J, Lee MS, Morabito M, Tsai LH: NUDEL is a novel Cdk5 substrate that associates with Lis1 and cytoplasmic dynein. Neuron 2000, 28:697-711.
42. Dujardin D, Vallee RB: Dynein at the cortex. Curr Opin Cell Biol 2002, 14:44-49. This review summarizes the evidence for cortical dynein.
56. Hoffmann B, Zuo W, Liu A, Morris NR: The Lis1-related protein NUDF of Aspergillus nidulans and its interaction partner NUDE bind directly to speci®c subunits of dynein and dynactin and to alpha- and gamma-tubulin. J Biol Chem 2001, 276:38877-38884. Although this paper deals only with Aspergillus proteins, their interactions are similar to those described for Lis1 and mammalian NUDE.
43. Jimbo T, Kawasaki Y, Koyama R, Sato R, Takada S, Haraguchi K, Akiyama T: Identi®cation of a link between the tumour suppressor APC and the kinesin superfamily. Nat Cell Biol 2002, 4:323-327. By demonstrating a link between a microtubule plus-end directed motor protein and the adenomatous polyposis coli (APC) protein, the authors provide a possible explanation for the location of APC at the plus ends of cytoplasmic microtubules (see also Mogensen et al. [2002] [39]). 44. Rosin-Arbesfeld R, Ihrke G, Bienz M: Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells. EMBO J 2001, 20:5929-5939.
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57. Dreger M, Bengtsson L, Schoneberg T, Otto H, Hucho F: Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane. Proc Natl Acad Sci USA 2001, 98:11943-11948. 58. Apel ED, Lewis RM, Grady RM, Sanes JR: Syne-1, a dystrophin- and Klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction. J Biol Chem 2000, 275:31986-31995.
Current Opinion in Cell Biology 2003, 15:54±59
Nuclear positioning: the means is at the ends N Ronald Morris
The nucleus, like other smaller organelles in the cell, is dynamic and can move about in the cytoplasm. In some cells, nuclear movements are concerned with mitosis or meiosis; in others, they are concerned with orienting nuclear divisions; and in still others, they deal with distributing nuclei through the cytoplasm. Recent interest in nuclear positioning has shown that nuclear movements are often mediated by the interactions of dynein and other proteins at the plus ends of astral microtubules with the cell cortex. How the microtubule minus ends interact with the nucleus also affects nuclear movements. Addresses Department of Pharmacology, UMDNJ ± Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA e-mail: [email protected]
Current Opinion in Cell Biology 2003, 15:54±59 0955-0674/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(02)00004-2 Abbreviations APC adenomatous polyposis coli MT microtubule MTOC microtubule-organizing center RNAi RNA-mediated interference SPB spindle pole body
Microtubules and nuclear positioning
Nuclear movements have been studied in a wide variety of eukaryotic cells, tissues, and organisms, and include such apparently diverse phenomena as the entry of mitotic nuclei into the bud in Saccharomyces cerevisiae [1±7,8], meiotic nuclear oscillations in Schizosaccharomyces pombe [9,10], pronuclear migration, rotation, and nuclear migration concurrent with cell migration in Caenorhabditis elegans [11±13,14,15], and nuclear migrations in Drosophila melanogaster [16±18], ®lamentous fungi [19,20,21,22,23,24,25] (Figure 1) and plants [26]. In some cases, like the cyclical nuclear movements seen in paraventricular brain neurons during development, in bipolar epithelial cells, and in the ommatidia of the Drosophila eye [18], the meaning of these nuclear movements can only be guessed at. Much of what we know about the mechanisms of nuclear movement comes from genetic studies in model organisms, speci®cally budding and ®ssion yeasts, ®lamentous fungi, Drosophila and Caenorhabditis. In animals and fungi, dynein, dynactin and other proteins found at the plus ends of microtubules (MTs) play a special role in this interaction. In higher plants, which lack dynein and its associated proteins [27], Current Opinion in Cell Biology 2003, 15:54±59
nuclear positioning is mediated by the preprophase MT band and actin [26]. This review will deal primarily with nuclear positioning in fungi and animal cells.
Nuclear positioning in fungi
The analysis of nuclear positioning in fungi has been extensive, in part because these organisms have excellent genetic systems and in part because the rigid morphologies of their cell walls provide landmarks against which the position of the nucleus can be charted. Mutations that affect nuclear movements in these organisms have mainly been identi®ed by direct visual inspection. The yeasts S. cerevisiae and S. pombe [1±7,8,9,10] and two ®lamentous fungi, Aspergillus nidulans and Neurospora crassa [19,20,21,22,23], provide the bulk of what we know about nuclear migration in the fungi. Signi®cant observations have also recently been made in Podospora anserina, and Ashbya gossypii [24,25]. Mitosis in S. cerevisiae provides a rough guide to nuclear movements in these fungi. In S. cerevisiae and other fungi, the nuclear microtubuleorganizing center (MTOC) is an organelle known as the spindle pole body (SPB) embedded in the nuclear envelope (Figure 2a). Astral MTs generated from the SPB continuously scan the mother cell cortex and the bud neck, cortex and tip. By interacting with these cortical sites they orient the mitotic spindle toward the bud, move the mitotic nucleus into the bud neck, and pull the daughter nucleus into the bud. Four different MTdependent motors, dynein and the Kar3p, Kip2p and Kip3p kinesins, are required for different steps in this process. Mutations in these motors also affect MT dynamics. A microtubule capture apparatus, in which the key component is Kar9p, is assembled at the bud tip with the help of the formin bni1, bud6, myosin and actin [1±3]. Kar9p captures and stabilizes MTs via a MT plus-end-associated protein, Bim1p/Yeb1p, a mammalian EB1 homologue [4,5]. Following this capture and stabilization MT shrinkage pulls the mitotic nucleus to the bud neck and a subsequent sliding interaction between MT-bound dynein and the bud cortex pulls the daughter nucleus into the bud [6]. The sliding interaction is mediated by Num1p, a cortical protein that interacts with Pac11p, the yeast dynein intermediate chain [7,8]. This general pattern of dynamic interactions between astral MTs and the cell cortex is also seen during mating in S. cerevisiae [28]. Nuclei move toward the tips of the mating cells before cell tip fusion, and following tip fusion move toward each other to achieve karyogamy. These movements are conspicuously coupled to MT plus-end growth and shrinkage [28]. A similar process occurs during meiotic prophase in the ®ssion yeast S. pombe, in which www.current-opinion.com
Nuclear positioning: the means is at the ends Morris 55
Figure 1
One of the most striking nuclear migration phenotypes is caused by mutations that affect dynein, dynactin, NUDF and other nuclear distribution proteins in the fungus Aspergillus nidulans. In the left panel, nuclei are distributed along the length of a young wild-type mycelium. A nuclear distribution mutant is shown on the right. Although this spore has produced two germ tubes (only one is clearly visible in the picture) and the nuclei have divided, they have not migrated out from the original spore portion of the mycelium. Nuclei are stained with DAPI.
the nucleus oscillates between the cell ends pulled by anteriorly oriented astral MTs from the SPB, which grow and shrink to pull the nucleus ®rst in one direction then the other [9,10]. These oscillations require dynein. Nuclear positioning at the center of interphase S. pombe cells has similarly been attributed to MT growth and shrinkage, but in this case has been ascribed to MTs pushing against the ends of the cells to move the nucleus [29]. Although it is not completely certain to what extent the motor activities of dynein and kinesins versus their effects on MT plus-end dynamics are responsible for nuclear positioning, it is clear from the experimental data, and from the fact that tubulin mutations affecting MT dynamics also affect nuclear movement [30,31], that growth and shrinkage of MTs can move nuclei. In multinucleate ®lamentous fungi such as Aspergillus and Neurospora, the nuclei are distributed more or less evenly along the hyphae. As in yeasts, the nuclei appear to be pulled along via astral MTs acting on their SPBs. Mutations in subunits of dynein [19,20,21,25], dynactin [19,21], the num1p-related protein apsA [24], kinesin [22], and in three additional proteins required for dynein function, NUDF (Pac1p in S. cerevisiae), NUDE and NUDC [20,32], cause migration to be abnormal (Figure 1). Mutations in these proteins also alter MT stability and plus-end dynamics [20,22]. Interestingly, green ¯uorescent protein (GFP) fusions of these same proteins are found at the plus ends of astral MTs, where one might expect to ®nd them if they were in¯uencing plus-end dynamics [19,20,21,23]. How these proteins affect MT plus-end dynamics is not understood.
affect nuclear movements in animal cells [11,12,15±17]. As in the fungi, GFP fusions have revealed that dynein and a set of dynein-interactive proteins are carried on the plus ends of astral MTs. The ®rst such protein to be observed was the mammalian cytoplasmic linker protein CLIP-170 [33,34,35]. CLIP-170 targets dynein and dynactin to the MT ends and links dynein to MTs [33,34,35]. Subsequently, many other proteins have been observed at the plus ends of MTs in mammalian systems including dynein [33,34], dynactin [34,35,36], Lis1 (the mammalian homologue of NUDF) [33], CLIP-115 (a relative of CLIP-170) [37,38], and CLASP proteins (which interact with CLIP-170 and CLIP-115) [38]. The CLASP proteins are of particular interest because they stabilize MTs and are speci®cally and uniquely found only on MTs oriented toward the leading edge of migrating cells. The human colon tumor suppressor adenomatous polyposis coli (APC) [39,40] and an associated protein EB1 [41], are also found at MT tips. Dynein [42] and APC [43] are associated with the cell cortex as well as with astral MTs and may act as astral MT attachment sites. How dynein, a plus-end-directed motor, and these other proteins come to be associated with MT plus ends are still matters for speculation. One such speculation has been that a plus-end-directed motor Ð a kinesin Ð brings these proteins to the MT tip. The fact that APC interacts with KAP3, a KIF3A/B kinesin-associated protein, has provided some support for this idea [43]. Because a dominant-negative KAP3 mutant interferes with APC plus-end localization, it has been suggested that the plus-end-directed KIP3A/B kinesin motor might target APC to MT ends.
Nuclear positioning in animal cells
Cortical capture proteins
Nuclear positioning in higher eukaryotes has some similarities to the fungal systems and some conspicuous differences. Mutations in dynein and several of the same proteins that affect nuclear migration in the fungi also www.current-opinion.com
By analogy with similar processes in yeast, it has been supposed that cortical APC, although not a Kar9p homologue, captures EB1 [44] and that cortical dynein interacts with MTs [42]. IQGAP1, a protein associated with Current Opinion in Cell Biology 2003, 15:54±59
56 Cell structure and dynamics
Figure 2
(a)
(b)
Current Opinion in Cell Biology
This highly schematized figure illustrates a significant difference in the organization of the nuclear MTOC and astral MTs between fungi and animal cells. (a) In fungi, the minus ends of astral MTs are rigidly anchored in the spindle pole body (blue), an organelle that is embedded in the nuclear envelope. The plus ends of the MTs, which are oriented toward the cell cortex, are dynamic, and proteins (green) concentrated at the plus ends interact with receptors (red) on the cell cortex. In S. cerevisiae, two such interactions have been described, MT Yeb1p with cortical Kar9p and MT dynein with cortical Num1p. (b) In animal cells, the minus ends of the astral MTs originate from a centrosomal MTOC. The plus ends of these astral MTs near the cell cortex are dynamic. Proteins (green) at the plus ends interact with receptors (red) on the cell cortex. These interactions, described in the text, are less well defined than in yeast. MTs from this MTOC may also interact with nuclear receptors, to couple the centrosome to the nucleus and position the centrosome (see [11]). Whether plus-end proteins are involved in this interaction, as hypothesized here, and, if so, whether they are the same plus-end proteins that interact with the cell cortex, remains to be determined.
cortical actin at the leading edge of migrating cells, has also been suggested as a cortical capture protein. IQGAP1 interacts with both CLIP-170 and with the small Rhofamily GTPases, Rac1 and Cdc42, which are important at the cortex for cell polarization and directed cell movement [45]. PLAC-24, a protein discovered by virtue of its interaction with the dynein intermediate chain, has also been localized to actin rich MT-interacting sites of cell±cell contact in epithelial cells [46]. It has been suggested that PLAC-24 is part of an evolutionarily conserved cortical MT binding complex. Current Opinion in Cell Biology 2003, 15:54±59
The centrosomal microtubule-organizing center
In animals, unlike fungi, the centrosomal MTOC at the minus ends of the astral MTs is not passively embedded in the nuclear envelope, but is a dynamic organelle attached to the nucleus by a dynein-dependant linkage [11] (Figure 2b). This is illustrated by the effect of RNAmediated interference (RNAi)-induced dynein de®ciency in C. elegans one-cell-stage embryos. In the presence of dynein heavy chain RNAi the centrosomes of the male pronucleus fail to separate to opposite sides of the www.current-opinion.com
Nuclear positioning: the means is at the ends Morris 57
nucleus, the pronuclei don't migrate, mitotic spindle orientation is abnormal, and most importantly the centrosomes detach from the pronucleus [11,15]. Similar defects in centrosomal positioning and nuclear attachment have been found in dynein de®cient Drosophila embryos [47]. The fact that dynein is found on the nuclear periphery in the C. elegans embryos suggests that MTOC positioning is caused by an interaction with dynein on the nuclear envelope [11]. In some cell types that migrate in tissue culture the centrosome re-orients toward the direction of cell locomotion [48,49±51,52]. For example, in a scratched astrocyte culture the cells moving into the cell-depleted gap produce a long anterior extension and the centrosomal MTOC, the Golgi apparatus and MTs all re-orient toward this extension [48]. In this system integrin-mediated recognition of lost cell±cell contacts recruit Cdc42 to the leading edge to activate a signal pathway that initiates an MTOC re-orientation [48]. The centrosome re-orientation requires dynein and dynactin [48,49]. However, in migrating PtK cells the centrosome lags behind the nucleus [50]. Thus the notion that the centrosome is positioned in migrating cells by astral MTs interacting with receptors at the leading edge of the cortex is probably an oversimpli®cation of a more varied and complex process. The data from PtK cells would seem to be equally compatible with an interaction between astral MTs and dynein associated with the nuclear envelope.
Nuclear migration and cell migration
Nuclear migration plays a role in some cell migrations. In C. elegans, for example, during embryonic development P cell nuclei migrate ventrally through an extended process. This is followed by retraction of the dorsal cytoplasm to leave the nucleus and cell body in a new position [13,14]. A similar process may occur during neuronal migration in humans. This possibility is based in part on the fact that lissencephaly, a disease in which neuronal migration to the cerebral cortex is retarded, is caused by haplode®ciency of Lis1, a homologue of the fungal nuclear migration gene NUDF, which is required for dynein function (see [20] for references). In migrating neonatal cerebellar neurons the centrosome re-orients in the direction of migration, and Lis1 co-localizes with dynein and NUDC around the centrosome and at neurite tips [52], suggesting that dynein and Lis1 mediate karyokinesis during cell migration. Nuclear migration in D. melanogaster oocytes also depends on Lis1 and dynein [16,17]. Co-localization and extensive physical interactions between Lis1, NUDC, NUDE, dynein, dynactin and CLIP-170, support the idea that Lis1 affects dynein function in animals [32,33,52,53±55,56]. A screen for C. elegans mutations that interfere with P cell and other nuclear migrations has produced two genes, UNC-83 and UNC-84, whose gene products interact with each other and bind to lamin in the nuclear envelope www.current-opinion.com
[13,14]. UNC-84 resembles the S. pombe sad1p SPB protein and a human inner nuclear membrane protein, suggesting that the role of UNC-84 in nuclear migration may be evolutionarily conserved [57]. Another dramatic nuclear migration that also involves a perinuclear protein occurs during photoreceptor cell development in the developing eye of D. melanogaster. Iterative up and down nuclear migrations involved in the morphogenesis of the photoreceptor fail to occur in the absence of a protein, Klarsicht [17], initially discovered as affecting lipid droplet movements in Drosophila embryos. Interestingly, there is a mammalian Klarsicht-related protein, Syne-1, which is speci®cally associated with nuclei clustered beneath the neuromuscular junction in muscle cells [58]. These nuclei migrate through a multinucleate myotube to accumulate at the neuromuscular junction, leading to the speculation that Syne-1 plays a role in this migration. Whether Klarsicht/Syne-1 is an integral nuclear membrane protein, and whether UNC-83/ UNC-84 interacts with MTs, anchors dynein to the nuclear envelope or acts by some other mechanism remains to be determined.
Conclusions
The systems that orient and move nuclei are moderately well conserved from fungi to animal cells. In both cases dynein, dynactin and other proteins on the plus ends of astral MTs appear to interact with proteins on the cell cortex to generate forces that act on the nuclear MTOC. Interactions between centrosomal MTs and the nuclear envelope may also position the centrosome in animal cells. Although the outlines of the mechanisms responsible for nuclear positioning are relatively well understood in S. cerevisiae, and partially understood in fungi, exactly how nuclei are moved and to what extent nuclear migration is coupled to cell migration is `early days' in animal systems. Much needs to be learned about the details of component interactions and the spatial and temporal regulation of nuclear migration in all systems.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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58 Cell structure and dynamics
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8.
Farkasovsky M, Kuntzel H: Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast. J Cell Biol 2001, 152:251-262. In addition to the interaction described in the title of this paper, the authors also show, as in Adames and Cooper (2000) [6] and Farkasovsky and Kuntzel (2000) [7], that the bud tip location of GFP±Num1p does not require Kar9p, Dyn1p or cytoplasmic MTs, but does require the formin Bni1p. 9.
Yamamoto A, Tsutsumi C, Kojima H, Oiwa K, Hiraoka Y: Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in ®ssion yeast. Mol Biol Cell 2001, 12:3933-3946. Nuclei of Schizosaccharomyces pombe undergo vigorous oscillations between the poles of the cell at meiotic prophase. GFP-tagged spindle pole body MTs lead these nuclear oscillations and interact distally with the cell cortex in wild-type cells, but not in dynein mutants. The authors propose a model in which nuclear oscillations are driven by cortical/ microtubule interactions and the dynamics of microtubule disassembly at the cortex. 10. Miki F, Okazaki K, Shimanuki M, Yamamoto A, Hiraoka Y, Niwa O: The 14-kDa dynein light chain-family protein Dlc1 is required for regular oscillatory nuclear movement and ef®cient recombination during meiotic prophase in Fission Yeast. Mol Biol Cell 2002, 13:930-946. Dlc1 belongs to the Tctex family of dynein light chains.
21. Zhang J, Han G, Xiang X: Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans. Mol Microbiol 2002, 44:381-392. The authors use mutations and GFP-labeled proteins to show that localization of the dynein intermediate and heavy chains to MT plus ends in A. nidulans requires both dynein subunits. They also demonstrate that heavy chain localization to MT plus ends requires NUDF, the A. nidulans homolog of mammalian Lis1. 22. Requena N, Alberti-Segui C, Winzenburg E, Horn C, Schliwa M, Philippsen P, Liese R, Fischer R: Genetic evidence for a microtubule-destabilizing effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution in Aspergillus nidulans. Mol Microbiol 2001, 42:121-132. 23. Lee IH, Kumar S, Plamann M: Null mutants of the Neurospora actin-related protein 1 pointed-end complex show distinct phenotypes. Mol Biol Cell 2001, 12:2195-2206. This paper demonstrates that the abnormal hyphal morphology of ropy mutants is not a necessary consequence of the nuclear migration defect seen in most of these mutants. Although mutations in the RO2 (p65), RO7 (Arp11), and RO12 (p25) subunits of the dynactin pointed end complex have abnormal curled hyphae, as do all the other Neurospora crassa ropy mutants, nuclear distribution is normal in RO12. 24. Graia F, Berteaux-Lecellier V, Zickler D, Picard M: ami1, an orthologue of the Aspergillus nidulans apsA gene, is involved in nuclear migration events throughout the life cycle of Podospora anserina. Genetics 2000, 155:633-646.
11. Gonczy P, Pichler S, Kirkham M, Hyman AA: Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J Cell Biol 1999, 147:135-150.
25. Alberti-Segui C, Dietrich F, Altmann-Johl R, Hoepfner D, Philippsen P: Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the ®lamentous ascomycete Ashbya gossypii. J Cell Sci 2001, 114:975-986. In contrast to other fungi in which dynein ablation causes nuclei to be clustered at the spore end of the hyphae, dynein ablation in Ashbyacauses nuclei to be clustered at the hyphal tips.
12. Yoder JH, Han M: Cytoplasmic dynein light intermediate chain is required for discrete aspects of mitosis in Caenorhabditis elegans. Mol Biol Cell 2001, 12:2921-2933.
26. Chytilova E, Macas J, Sliwinska E, Rafelski SM, Lambert GM, Galbraith DW: Nuclear dynamics in Arabidopsis thaliana. Mol Biol Cell 2000, 11:2733-2741.
13. Starr DA, Hermann GJ, Malone CJ, Fixsen W, Priess JR, Horvitz HR, Han M: unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration. Development 2001, 128:5039-5050. 14. Lee KK, Starr D, Cohen M, Liu J, Han M, Wilson KL, Gruenbaum Y: Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans. Mol Biol Cell 2002, 13:892-901. This interesting paper establishes a physical basis for the nuclear envelope localization of UNC-84. 15. Gonczy P, Echeverri C, Oegema K, Coulson A, Jones SJ, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E et al.: Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 2000, 408:331-336. 16. Swan A, Nguyen T, Suter B: Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning. Nat Cell Biol 1999, 1:444-449. 17. Lei Y, Warrior R: The Drosophila lissencephaly1 (DLis1) gene is required for nuclear migration. Dev Biol 2000, 226:57-72. 18. Mosley-Bishop KL, Li Q, Patterson L, Fischer JA: Molecular analysis of the Klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye. Curr Biol 1999, 9:1211-1220. 19. Xiang X, Han G, Winkelmann DA, Zuo W, Morris NR: Dynamics of cytoplasmic dynein in living cells and the effect of a mutation in the dynactin complex actin-related protein Arp1. Curr Biol 2000, 10:603-606. 20. Han G, Liu B, Zhang J, Zuo W, Morris NR, Xiang X: The Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics. Curr Biol 2001, 11:719-724. In addition to reporting that Aspergillus cytoplasmic dynein heavy chain and NUDF localize to microtubule ends and affect microtubule dynamics, this paper also contains useful references to earlier papers demonstrating that NUDF is required for dynein function. Current Opinion in Cell Biology 2003, 15:54±59
27. Lawrence CJ, Morris NR, Meagher RB, Dawe RK: Dyneins have run their course in plant lineage. Traf®c 2001, 2:362-363. 28. Maddox P, Chin E, Mallavarapu A, Yeh E, Salmon ED, Bloom K: Microtubule dynamics from mating through the ®rst zygotic division in the budding yeast Saccharomyces cerevisiae. J Cell Biol 1999, 144:977-987. 29. Tran PT, Marsh L, Doye V, Inoue S, Chang F: A mechanism for nuclear positioning in ®ssion yeast based on microtubule pushing. J Cell Biol 2001, 153:397-411. Using GFP±tubulin to visualize microtubules (MTs), the authors demonstrate that cytoplasmic MTs emanating from the nucleus dynamically sense the positions of the ends of the cell and maintain the nucleus in the center of the cell by pushing against the ends. 30. Caron JM, Vega LR, Fleming J, Bishop R, Solomon F: Single site alpha-tubulin mutation affects astral microtubules and nuclear positioning during anaphase in Saccharomyces cerevisiae: possible role for palmitoylation of alpha-tubulin. Mol Biol Cell 2001, 12:2672-2687. 31. Dougherty CA, Sage CR, Davis A, Farrell KW: Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis. Biochemistry 2001, 40:15725-15732. This tubulin mutation, like the one described in Tran et al. (2001) [29] affects nuclear positioning. The import of these two papers for dyneinmediated nuclear positioning is that because microtubule dynamics affect nuclear positioning, the effect of dynein on positioning could just as well be mediated via an effect of dynein on microtubule dynamics as by dynein motor activity per se. 32. E®mov VP, Morris NR: The Lis1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein. J Cell Biol 2000, 150:681-688. 33. Coquelle FM, Caspi M, Cordelieres FP, Dompierre JP, Dujardin DL, Koifman C, Martin P, Hoogenraad CC, Akhmanova A, Galjart N et al.: Lis1, CLIP-170's key to the dynein/dynactin pathway. Mol Cell Biol 2002, 22:3089-3102. www.current-opinion.com
Nuclear positioning: the means is at the ends Morris 59
This paper provides evidence that Lis1 acts as an adaptor between CLIP170 and cytoplasmic dynein by demonstrating that they colocalize and interact physically. 34. Vaughan KT, Tynan SH, Faulkner NE, Echeverri CJ, Vallee RB: Colocalization of cytoplasmic dynein with dynactin and CLIP-170 at microtubule distal ends. J Cell Sci 1999, 112:1437-1447. 35. Valetti C, Wetzel DM, Schrader M, Hasbani MJ, Gill SR, Kreis TE, Schroer TA: Role of dynactin in endocytic traf®c: effects of dynamitin overexpression and colocalization with CLIP-170A. Mol Biol Cell 1999, 10:4107-4120. 36. Vaughan PS, Miura P, Henderson M, Byrne B, Vaughan KT: A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport. J Cell Biol 2002, 158:305-319. This exciting report shows that recruitment of dynactin P150glued to microtubule plus ends is modulated by phosphorylation. Of particular interest is a demonstration that interactions between microtubule plus ends and Golgi membranes precede centripetal, dynein-mediated membrane transport. 37. Hoogenraad CC, Akhmanova A, Grosveld F, De Zeeuw CI, Galjart N: Functional analysis of CLIP-115 and its binding to microtubules. J Cell Sci 2000, 113:2285-2297. 38. Akhmanova A, Hoogenraad CC, Drabek K, Stepanova T, Dortland B, Verkerk T, Vermeulen W, Burgering BM, De Zeeuw CI, Grosveld F, Galjart N: Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile ®broblasts. Cell 2001, 104:923-935. The CLASP proteins are found on distal segments of microtubules oriented toward the leading edge of migrating cells. The authors provide evidence that CLASPs regulate MT dynamics in response to positional clues. 39. Mogensen MM, Tucker JB, Mackie JB, Prescott AR, Nathke IS: The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells. J Cell Biol 2002, 157:1041-1048. It had previously been shown (see Mimori-Kiyosue et al. [2000] [41]) that EB1, a protein associated with the adenomatous polyposis coli (APC) protein, is found at the plus ends of microtubules. This paper rounds out the picture by showing that APC co-locates with EB1 at microtubule plus ends in polarized epithelial cells in which microtubule polarity has been de®nitively established by hook decoration. The authors show that in mice heterozygous for APC, there are fewer microtubule bundles, suggesting that the presence of APC stabilizes microtubules. 40. Zumbrunn J, Kinoshita K, Hyman AA, Nathke IS: Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation. Curr Biol 2001, 11:44-49.
45. Fukata M, Watanabe T, Noritake J, Nakagawa M, Yamaga M, Kuroda S, Matsuura Y, Iwamatsu A, Perez F, Kaibuchi K: Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170. Cell 2002, 109:873-885. This beautiful paper demonstrates that IQGAP1, an effector of the Rac1 and Cdc42 GTPases, interact with the microtubule plus-end protein CLIP-170 to target the plus ends of cytoplasmic microtubules to the polarized cell cortex. 46. Karki S, Ligon LA, DeSantis J, Tokito M, Holzbaur EL: PLAC-24 is a cytoplasmic dynein-binding protein that is recruited to sites of cell±cell contact. Mol Biol Cell 2002, 13:1722-1734. 47. Robinson JT, Wojcik EJ, Sanders MA, McGrail M, Hays TS: Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila. J Cell Biol 1999, 146:597-608. 48. Etienne-Manneville S, Hall A: Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell 2001, 106:489-498. Cells migrating into a wound re-orient their microtubule organizing center and microtubules by a dynein-requiring process initiated by the interaction of integrins with the extracellular matrix. 49. Palazzo AF, Cook TA, Alberts AS, Gundersen GG: mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat Cell Biol 2001, 3:723-729. 50. Yvon AM, Walker JW, Danowski B, Fagerstrom C, Khodjakov A, Wadsworth P: Centrosome reorientation in wound-edge cells is cell type speci®c. Mol Biol Cell 2002, 13:1871-1880. 51. Chevrier V, Piel M, Collomb N, Saoudi Y, Frank R, Paintrand M, Narumiya S, Bornens M, Job D: The Rho-associated protein kinase p160ROCK is required for centrosome positioning. J Cell Biol 2002, 157:807-817. 52. Aumais JP, Tunstead JR, McNeil RS, Schaar BT, McConnell SK, Lin SH, Clark GD, Yu-Lee LY: NUDC associates with Lis1 and the dynein motor at the leading pole of neurons. J Neurosci 2001, 21:RC187. NUDC co-localizes with Lis1 and the cytoplasmic dynein intermediate chain in a polarized manner at the microtubule organizing center in migrating cerebellar granule cells. It also co-immunoprecipitates with CDIC and cytoplasmic dynein. These data suggest that a functional interaction of NUDC with Lis1 and dynein underlies neuronal migration. 53. Kitagawa M, Umezu M, Aoki J, Koizumi H, Arai H, Inoue K: Direct association of Lis1, the lissencephaly gene product, with a mammalian homologue of a fungal nuclear distribution protein, rNUDE. FEBS Lett 2000, 479:57-62. 54. Tai CY, Dujardin DL, Faulkner NE, Vallee RB: Role of dynein, dynactin, and CLIP-170 interactions in Lis1 kinetochore function. J Cell Biol 2002, 156:959-968.
41. Mimori-Kiyosue Y, Shiina N, Tsukita S: The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr Biol 2000, 10:865-868.
55. Niethammer M, Smith DS, Ayala R, Peng J, Ko J, Lee MS, Morabito M, Tsai LH: NUDEL is a novel Cdk5 substrate that associates with Lis1 and cytoplasmic dynein. Neuron 2000, 28:697-711.
42. Dujardin D, Vallee RB: Dynein at the cortex. Curr Opin Cell Biol 2002, 14:44-49. This review summarizes the evidence for cortical dynein.
56. Hoffmann B, Zuo W, Liu A, Morris NR: The Lis1-related protein NUDF of Aspergillus nidulans and its interaction partner NUDE bind directly to speci®c subunits of dynein and dynactin and to alpha- and gamma-tubulin. J Biol Chem 2001, 276:38877-38884. Although this paper deals only with Aspergillus proteins, their interactions are similar to those described for Lis1 and mammalian NUDE.
43. Jimbo T, Kawasaki Y, Koyama R, Sato R, Takada S, Haraguchi K, Akiyama T: Identi®cation of a link between the tumour suppressor APC and the kinesin superfamily. Nat Cell Biol 2002, 4:323-327. By demonstrating a link between a microtubule plus-end directed motor protein and the adenomatous polyposis coli (APC) protein, the authors provide a possible explanation for the location of APC at the plus ends of cytoplasmic microtubules (see also Mogensen et al. [2002] [39]). 44. Rosin-Arbesfeld R, Ihrke G, Bienz M: Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells. EMBO J 2001, 20:5929-5939.
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57. Dreger M, Bengtsson L, Schoneberg T, Otto H, Hucho F: Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane. Proc Natl Acad Sci USA 2001, 98:11943-11948. 58. Apel ED, Lewis RM, Grady RM, Sanes JR: Syne-1, a dystrophin- and Klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction. J Biol Chem 2000, 275:31986-31995.
Current Opinion in Cell Biology 2003, 15:54±59