Microtubule Nucleation at the Centrosome and Beyond

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Microtubule nucleation at the centrosome and beyond

Sabine Petry and Ronald D. Vale

The overall organization of the microtubule cytoskeleton depends critically on where and when new microtubules are nucleated. Here we review recent work on factors that are involved in localizing and potentially activating γ-tubulin, the key microtubule nucleating protein.

Microtubules, 25 nm polar filaments assembled at the nuclear envelope7, the Golgi apparatus8,9, lar features with the γ-TuRC ring18. The helical from α/β-tubulin heterodimers, permeate the pre-existing microtubules10,11 and the plasma symmetry made it possible to apply averaging cytoplasm of virtually all eukaryotic cells. They membrane12. Collectively, these non-centrosomal techniques, resulting in a medium-resolution serve as the chief structural component of the sites seem to play an important role in governing cryo-EM reconstruction at 8 Å; this revealed mitotic spindle in dividing cells and provide cellular microtubule architecture. new details on how γ-tubulin, GCP2 and GCP3 roadways for motor-driven transport to position The best-characterized microtubule nucleat- are arranged within γ-TuSC16. In contrast, much the majority of membrane-bounded organelles in ing factors are protein complexes of γ-tubulin less is known about the architecture of γ-TuRC. animal cells1. Microtubules also work in conjunc- and several associated proteins. The yeast In 2000, three landmark papers reported that it tion with the actin cytoskeleton and signalling γ-tubulin small complex (γ-TuSC) consists of 2 forms a cone-like cap at the microtubule minus systems to establish and maintain cell polarity2. γ-tubulin molecules and one each of γ-tubulin end following microtubule nucleation19–21. Microtubule assembly occurs in vitro when the complex proteins (GCP) 2 and 3 (refs. 13–15), Based on prior results and gold labelling of concentrations of pure α/β-tubulin heterodimers and is capable of moderately facilitating micro- individual subunits, the γ-TuRC-specific subu- exceed a ‘critical concentration’. However, they tubule nucleation16. In higher eukaryotes, the nits (GCP4, 5 and 6), which are essential for rarely spontaneously assemble in cells and instead larger γ-tubulin ring complex (γ-TuRC), named γ-TuRC formation, were first hypothesized to require a nucleating factor to initiate polymeriza- for its characteristic shape observed in negative- bind to GCP2 and 3 to form the distal tip of the tion. Microtubules are nucleated from the centro- stain electron microscopy (EM) images17, is a γ-TuRC cap. However, the crystal structure of some, which was originally believed to be the sole marginally more efficient microtubule nuclea- GCP4 (ref. 22) indicates another possibility. All cellular structure from which microtubules are tor14. Both the γ-TuSC and γ-TuRC complexes GCPs are characterized by an N-terminal Grip1 nucleated and was thus termed the microtubule are thought to form a ring-like template that and a C-terminal Grip2 motif, and GCP4 can organizing centre (MTOC). Microtubules were binds the α-tubulin subunit, which is exposed at bind directly to γ-tubulin in vitro via its Grip2 proposed to then dissociate or sever from the cen- the microtubule minus end. Thus, microtubules motif 22. This suggests that GCP4, and perhaps trosome for transport to other locations in the cell grow with a defined polarity from γ-tubulin also GCP5 and 6, which are closely related to by molecular motors. Although severing from the complexes, with their minus ends anchored on GCP4 in their sequence and domain organiza- centrosome and motor-driven transport are likely the ring of γ-tubulin subunits and the plus end of tion, might be directly incorporated into the to occur in many cells3, it is becoming clear that the microtubule extending into the cytoplasm. γ-TuRC ring and contact γ-tubulin, instead microtubule nucleation also takes place in other In this Perspective, we focus on the latest of only forming the distal end of the complex cellular locations. Indeed, many cells (egg meiotic research and emerging questions concerning (Fig. 1). Consistent with this idea, modelling of and plant cells, for instance) lack classical cen- the regulation of γ-tubulin activity. Recent the GCP4 crystal structure into the EM recon- trosomes or can function without them. The list work has uncovered some of the factors that struction of the yeast γ-TuSC provided a good of non-centrosomal MTOCs is now considerable, dock γ-tubulin to different structures in the fit and revealed a potential hinge point in the and still expanding: during cell division, micro- cell. Furthermore, purified γ-tubulin complexes middle of the structure. Straightening this hinge tubules originate from spindle microtubules4, seem not to be constitutively active, suggesting might decrease the spacing between γ-tubulin kinetochores5 and in the vicinity of chromatin6; that the efficiency of γ-tubulin-mediated micro- molecules within the γ-TuSC ring and bring and during interphase, microtubules are made tubule nucleation is tuned by regulatory factors, the complex into a hypothesized nucleation- a few of which have been identified. competent conformation22, which would be Sabine Petry is in the Department of Molecular 23 Biology, Princeton University, Princeton, New Jersey assumed when bound to a microtubule . When 08540, USA. Ronald D. Vale is in the Department of Structural insight into γ-TuSC and γ-TuRC γ-TuSC is artificially trapped in this closed state, Cellular and Molecular Pharmacology and the Howard Under certain conditions, the yeast γ-TuSC its nucleation capacity is increased, further sup- Hughes Medical Institute, University of California, 23 San Francisco 94158-2517, California, USA. tetramer assembles into transient spiral-like porting the idea of this allosteric switch . An e-mail: [email protected] structures, in which the first turn shares simi- even higher increase in microtubule nucleation

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of Mto1 and Mto2, along with a γtubulin com- plex26. Lynch et al. also showed that multimeri- ? γ‐tubulin zation of Mto1/2 may induce multimerization Nuclear GCP4 TPX2 envelope ? 26 GCP6 GCP3 of γ-TuSC . However, how and to what extent ? GCP5 GCP2 Augmin the Mto1/2 complex activates yeast γ-tubulin Ninein Core subunits required remains to be determined. for ring assembly Pericentrin In summary, the microtubule nucleation NEDD1 and localization of the yeast γ-tubulin complex NME7 Direct binding partners Mzt1/GCP9 Microtubule is regulated by interacting factors and activat- (mitosis) LGALS3BP Mzt2/GCP8 ing phosphorylations. It is currently not clear (interphase) whether individual proteins encode all these CDK5RAP2 functionalities (for example, Spc110), or whether Golgi apparatus Myomegalin multiple activating factors are required simulta- Cep192 Ninein neously. It will be important to assess the extent AKAP450 Pericentrin to which these factors activate γTuSC on their own versus in various combinations, and, most AKAP450AKAP450 Centrosome importantly, how activation is achieved. This will GM130 require quantitative in vitro studies to comple- ment the elegant in vivo studies that have been performed in yeast. Finally, an EM structure of Figure 1 A schematic diagram of γ-TuRC composition and its connections to various microtubule nucleation pathways in an animal cell. Inner central circle: The core subunits γ-tubulin and GCP2–6 are required for a fully activated yeast γ-TuSC assembly, in con- γ-TuRC ring formation. Recent structural evidence suggests that GCP4 to 6 may be directly incorporated junction with the current medium-resolution into the γ-TuRC ring instead of forming only its very tip. However, the exact location of GCP4–6 is not structure of the yeast γ-TuSC spiral, will provide known (question marks). Outer central circle: Other proteins have been co-purified with -TuRC and thus γ useful insights into the mechanism of activation. directly bind γ-TuRC in vitro. These proteins may not be constitutive subunits, because it is not yet clear whether all γ-TuRC populations contain them. These include the kinase NME7, CDK5RAP2, Mzt1, Mzt2 and NEDD1, which are described in the main text, as well as the protein LGALS3BP, the precise role of Localization and activation factors of which remains to be defined. All other indicated proteins have been shown to target γ-TuRC to different higher eukaryote γ-tubulin complexes MTOCs (nuclear envelope, Golgi apparatus, centrosome and MTs). Dashed lines indicate an interaction with Higher eukaryotic cells can have up to 1,000 γ-TuRC where the interacting subunit is not yet known, whereas solid arrows indicate recruitment through an identified protein or subunit. Interactions or recruitment can occur for the whole MTOC (line/arrow from times more microtubules than yeast. Not surpris- MTOC border) or with individual proteins (line/arrow pointing to the protein name). ingly, the regulation of microtubule nucleation seems more complex. Besides the GRIP-motif- activity could be achieved with tubulin of the discovered Spc110/Pcp1 motif (SPM), which is containing GCPs that build γ-TuRC, additional same species as γ-TuRC (Saccharomyces cerevi- also present in the fission yeast spindle body proteins co-purify with the complex, many siae) instead of porcine brain tubulin, which is protein Pcp1 and human pericentrin25. Both the of which may play a role in its localization used traditionally23. However, higher-resolution CM1 and SPM motifs, and phosphorylation by and activation (Fig. 1 and Table 1). Perhaps structures of the complete γ-TuRC (similar to the cell-cycle-regulating kinases Mps1 and Cdk the best studied is the WD-domain protein of those achieved for the γ-TuSC spiral), both in the central region of Spc110, are important 71 kDa (Dgp71WD), which co-purifies with before and after microtubule nucleation, will be for promoting Spc110 oligomerization, but only Drosophila melanogaster γTuRC. Depletion of needed to understand how GCP4, 5 and 6 are phosphorylation and the SPM motif seem to this protein, or its Xenopus laevis orthologue arranged in the complex, and how γ-TuRC facili- control microtubule nucleation25. NEDD1, impairs γ-tubulin recruitment to spin- tates microtubule nucleation (Table 1). Other proteins that interact with γ-tubulin dle microtubules27,28. Depletion or inhibition complexes contain only the CM1 motif; for exam- of the human orthologue GCP-WD abolishes Localization and activation factors for ple, Spc72, Mto1, centrosomin, CDK5RAP2 and γ-TuRC localization and microtubule nucleation yeast γ-tubulin complexes myomegalin25. Among the best characterized of at the centrosome and chromatin28,29. Differential γ-Tubulin complexes must be correctly local- these is the Schizosaccharomyces pombe Mto1 phosphorylation is likely to control the sites to ized and activated in cells. This process is protein, which forms a complex with Mto2. which NEDD1 recruits γ-TuRC30,31, yet it remains perhaps best understood in yeast, which has The C-terminus of Mto1 localises the γ-tubulin unclear whether GCP-WD also stimulates the a relatively simple microtubule cytoskeleton complex to several cytoplasmic MTOCs and microtubule nucleation activity of γ-TuRC. A made up of approximately 40 filaments. Spc110 the spindle pole body, and its Nterminal CM1 second localization factor pair is MOZART2A/B appears to be a critical protein both for local- motif, in conjunction with Mto2, also pro- (mitotic-spindle organizing protein associ- izing and activating the complex, as shown in motes the microtubule nucleation activity of ated with a ring of γtubulin, 16.2 kDa), which S. cerevisiae. The N-terminus of Spc110, which γ-tubulin complexes26. The roles in recruitment has recently been renamed GCP8A/B. These induces a ring-like assembly of γ-TuSC as dis- and activation of γ-tubulin could be separated highly related proteins (98% sequence identity) cussed above18, contains two conserved motifs in minimal Mto1/2 versions, and are therefore were co-purified with γ-TuRC from human important for microtubule nucleation activa- independent of one another26. Active nucleation cells in two independent studies32,33. GCP8B tion: the conserved CM1 motif 24 and a newly complexes are likely to contain 13 copies each co-localizes with γ-TuRC and is necessary for its

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Table 1 Overview of γ-TuRC complexes and the assays that have been performed Co-purification γ-Tubulin complex Subunit Binding site on γ-TuRC Effect on in vitro nucleation activity with γ-TuRC γ-Tubulin • First ring layer and GCPs

γ-TuSC Core GCP2 • γ-Tubulin, GCP3 γ-TuSC sufficient for nucleation γ-TuRC with GCP3 • γ-Tubulin, GCP2 subunits required for GCP4 • γ-Tubulin, other? ring assembly GCP5 • ND γ-TuRC nucleation greater than γ-TuSC Extended γ-TuRC with GCP6 • ND direct binding NEDD1 • ND ND partners NME7 • ND 2.5-fold enhancement for γ-TuRC CDK5RAP2 • ND 7.5-fold enhancement for γ-TuRC Mzt2/GCP8 • ND ND Mzt1/GCP9 • GCP3, other? ND LGALS3BP • ND ND For γ-TuSC, co-purification has been achieved, its in vitro nucleation activity has been tested, the binding sites for subunits γ-tubulin, GCP2 and GCP3 have been identified, and a medium- resolution structure has been solved by electron microscopy (EM). In contrast, the exact binding sites for GCP4, 5 and 6 are not clear, and the existing EM structure is at low resolution. Only a few co-purified binding partners have been tested for the activity effect on γ-TuRC, and it remains unclear how they interact with the complex. So far, NME740 and CDK5RAP239 have been shown to enhance the MT nucleation activity of γ-TuRC, but the mechanisms remain unclear. It is also unknown whether the activation mechanism is conserved for all MT nucleation pathways (see Fig. 1). ND, not determined. recruitment to interphase centrosomes down- complex binding domain (termed γ-TuNA; generated at the nuclear membrane play a role stream of GCP-WD33. MOZART1 (renamed γ-TuRC-mediated nucleation activator), which in establishing nuclear shape and positioning GCP9), a third localization factor that shares is also conserved in the γ-tubulin complex nuclear pore complexes43. However, in both weak sequence similarity to MOZART2, also tethering proteins centrosomin (Drosophila) plant and animal cells, the overall mechanisms seems to be involved in γ-tubulin recruitment and Mto1p and Pcp1p (S. pombe)39. Currently, of γ-tubulin localization and activation at the to centrosomes in mitotic cells, and its depletion there is little understanding of the mechanism of nuclear membrane remain poorly understood. leads to mitotic spindle defects32. γ-TuRC activation and localization by this very Other proteins implicated in the recruitment small domain. Golgi apparatus. The Golgi apparatus of animal of γ-TuRC to centrosomes include pericen- cells typically faces the leading edge or the axis of trin34 and AKAP450 (also known as CGNAP or Non-centrosomal microtubule nucleation secretion of the cell. Microtubules can be gener- AKAP9)35. These two proteins share a so-called centres ated from the Golgi in a γ-tubulin-dependent PACT domain at their C-terminus for centro- The majority of work on microtubule nucleation manner; some interphase cells, such as RPE-1 some targeting, and they interact with γ-TuRC has involved centrosomes or spindle pole bodies. cells, generate about half of their microtubule through their N-terminus36. Furthermore, However, it is clear that microtubules arise from cytoskeleton from Golgi MTOCs8,9. Golgi- Cep192 recruits GCP-WD/NEDD137, and membranous organelles, chromosomes and nucleated microtubules have been proposed to ninein-like proteins play a role in γ-TuRC other sites in the cell, in a γ-tubulin-dependent play an important role in the fusion of mem- recruitment to centrosomes during interphase38. manner. Here, we briefly describe some of the branes into a continuous Golgi ribbon46. On a A γ-TuRC-associated protein with nucleation- best-understood non-centrosomal microtubule molecular level, several proteins that anchor activating function is the nucleoside-diphosphate nucleation processes in animal cells. γ-TuRC to pericentriolar material also co- kinase NME732,33,39. This protein localizes to most localize with the cis-Golgi, such as myomega- sites of interphase microtubule nucleation, and to Nuclear membrane. Several cell types exhibit lin47. The cis-Golgi-specific protein GM130 both centrosomes and the mitotic spindle dur- microtubule nucleation at the nuclear mem- recruits AKAP45048, which in turn recruits ing mitosis; yet its depletion does not interfere brane, and this is best characterized in skeletal CDK5RAP249. Therefore, GM130 may be a key with the centrosomal localization of γ-TuRC (or muscle cells7. γ-Tubulin and the centrosomal regulatory factor of the Golgi MTOC; however, CDK5RAP2; see below)40. NME7 increases the proteins pericentrin and ninein are redistributed details of how microtubule nucleation from the nucleation capacity of γ-TuRC in vitro by ~2.5- to the nuclear envelope following skeletal muscle Golgi is regulated and activated remain unclear. fold, which is dependent on its kinase activity differentiation41, and nuclei of non-muscle cells with as-yet unknown targets. Thus, NME7 is a acquire the ability to bind centrosomal proteins Chromatin and kinetochores. Ran-GTP greatly mild modulator of γ-TuRC activity. when fused to differentiating myoblasts42. In activates microtubule generation near chroma- The best characterized activator factor for addition, GCP9 is important for γ-tubulin locali- tin6,50 by releasing a few dozen spindle assembly γ-TuRC is CDK5RAP2, which binds to it and zation to the nuclear envelope43, and local micro- factors (SAFs) after nuclear envelope breakdown, is involved in its centrosomal attachment39. tubule formation can be blocked by an inhibitory which were proposed to contribute to microtu- In vitro, CDK5RAP2 activates γ-TuRC-mediated γ-tubulin antibody41. The nuclei of plant cells bule formation and stabilization50,51. However, microtubule nucleation by ~7-fold39. The activa- also bind γ-tubulin and GCP2/3 (ref. 44) and their exact function remains unclear. In particu- tion function of the large CDK5RAP2 protein are able to nucleate microtubules from their sur- lar, whether they stabilize or generate microtu- is contained within in the ~5.5 kDa γ-tubulin faces45. It has been suggested that microtubules bules, and whether they belong to overlapping

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© 2015 Macmillan Publishers Limited. All rights reserved PERSPECTIVE or separate microtubule nucleation pathways, is possible to demonstrate directly that newly nucleating activity in vitro72. Similarly, the pro- not known. The chromosomal passenger com- forming microtubules grew from the wall of tein TPX2 can nucleate microtubules from puri- plex (CPC) can compensate for the Ran-GTP pre-existing ones4. The branch angle between fied tubulin in vitro73. However, the promotion gradient under certain conditions by generating the daughter and mother microtubule was of microtubule nucleating activity of these fac- microtubules from centromeric chromatin52. usually shallow, and in many cases the newly tors is not fully γ-tubulin-independent7271, and Both the Ran-GTP and CPC pathways, along nucleated microtubules grew parallel to the recent evidence supports a model in which these with the Nup107-160 complexes53, contribute to template microtubule. Therefore, this nucleating microtubule-associated proteins control the microtubule formation at kinetochores54, which mechanism preserves the polarity of the tem- kinetics of microtubule nucleation from γ-TuRC may assist in kinetochore capture54. plate filament. Microtubule-dependent micro- or pre-existing microtubule plus ends74, leaving tubule nucleation provides an effective means of open the question of whether they act as true Microtubule-dependent microtubule nuclea- amplifying their numbers quickly with the same nucleators. Finally, microtubules themselves tion. In addition to nucleation from large orga- polarity, which appears to be important for the could serve as seeds for nucleation, which can nelles such as centrosomes, nuclei and the Golgi, organization of a large and polar microtubule- be generated through the severing activity of microtubules also can be nucleated from a rather rich structure such as a spindle. The cell-free katanin or spastin75. simple source: a pre-existing microtubule. This Xenopus extract assay also allowed the identifi- Many of the upcoming challenges for under- was initially observed in interphase plant and cation of γ-tubulin, augmin and TPX2 (targeting standing the mechanism of microtubule nuclea- yeast cells. In plant cells, microtubules were seen factor of Xlkp2) as key molecular factors needed tion will require biochemistry and structural to branch off existing microtubules at the cell for microtubule branching. How augmin, TPX2 studies. The current structural work on γ-TuSC cortex with a characteristic angle of ~40° and 0°, and γ-TuRC work together to induce branching has been very illuminating, but a high-resolu- in a γ-tubulin-dependent manner10,55, whereas microtubule nucleation, and the structure of the tion view of the ‘active nucleating state’ of both in S pombe, microtubule nucleation produces protein complex at the branch point, remain γ-TuSC and γ-TuRC is still needed. In addition, daughter microtubules that are anti-parallel to important and unanswered questions. genetics and biochemistry have identified many the mother microtubule (at a 180° angle)11. interacting proteins of these complexes, high- Shortly after these observations, new evidence Conclusion and outlook lighted in this review, which have been impli- indicated that microtubules are generated within Interesting parallels can be drawn between cated in localizing and/or activating γ-tubulin. the body of spindles56,57 and not only at cen- actin and microtubule nucleation. Although However, little is known about the upstream trosomes and chromosomes, as was previously the actin nucleator Arp2/3 was discovered mechanisms ensuring that these proteins acti- thought. As γ-tubulin was localized to the body after γ-tubulin67, the complete Arp2/3 complex vate microtubule nucleation at the correct loca- of the spindle, it was a candidate for nucleat- has been crystallized, its structure on a mother tion and time. There are clear indications that ing these new microtubules, and several genes filament has been deduced by high-resolution signalling pathways involving phosphorylation were found to specifically target γ-tubulin to cryo-EM, and the mechanism of activating cascades or Ran-GTP impinge on γ-tubulin spindle microtubules but not to centrosomes58. Arp2/3 by WAVE and WASp complexes has complexes, but the details are crudely under- These proteins were shown to form an 8-subu- been dissected in great detail68. In addition, stood51. Whether proteins other than γ-tubulin nit complex (called augmin) in Drosophila59 and several other actin nucleators (such as formins can nucleate microtubules in cells also remains human cells60,61. Recently, progress has been and SPIRE) and their mechanisms have been an open question. The field will need to address made in deciphering the interactions of subu- uncovered68. This work on actin nucleation these questions to further understand how nits within a recombinant augmin complex62. foreshadows what remains to be discovered microtubule nucleation is initiated and regu- Depletion of augmin gives rise to spindle defects regarding microtubule nucleation. lated to ensure that it occurs at the correct time in animal cells and a reduction in microtubule Although γ-tubulin is generally regarded as and place to create the microtubule networks density within the spindle60,61. Although absent the universal microtubule nucleator, it remains fundamental to the activities of eukaryotic cells. from both S. pombe and S. cerevisiae, augmin possible that additional microtubule nucle- ADDITIONAL INFORMATION also is found in plants and plays an important ating proteins exist. For example, depletion Reprints and permissions information is available role in the formation of the plant spindle and of γ-tubulin by RNA interference (RNAi) in online at www.nature.com/reprints. Correspondence the microtubule-rich phragmoplast63–65, and in Caenorhabditis elegans69 and Drosophila inter- should be addressed to S.P. 70 triggering microtubule-dependent microtubule phase cells still allowed for the formation of an COMPETING FINANCIAL INTERESTS nucleation at the plant cortex66. microtubule cytoskeleton, albeit in an abnormal The authors declare no competing financial interests. Although it is clear that new microtubules form and with reduced microtubule filament 1. De Forges, H., Bouissou, A. & Perez, F. Interplay arise within the spindle, the very high micro- numbers. However, it is difficult to ascertain between microtubule dynamics and intracellu- tubule density precluded direct observations whether the microtubules present in γ-tubulin lar organization. Int. J. Biochem. Cell Biol. 44, 266–274 (2012). of whether new microtubules were being RNAi were created from residual γ-tubulin or 2. Akhshi, T. K., Wernike, D. & Piekny, A. Microtubules and actin crosstalk in cell migration and division. templated from pre-existing ones. Cell-free resulted from the activity of another nucleating Cytoskeleton 71, 1–23 (2014). extracts of Xenopus eggs in conjunction with process. A candidate for an alternative nucle- 3. Sharp, D. J. & Ross, J. L. Microtubule-severing enzymes at the cutting edge. J. Cell Sci. 125, total internal fluorescence (TIRF) microscopy ating factor is the microtubule polymerase 2561–2569 (2012). circumvented this roadblock. By simultaneously XMAP215, which can partially rescue the deple- 4. Petry, S., Groen, A. C., Ishihara, K., Mitchison, T. J. 71 & Vale, R. D. Branching microtubule nucleation in imaging microtubules with fluorescent tubulin tion of γ-tubulin in acentrosomal spindles Xenopus egg extracts mediated by augmin and TPX2. and newly growing microtubule ends, it was and has been shown to promote microtubule Cell 152, 768–777 (2013).

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