Proteome Analysis of the Human Mitotic Spindle

Abstract

The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. Malfunctioning of the spindle can lead to chromosome missegregation and hence result in aneuploidy, a hallmark of most human cancers. Despite major progress in deciphering the temporal and spatial regulation of the mitotic spindle, its composition and function are not fully understood. A more complete inventory of spindle components would therefore constitute an important advance. Here we describe the purification of human mitotic spindles and their analysis by MS/MS. We identified 151 proteins previously known to associate with the spindle apparatus, centrosomes, and/or kinetochores and 644 other proteins, including 154 uncharacterized components that did not show obvious homologies to known proteins and did not contain motifs indicative of a particular localization. Of these uncharacterized proteins, 17 were tagged and localized in transfected mitotic cells, resulting in the identification of six genuine spindle components (KIAA0008, CdcA8, KIAA1187, FLJ12649, FLJ90806, and C20Orf129). This study illustrates the strength of a proteomic approach for the analysis of isolated human spindles and identifies several novel spindle components for future functional studies. The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. Malfunctioning of the spindle can lead to chromosome missegregation and hence result in aneuploidy, a hallmark of most human cancers. Despite major progress in deciphering the temporal and spatial regulation of the mitotic spindle, its composition and function are not fully understood. A more complete inventory of spindle components would therefore constitute an important advance. Here we describe the purification of human mitotic spindles and their analysis by MS/MS. We identified 151 proteins previously known to associate with the spindle apparatus, centrosomes, and/or kinetochores and 644 other proteins, including 154 uncharacterized components that did not show obvious homologies to known proteins and did not contain motifs indicative of a particular localization. Of these uncharacterized proteins, 17 were tagged and localized in transfected mitotic cells, resulting in the identification of six genuine spindle components (KIAA0008, CdcA8, KIAA1187, FLJ12649, FLJ90806, and C20Orf129). This study illustrates the strength of a proteomic approach for the analysis of isolated human spindles and identifies several novel spindle components for future functional studies. During mitosis, the two newly forming daughter cells must receive one copy of each chromosome. To accomplish this task, the mitotic spindle pulls sister chromatids toward opposite poles of the dividing cell. This microtubule-based structure comprises dynamic polymers made of αβ-tubulin heterodimers, associated with a large variety of microtubule-associated proteins (1Kline-Smith S.L. Walczak C.E. Mitotic spindle assembly and chromosome segregation: refocusing on microtubule dynamics..Mol. Cell. 2004; 15: 317-327Google Scholar, 2Cassimeris L. Spittle C. Regulation of microtubule-associated proteins..Int. Rev. Cytol. 2001; 210: 163-226Google Scholar, 3Compton D.A. Spindle assembly in animal cells..Annu. Rev. Biochem. 2000; 69: 95-114Google Scholar, 4Nogales E. Structural insights into microtubule function..Annu. Rev. Biochem. 2000; 69: 277-302Google Scholar, 5Gadde S. Heald R. Mechanisms and molecules of the mitotic spindle..Curr. Biol. 2004; 14: R797-R805Google Scholar). At the transition from interphase to mitosis, the microtubule network undergoes a profound morphological change. In particular, the microtubule organizing centers of animal cells, the centrosomes, position to opposite sides of the nucleus and increase their microtubule nucleation capacity. Following nuclear envelope breakdown, microtubules emanating from the centrosomes capture each chromosome at the kinetochore, a protein complex assembled on centromeric DNA (6Biggins S. Walczak C.E. Captivating capture: How microtubules attach to kinetochores..Curr. Biol. 2003; 13: R449-R460Google Scholar). Appropriate bipolar attachment of sister chromatids is monitored by a surveillance mechanism, the spindle checkpoint (7Musacchio A. Hardwick K.G. The spindle checkpoint: Structural insights into dynamic signalling..Nat. Rev. Mol. Cell. Biol. 2002; 3: 731-741Google Scholar). Once all kinetochores are attached to microtubules emanating from opposite poles, the connection between sister chromatids is severed and chromatids are pulled apart. In addition to its central role in chromosome segregation, the mitotic spindle also determines the positioning and orientation of the cleavage plane. Therefore, proper positioning of the mitotic spindle is of particular importance for asymmetric cell divisions during development (8Glotzer M. Animal cell cytokinesis..Annu. Rev. Cell Dev. Biol. 2001; 17: 351-386Google Scholar, 9Scholey J.M. Brust-Mascher I. Mogilner A. Cell division..Nature. 2003; 422: 746-752Google Scholar). A large number of proteins associate with the mitotic spindle and regulate its dynamic formation and function. Stabilizing and destabilizing proteins control the high turnover rate of mitotic microtubules, which have a half-life of less than 60 s (3Compton D.A. Spindle assembly in animal cells..Annu. Rev. Biochem. 2000; 69: 95-114Google Scholar). Furthermore, motor proteins of the kinesin and dynein families play crucial roles in the formation of a bipolar mitotic spindle (10Sharp D.J. Rogers G.C. Scholey J.M. Microtubule motors in mitosis..Nature. 2000; 407: 41-47Google Scholar, 11Wittmann T. Hyman A. Desai A. The spindle: A dynamic assembly of microtubules and motors..Nat. Cell Biol. 2001; 3: E28-E34Google Scholar). By interacting with microtubules during early mitosis, they push the spindle poles apart, then play important roles in chromosome-capture by microtubules and power chromosome movement throughout mitosis. Finally, the spindle harbors several regulatory proteins, notably protein kinases and phosphatases (12Nigg E.A. Mitotic kinases as regulators of cell division and its checkpoints..Nat. Rev. Mol. Cell. Biol. 2001; 2: 21-32Google Scholar), which coordinate spindle function in time and space. Although our understanding of microtubule dynamics and spindle formation has greatly advanced during the last two decades, the complexity of the spindle continues to hamper its investigation. A more complete inventory of the mitotic spindle may thus contribute to a better understanding of how exactly the spindle is assembled, what role it plays in the spindle checkpoint, and how it induces cleavage furrow formation. Recent proteomic studies have begun to address the composition of the human centrosome (13Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling..Nature. 2003; 426: 570-574Google Scholar), the spindle midbody in hamster cells (14Skop A.R. Liu H. Yates III, J. Meyer B.J. Heald R. Dissection of the mammalian midbody proteome reveals conserved cytokinesis mechanisms..Science. 2004; 305: 61-66Google Scholar), and in vitro-assembled spindle structures (15Mack G.J. Compton D.A. Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein..Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14434-14439Google Scholar, 16Liska A.J. Popov A.V. Sunyaev S. Coughlin P. Habermann B. Shevchenko A. Bork P. Karsenti E. Shevchenko A. Homology-based functional proteomics by mass spectrometry: Application to the Xenopus microtubule-associated proteome..Proteomics. 2004; 4: 2707-2721Google Scholar). Here we describe a comprehensive proteomic study of human spindles isolated from synchronized HeLa S3 cells (17Hunt D.F. Michel H. Dickinson T.A. Shabanowitz J. Cox A.L. Sakaguchi K. Appella E. Grey H.M. Sette A. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad..Science. 1992; 256: 1817-1820Google Scholar, 18Peng J. Gygi S.P. Proteomics: The move to mixtures..J. Mass Spectrom. 2001; 36: 1083-1091Google Scholar, 19Wolters D.A. Washburn M.P. Yates III, J.R. . An automated multidimensional protein identification technology for shotgun proteomics..Anal. Chem. 2001; 73: 5683-5690Google Scholar). MS identified a total of 795 proteins, including 151 previously known spindle-associated components. Of 17 previously uncharacterized proteins analyzed further, six were found to be bona fide spindle components. HeLa S3 cells were maintained in Dulbeccos modified Eagles medium containing 10% FCS, 50 U/ml penicillin, and 50 μg/ml streptomycin. Cells were grown at 37 °C in a humidified incubator with a 5% CO2 atmosphere. HeLa S3 cells were first synchronized at the G1/S phase boundary by treatment for 16 h with 1.6 μg/ml aphidicolin. They were then released for 14 h into medium containing 40 ng/ml nocodazole to block them in mitosis. Mitotic cells were harvested by shake-off, followed by centrifugation at 300 × g, washed twice with PBS, and released into normal medium for 30–40 min, until most of them had reached metaphase. (This was controlled by immunofluorescence analysis of aliquots of 4,6-diamidino-2-phenylindole (DAPI) 1The abbreviations used are: DAPI4,6-diamidino-2-phenylindoleMTmicrotubuleDICdifferential interference contrastcccoiled-coilPIPESpiperazine-1,4-bis(2-ethanesulfonic acid). -stained cells for metaphase plate formation). Microtubules were subsequently stabilized by addition of 5 μg/ml taxol to the medium for 3 min. Cells were then harvested, washed with PBS containing 2 μg/ml latrunculin B, 1 mm PMSF, 5 μg/ml taxol, and then incubated for 15 min at 37 °C in lysis buffer: 100 mm piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), pH 6.9, 1 mm MgSO4, 2 mm EGTA, 0.5% Nonidet P-40, 5 μg/ml taxol, 2 μg/ml latrunculin B, including nucleases (200 μg/ml DNAseI, 10 μg/ml RNase A, 1 U/ml micrococcal nuclease, 20 U/ml benzoase), protease inhibitors (1 μg/ml pepstatin, 1 μl/ml leupeptin, 1 μg/ml aprotinin, 1 mm PMSF), and 20 mm β-glycerolphosphate as a phosphatase inhibitor. Lysed cells were harvested by centrifugation at 700 × g for 2 min and resuspended in the same buffer, incubated for 5 min, and harvested again by centrifugation. Spindles were subsequently isolated by incubating the lysed cell “ghosts” in isolation buffer (1 mm PIPES, pH 6.9, 5 μg/ml taxol) for 5–10 min (until differential interference contrast (DIC) microscopy revealed spindles to be free from intermediate filaments) and collected by sedimentation at 1,500 × g for 3 min. If required, this step was repeated once. Mitotic spindles were resuspended in 0.1 m glycine, pH 2.8, and sonified for 30 s in an ultrasonic water bath, followed by acetone precipitation. Proteins were then heated at 94 °C for 5 min in SDS-PAGE sample buffer before loading onto NuPAGE gradient gels (Invitrogen, San Diego, CA). 4,6-diamidino-2-phenylindole microtubule differential interference contrast coiled-coil piperazine-1,4-bis(2-ethanesulfonic acid). For MS analysis, NuPAGE gradient gels were used in a Bis-Tris buffer system according to the manufacturer’s instructions (Invitrogen). Gels were stained with 0.5% Coomassie in 50% methanol/10% acetic acid and destained in 50% methanol/10% acetic acid. For Western blotting, proteins were separated in homemade gels and subsequently transferred to nitrocellulose filters (Schleicher & Schuell, Keene, NH) using a semi-dry blotting device (Hoefer). Filters were blocked in PBS buffer containing 0.05% Tween 20 and 5% milk and subsequently probed with antibodies diluted in the same buffer. Antibodies used were: monoclonal anti-Plk1 (PL2) (culture supernatant 1:10) (20Golsteyn R.M. Schultz S.J. Bartek J. Ziemiecki A. Ried T. Nigg E.A. Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5..J. Cell Sci. 1994; 107: 1509-1517Google Scholar), monoclonal anti-α-tubulin (1:4,000; Sigma-Aldrich, St. Louis, MO), anti-Hec1 (1:1,000) (21Martin-Lluesma S. Stucke V.M. Nigg E.A. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2..Science. 2002; 297: 2267-2270Google Scholar), anti-Eg5 (2 μg/ml) (22Blangy A. Lane H.A. d’Herin P. Harper M. Kress M. Nigg E.A. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo..Cell. 1995; 83: 1159-1169Google Scholar), anti-Asf1 (3 μg/ml) (23Sillje H.H. Nigg E.A. Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases..Curr. Biol. 2001; Scholar). with antibodies were by was as A. M. Mann M. Mass of proteins Chem. Scholar), and were and using J. Mann M. and for and sample in proteomics..Anal. Chem. 2003; Scholar). The was analyzed with a system to a mass The was as by E. G.J. and to and protein Sci. 2002; Scholar). were in 5 of acid and by the onto a with phase at a rate of 2 The was to a pulled with This had an of and a of and was as by J. J.S. Mann M. with for A. 2002; Scholar). Peptides were separated by a gradient of between buffer A 0.5% acid) and buffer 0.5% acid) at a rate of the analysis MS were followed by in were the Mass the human using for and mass Proteins identified by two more with a of than 50 by one with a of than 60 were all proteins were of For the were from the and HeLa and/or from the DNA KIAA1187, The was assembled of two and and was assembled from and a from a HeLa CA). DNA were by using and into a with a an HeLa S3 cells were grown on and transfected with of DNA using according to the manufacturer’s h cells were washed with PBS and for 10 min in buffer mm PIPES, pH 10 mm EGTA, 1 mm were washed with PBS and incubated with anti-Hec1 followed by and in PBS buffer containing were washed with PBS and onto microscopy was with a II and an × were with a device and and were with To mitotic spindles from human cells to high and in for mass analysis, previously were Proteins associated with the microtubules of the mammalian mitotic Scholar, R. The mitotic spindle of hamster cells isolated in Cell Sci. Scholar, identification of microtubule-associated proteins by of Scholar). The the of microtubules by taxol and is in HeLa S3 cells were first synchronized at the G1/S phase transition by an Following from this cells were with of nocodazole microtubule these spindle cells were in as a result of spindle checkpoint The cells were then harvested by and released from the nocodazole block formation of mitotic most cells had reached a metaphase taxol was to To to be used as a high of nocodazole was resulting in microtubule Cells were then lysed and with to chromosomes from the microtubule network and with latrunculin to the which would the M. S.L. I. of by latrunculin Scholar). Finally, of the lysed cell “ghosts” in buffer in of the intermediate the mitotic spindles Spindles were collected by centrifugation. of the spindle by microscopy the of the 2 from and metaphase be free of other spindle structures be the isolation was in the of nocodazole not In analysis of the spindle on Coomassie gels a of and other proteins, with the control The of spindle components was by Western analysis an not of also of the microtubule-associated motor (22Blangy A. Lane H.A. d’Herin P. Harper M. Kress M. Nigg E.A. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo..Cell. 1995; 83: 1159-1169Google Scholar), the mitotic H.H. Nigg E.A. kinases and the of cell Rev. Mol. Cell. Biol. 2004; Scholar), and the kinetochore protein Hec1 (21Martin-Lluesma S. Stucke V.M. Nigg E.A. Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2..Science. 2002; 297: 2267-2270Google Scholar). of these proteins were in the control the chromatin assembly factors and (23Sillje H.H. Nigg E.A. Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases..Curr. Biol. 2001; analyzed as were from the spindle as For the identification of proteins the spindle proteins were separated by on a SDS-PAGE gradient Coomassie each was into 20 protein mass The proteins were then with and were identified by of several spindle in the identification of an for the of these 3 illustrates of one from and one from FLJ12649, two newly identified spindle proteins show and of thus protein To of the identified proteins, a was in and was for using a of the in the identification of a total of 795 proteins Of proteins were identified by more than proteins were identified by a and proteins were identified by two for the of the and were before the proteins were Of the 795 proteins, 151 were previously to associate with mitotic spindle structures and/or microtubules and hence be as genuine spindle components the previously known spindle proteins for of all proteins, the of the 30 proteins in the MS analysis were genuine spindle a high of spindles in the analyzed The of known spindle components not major proteins and Eg5, also regulatory proteins, including protein kinases and and phosphatases and components of the spindle checkpoint and This that the spindle analyzed proteins to kinetochores and centrosomes in addition to microtubule-associated the of the of the interphase centrosome were not which that centrosome proteins had to spindle were during spindle that centrosomes contribute a to the spindle, proteins may have to The proteins were into on the of and/or the of particular and/or with previously known acid proteins, proteins, 50 proteins, and a of proteins with and/or to The 154 previously uncharacterized proteins This was most to novel spindle components. This not that bona fide spindle components also be in the other For the comprises 10 and it is that of these associate with kinetochores during mitosis, as by studies on the nuclear I. A. M. J. The including is as one to kinetochores in Biol. Cell. 2004; 15: Scholar). In microtubules not play a role in sister segregation, also as major for the of E. Microtubules and microtubule-associated Cell Biol. 1995; Scholar, of with in assembled 14: Scholar), M. R. R. H. a novel microtubule motor protein for of 1994; A.J. B. S.J. association of a microtubule motor during in Cell Biol. Scholar), E. S. K. P. movement of in for and Biol. Cell. 2004; 15: Scholar), and localization and the Cell Biol. 2004; Scholar). the identification of a high number of and proteins that these proteins interact with the mitotic spindle in The that the spindle contain more proteins than a first step toward this several previously uncharacterized proteins were on the of 17 novel were for characterization were to a and transfected into HeLa S3 The tagged proteins were then localized by immunofluorescence Of the 17 proteins six localization to spindle structures 5 and For of these proteins, and their were by other in the of this to spindle microtubules and a that is found in a number of proteins Identification of a novel cell cycle in human 2003; Scholar). function is as in it a from the spindle was to a complex with the kinetochore proteins and and, as it with Hec1 at the kinetochores R. H. Identification of two novel components of the human kinetochore Biol. Chem. 2004; Scholar, Shabanowitz J. D.F. G.J. 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The chromosomal complex is for microtubule and spindle 2004; of 17 in a spindle localization of novel spindle proteins were in HeLa S3 cells for with the proteins were stained with and spindles were stained with was used to the DNA with are at the 5 The other novel spindle proteins identified FLJ12649, KIAA1187, and have not previously and localized to microtubules throughout and not proteins coiled-coil motifs with to structures in the microtubule-associated protein they are of these of and in microtubule in interphase cells not that these proteins to The novel localized to spindle microtubules with a toward the spindle poles this protein an and a with to a The of this protein were to be during A.J. Identification of in the human cell cycle and their in Biol. Cell. 2002; 13: Scholar), with a mitotic function for the of at spindle proteins is known to be cell cycle the analysis of constitute a for the of spindle The of this approach is by the of the approach not be to the class of 154 uncharacterized proteins, their in is A.J. Identification of in the human cell cycle and their in Biol. Cell. 2002; 13: Scholar, T. J.M. J. The in the of human to Scholar). were for of the 17 proteins that were Of proteins, six spindle protein and cell cycle regulation of their proteins did spindle proteins and and the A.J. Identification of in the human cell cycle and their in Biol. Cell. 2002; 13: Scholar, by Cell cycle 2001; Scholar). with it is to the of using cell cycle regulation of as a We have also the of Analysis of the structures of the 17 analyzed proteins that were this of the six newly identified spindle proteins all proteins identified in our spindle inventory were analyzed for the of the at we found that 50% of the known spindle proteins one more of the 644 other proteins this The of structures therefore to constitute a for the number of spindle proteins for future for was found to be more in known spindle proteins than in other components of our of the known spindle proteins in the of proteins with other were by more than two of the previously proteins that were for localization all of as bona fide spindle components were identified by more of the proteins that did not to the spindle were identified by one the number of identified for a protein may constitute a for which proteins are spindle components. is for all complex an inventory of the of the mitotic spindle an important for functional studies. analysis has to the identification of several novel spindle components and, in an of for future of the proteomic approach also it to the in spindle composition during mitotic that spindle proteins cell L. Spittle C. Regulation of microtubule-associated proteins..Int. Rev. Cytol. 2001; 210: 163-226Google Scholar), a better understanding of spindle regulation and dynamics would greatly from the identification and functional analysis of spindle components. notably and be to analysis of the spindle for a analysis of the temporal and spatial regulation of spindle assembly and function during cell We and for We the DNA and the for