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Dissecting the mechanisms of cell division

  • Joseph Y. Ong
    Affiliations
    Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095
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  • Jorge Z. Torres
    Correspondence
    To whom correspondence should be addressed. Tel.: 310-206-2092 ; Fax: 310-206-5213;.
    Affiliations
    Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095

    The Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, California 90095

    Molecular Biology Institute, UCLA, Los Angeles, California 90095
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  • Author Footnotes
    2 The abbreviations used are: CDKcyclin-dependent kinaseAPC/Canaphase-promoting complex/cyclosomeMPFmaturation-promoting factorROSreactive oxygen species.
Open AccessPublished:June 07, 2019DOI:https://doi.org/10.1074/jbc.AW119.008149
      Cell division is a highly regulated and carefully orchestrated process. Understanding the mechanisms that promote proper cell division is an important step toward unraveling important questions in cell biology and human health. Early studies seeking to dissect the mechanisms of cell division used classical genetics approaches to identify genes involved in mitosis and deployed biochemical approaches to isolate and identify proteins critical for cell division. These studies underscored that post-translational modifications and cyclin–kinase complexes play roles at the heart of the cell division program. Modern approaches for examining the mechanisms of cell division, including the use of high-throughput methods to study the effects of RNAi, cDNA, and chemical libraries, have evolved to encompass a larger biological and chemical space. Here, we outline some of the classical studies that established a foundation for the field and provide an overview of recent approaches that have advanced the study of cell division.

      Introduction to cell division

      Cell division, or mitosis, is the process by which a mother cell divides its nuclear and cytoplasmic components into two daughter cells. Mitosis is divided into four major phases: prophase, metaphase, anaphase, and telophase. Careful regulation of the cell division program is crucial for proper cell growth, development, and gametogenesis. Dysfunction or misregulation of cell division can lead to growth defects (
      • Tomkins D.J.
      • Sisken J.E.
      Abnormalities in the cell-division cycle in Roberts syndrome fibroblasts: a cellular basis for the phenotypic characteristics?.
      ,
      • Hung C.Y.
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      • Baker J.D.
      • Bauer J.W.
      • Gussoni E.
      • Hainzl S.
      • Klausegger A.
      • Lorenzo J.
      • Mihalek I.
      • Rittinger O.
      • Tekin M.
      • Dallman J.E.
      • Bodamer O.A.
      A defect in the inner kinetochore protein CENPT causes a new syndrome of severe growth failure.
      ) and proliferative diseases like cancer (
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of Cancer: the next generation.
      ) and aging-related diseases (
      • Macedo J.C.
      • Vaz S.
      • Bakker B.
      • Ribeiro R.
      • Bakker P.L.
      • Escandell J.M.
      • Ferreira M.G.
      • Medema R.
      • Foijer F.
      • Logarinho E.
      FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence.
      ), including Alzheimer's disease (
      • Yang Y.
      • Varvel N.H.
      • Lamb B.T.
      • Herrup K.
      Ectopic cell cycle events link human Alzheimer's disease and amyloid precursor protein transgenic mouse models.
      ). Therefore, analyses of the pathways and mechanisms that promote proper cell division are important avenues through which we can understand cell regulation and its misregulation in human disease.
      Cell division is driven by two main modes of post-translational modifications. First, protein kinases like cyclin-dependent kinases (CDKs)
      The abbreviations used are: CDK
      cyclin-dependent kinase
      APC/C
      anaphase-promoting complex/cyclosome
      MPF
      maturation-promoting factor
      ROS
      reactive oxygen species.
      (
      • Peter M.
      • Nakagawa J.
      • Dorée M.
      • Labbé J.C.
      • Nigg E.A.
      Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase.
      ,
      • Bischoff J.R.
      • Friedman P.N.
      • Marshak D.R.
      • Prives C.
      • Beach D.
      Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2.
      ) and Polo-like kinases (
      • Fu Z.
      • Malureanu L.
      • Huang J.
      • Wang W.
      • Li H.
      • van Deursen J.M.
      • Tindall D.J.
      • Chen J.
      Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progression.
      ) phosphorylate their substrates to modify their activity or stability; this modification is opposed by protein phosphatase–mediated dephosphorylation (for example, Cdc25 (
      • Lammer C.
      • Wagerer S.
      • Saffrich R.
      • Mertens D.
      • Ansorge W.
      • Hoffmann I.
      The cdc25B phosphatase is essential for the G2/M phase transition in human cells.
      ) and various PP2A (
      • Torres J.Z.
      • Ban K.H.
      • Jackson P.K.
      A specific form of phosphoprotein phosphatase 2 regulates anaphase-promoting complex/cyclosome association with spindle poles.
      ) complexes). Second, E3 ubiquitin ligases like the anaphase-promoting complex/cyclosome (APC/C) (
      • Davey N.E.
      • Morgan D.O.
      Building a regulatory network with short linear sequence motifs: lessons from the Degrons of the anaphase-promoting complex.
      ) and Cullin 1-based SCF (Skp-Cullin-F box) (
      • Yu Z.K.
      • Gervais J.L.
      • Zhang H.
      Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins.
      ) complexes ubiquitylate their substrates and target them for proteasomal degradation; this modification is opposed by deubiquitylases such as USP37 (
      • Huang X.
      • Summers M.K.
      • Pham V.
      • Lill J.R.
      • Liu J.
      • Lee G.
      • Kirkpatrick D.S.
      • Jackson P.K.
      • Fang G.
      • Dixit V.M.
      Deubiquitinase USP37 is activated By CDK2 to antagonize APCCDH1 and promote S phase entry.
      ) and Cezanne (
      • Bonacci T.
      • Suzuki A.
      • Grant G.D.
      • Stanley N.
      • Cook J.G.
      • Brown N.G.
      • Emanuele M.J.
      Cezanne/OTUD7B is a cell cycle-regulated deubiquitinase that antagonizes the degradation of APC/C substrates.
      ). Spatiotemporal control of when these post-translational modifications occur gives rise to the ordered events of cell division. Our current understanding of key regulators of cell division is founded upon many classical genetic and biochemical studies aimed at understanding the cell cycle. We begin by highlighting some of these seminal studies, transition to discussing modern techniques and approaches used to dissect the mechanisms of cell division, and conclude with future directions and perspectives on the cell division field.

      Classical studies of cell division: post-translational regulation

      Early cell cycle studies established that phosphorylation was important for cell division. These studies assessed the DNA content, size, and doubling time of mutant strains of the yeast Schizosaccharomyces pombe to identify genes, termed cell division cycle (cdc) genes (
      • Hartwell L.H.
      • Mortimer R.K.
      • Culotti J.
      • Culotti M.
      Genetic control of the cell division cycle in yeast: V. genetic analysis of cdc mutants.
      ). One of the first cdc genes to be characterized was cdc9-50, later renamed WEE1 (
      • Nurse P.
      Genetic control of cell size at cell division in yeast.
      ). WEE1-mutant yeast divided at a smaller size than their WT counterparts, suggesting that loss of Wee1p activity accelerated mitotic entry and that Wee1p was an inhibitor of mitosis. Later, it was discovered that overexpression of the S. pombe gene cdc25, determined to encode a protein phosphatase (
      • Lee M.S.
      • Ogg S.
      • Xu M.
      • Parker L.L.
      • Donoghue D.J.
      • Maller J.L.
      • Piwnica-Worms H.
      cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2.
      ), led to increased rates of mitotic entry (
      • Russell P.
      • Nurse P.
      cdc25+ functions as an inducer in the mitotic control of fission yeast.
      ). Moreover, Wee1p and Cdc25p worked in opposition to each other, suggesting a balancing act between these two proteins to regulate the initiation of mitosis (
      • Fantes P.
      Epistatic gene interactions in the control of division in fission yeast.
      ). The cloning of WEE1 indicated that it resembled a protein kinase (
      • Russell P.
      • Nurse P.
      Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog.
      ), suggesting that phosphorylation could regulate cell division. This analysis also suggested that a common substrate of Cdc25p and Wee1p was Cdc2p, a protein kinase (
      • Hindley J.
      • Phear G.A.
      Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases.
      ) known to be involved in the initiation of DNA replication (Cdc2p in S. pombe and Cdc28p in Saccharomyces cerevisiae, now known as CDK1 in humans) (
      • Conrad M.N.
      • Newlon C.S.
      Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome.
      ). The possibility that Wee1p and Cdc25p worked in opposition to each other at the biochemical level was later confirmed when it was shown that Wee1p phosphorylated and inactivated Cdc2p (
      • Lundgren K.
      • Walworth N.
      • Booher R.
      • Dembski M.
      • Kirschner M.
      • Beach D.
      mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2.
      ) and that Cdc25p dephosphorylated and activated Cdc2p (
      • Lee M.S.
      • Ogg S.
      • Xu M.
      • Parker L.L.
      • Donoghue D.J.
      • Maller J.L.
      • Piwnica-Worms H.
      cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2.
      ). Thus, the ability of Cdc2p to regulate mitotic entry depended on its phosphorylation state (
      • Gould K.L.
      • Nurse P.
      Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis.
      ), a theme that has now extended to other mitotic kinases.
      Meanwhile, parallel studies in frog oocytes demonstrated that a cytoplasmic substance, termed maturation-promoting factor (MPF), regulated the initiation of meiosis (
      • Hara K.
      • Tydeman P.
      • Kirschner M.
      A cytoplasmic clock with the same period as the division cycle in Xenopus eggs.
      ,
      • Masui Y.
      • Markert C.L.
      Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes.
      ). Curiously, the levels of MPF seemed to go up and down during the different phases of meiosis (
      • Gerhart J.
      • Wu M.
      • Kirschner M.
      Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs.
      ). Purification of MPF (
      • Lohka M.J.
      • Hayes M.K.
      • Maller J.L.
      Purification of maturation-promoting factor, an intracellular regulator of early mitotic events.
      ) suggested that this protein complex contained two proteins: a protein kinase of ∼32 kDa, later identified to be a homologue of S. pombe Cdc2p (
      • Dunphy W.G.
      • Brizuela L.
      • Beach D.
      • Newport J.
      The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis.
      ), and a protein of ∼45 kDa, later identified to be cyclin B (
      • Draetta G.
      • Luca F.
      • Westendorf J.
      • Brizuela L.
      • Ruderman J.
      • Beach D.
      cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF.
      ). The interaction between the kinase Cdc2p and cyclins, a class of proteins named because their protein levels cycled with each mitotic division in sea urchins and clams (
      • Evans T.
      • Rosenthal E.T.
      • Youngblom J.
      • Distel D.
      • Hunt T.
      Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division.
      ), became a key resource for understanding the mechanisms of cell division. The discovery of CDK2 and CDK2–cyclin A complexes (
      • Pines J.
      • Hunter T.
      Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B.
      ,
      • Tsai L.-H.
      • Harlow E.
      • Meyerson M.
      Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase.
      ) and Cdc2–cyclin A and Cdc2–cyclin B complexes (
      • Draetta G.
      • Luca F.
      • Westendorf J.
      • Brizuela L.
      • Ruderman J.
      • Beach D.
      cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF.
      ,
      • Labbé J.C.
      • Capony J.P.
      • Caput D.
      • Cavadore J.C.
      • Derancourt J.
      • Kaghad M.
      • Lelias J.M.
      • Picard A.
      • Dorée M.
      MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B.
      ) suggested that different cyclin-kinase pairs could regulate different aspects of mitotic entry and progression (
      • Pines J.
      • Hunter T.
      Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B.
      ). Subsequent studies in model organisms demonstrated that, among its many substrates, Cdc2 phosphorylated nuclear lamins for nuclear envelope breakdown (
      • Dessev G.
      • Iovcheva-Dessev C.
      • Bischoff J.R.
      • Beach D.
      • Goldman R.
      A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.
      ,
      • Peter M.
      • Nakagawa J.
      • Dorée M.
      • Labbé J.C.
      • Nigg E.A.
      In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase.
      ) and cytoskeletal elements for important morphological changes during mitosis (
      • Chou Y.H.
      • Ngai K.L.
      • Goldman R.
      The regulation of intermediate filament reorganization in mitosis. p34cdc2 phosphorylates vimentin at a unique N-terminal site.
      ,
      • Yamashiro S.
      • Yamakita Y.
      • Hosoya H.
      • Matsumura F.
      Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis.
      ). The ability of cyclins and their kinases to mediate mitotic entry and progression has become the engine that drives cell division.
      Similar to phosphorylation and protein kinases, ubiquitylation and E3 ubiquitin ligases play important roles in cell division (
      • Ong J.Y.
      • Torres J.Z.
      E3 ubiquitin ligases in cancer and their pharmacological targeting.
      ). For example, the cycling levels of cyclin B were partially explained by the ubiquitination (
      • Hershko A.
      • Ganoth D.
      • Pehrson J.
      • Palazzo R.E.
      • Cohen L.H.
      Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts.
      ,
      • Glotzer M.
      • Murray A.W.
      • Kirschner M.W.
      Cyclin is degraded by the ubiquitin pathway.
      ) and subsequent degradation of cyclin B by the APC/C (
      • King R.W.
      • Peters J.M.
      • Tugendreich S.
      • Rolfe M.
      • Hieter P.
      • Kirschner M.W.
      A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.
      ,
      • Sudakin V.
      • Ganoth D.
      • Dahan A.
      • Heller H.
      • Hershko J.
      • Luca F.C.
      • Ruderman J.V.
      • Hershko A.
      The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.
      ). Degradation of Emi1 (
      • Margottin-Goguet F.
      • Hsu J.Y.
      • Loktev A.
      • Hsieh H.M.
      • Reimann J.D.
      • Jackson P.K.
      Prophase destruction of Emi1 by the SCF (βTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase.
      ) and Wee1 (
      • Watanabe N.
      • Arai H.
      • Nishihara Y.
      • Taniguchi M.
      • Watanabe N.
      • Hunter T.
      • Osada H.
      M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP.
      ) via ubiquitylation of the Cul1-based SCF (Skp-Cullin-F box) complex is necessary for proper mitotic exit. Whereas phosphatases (such as Wee1 or PP2A (
      • Torres J.Z.
      • Ban K.H.
      • Jackson P.K.
      A specific form of phosphoprotein phosphatase 2 regulates anaphase-promoting complex/cyclosome association with spindle poles.
      )) have been well studied as antagonizers of cell division kinases, the role of deubiquitinating enzymes and the identification of their substrates remain to be fully explored (
      • Mapa C.E.
      • Arsenault H.E.
      • Conti M.M.
      • Poti K.E.
      • Benanti J.A.
      A balance of deubiquitinating enzymes controls cell cycle entry.
      ).
      Beyond these classical genetic and biochemical studies, modern approaches aimed at dissecting the mechanisms of cell division have greatly advanced our understanding of this dynamic process. Here, we present a broad overview of recent approaches that take a comprehensive and “-omics” view to identify novel components critical for cell division, to understand the function of the cell division machinery, and to analyze the pathways and other novel factors that contribute to cell division.

      Genetic dissection of cell division

      Although the aforementioned traditional yeast mutagenesis studies were seminal to the field of cell division, in the era of modern genomics, genetic analyses of cell division have become more targeted and efficient. The availability of RNAi and CRISPR-Cas9 gRNA (
      • McKinley K.L.
      • Cheeseman I.M.
      Large-scale analysis of CRISPR/Cas9 cell-cycle knockouts reveals the diversity of p53-dependent responses to cell-cycle defects.
      ) libraries has made studying gene expression knockdowns a viable option for discovering novel genes involved in cell division (Fig. 1, upper left). Approaches that screen these libraries are usually coupled with a high-throughput method of multiparametric data analysis, such as assessing mitotic progression via microscopy and DNA content or via the HeLa fluorescence ubiquitination cell cycle indicator (FUCCI) cell lines, which change color based on the cell cycle phase (
      • Sakaue-Sawano A.
      • Kurokawa H.
      • Morimura T.
      • Hanyu A.
      • Hama H.
      • Osawa H.
      • Kashiwagi S.
      • Fukami K.
      • Miyata T.
      • Miyoshi H.
      • Imamura T.
      • Ogawa M.
      • Masai H.
      • Miyawaki A.
      Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.
      ). As an example, our group performed an siRNA screen to assess the importance of ∼600 mitotic microtubule-associated proteins for their function in cell division and used high content imagers to quantify the mitotic index and apoptotic index of each knockdown (
      • Torres J.Z.
      • Summers M.K.
      • Peterson D.
      • Brauer M.J.
      • Lee J.
      • Senese S.
      • Gholkar A.A.
      • Lo Y.-C.
      • Lei X.
      • Jung K.
      • Anderson D.C.
      • Davis D.P.
      • Belmont L.
      • Jackson P.K.
      The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly.
      ). Through this approach, we discovered StarD9, a novel protein involved in centrosome cohesion and whose depletion led to a dynamic unstable mitotic arrest (
      • Torres J.Z.
      • Summers M.K.
      • Peterson D.
      • Brauer M.J.
      • Lee J.
      • Senese S.
      • Gholkar A.A.
      • Lo Y.-C.
      • Lei X.
      • Jung K.
      • Anderson D.C.
      • Davis D.P.
      • Belmont L.
      • Jackson P.K.
      The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly.
      ). Combined with microscopy and computer-aided imaging, siRNA screens have now analyzed the importance of ∼22,000 genes for cell division, uncovering novel proteins critical for this process (
      • Neumann B.
      • Walter T.
      • Hériché J.-K.
      • Bulkescher J.
      • Erfle H.
      • Conrad C.
      • Rogers P.
      • Poser I.
      • Held M.
      • Liebel U.
      • Cetin C.
      • Sieckmann F.
      • Pau G.
      • Kabbe R.
      • Wünsche A.
      • et al.
      Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
      ).
      Figure thumbnail gr1
      Figure 1Overview of approaches used to dissect the mechanisms of cell division. Multiple approaches have been used to dissect the mechanisms of cell division, including genetic, proteomic, chemical, structural, and computational approaches. Figure contains the structure of the MIND complex from Kluyveromyces lactis (
      • Dimitrova Y.N.
      • Jenni S.
      • Valverde R.
      • Khin Y.
      • Harrison S.C.
      Structure of the MIND complex defines a regulatory focus for yeast kinetochore assembly.
      ) (Protein Data Bank code 5T58 (
      • Berman H.M.
      • Westbrook J.
      • Feng Z.
      • Gilliland G.
      • Bhat T.N.
      • Weissig H.
      • Shindyalov I.N.
      • Bourne P.E.
      The Protein Data Bank.
      ), created using the NGL Viewer (
      • Rose A.S.
      • Bradley A.R.
      • Valasatava Y.
      • Duarte J.M.
      • Prlic A.
      • Rose P.W.
      NGL viewer: web-based molecular graphics for large complexes.
      )). Examples of Plk1-interacting proteins are Bub1 (
      • Jia L.
      • Li B.
      • Yu H.
      The Bub1–Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation.
      ), Cdh1 (
      • Bassermann F.
      • Frescas D.
      • Guardavaccaro D.
      • Busino L.
      • Peschiaroli A.
      • Pagano M.
      The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint.
      ), and Chk2 (
      • van Vugt M.A.
      • Gardino A.K.
      • Linding R.
      • Ostheimer G.J.
      • Reinhardt H.C.
      • Ong S.-E.
      • Tan C.S.
      • Miao H.
      • Keezer S.M.
      • Li J.
      • Pawson T.
      • Lewis T.A.
      • Carr S.A.
      • Smerdon S.J.
      • Brummelkamp T.R.
      • Yaffe M.B.
      A mitotic phosphorylation feedback network connects Cdk1, Plk1, 53BP1, and Chk2 to inactivate the G(2)/M DNA damage checkpoint.
      ). Examples of PLK1 substrates are FOXM1 (
      • Fu Z.
      • Malureanu L.
      • Huang J.
      • Wang W.
      • Li H.
      • van Deursen J.M.
      • Tindall D.J.
      • Chen J.
      Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progression.
      ), Cdc25C (
      • Toyoshima-Morimoto F.
      • Taniguchi E.
      • Nishida E.
      Plk1 promotes nuclear translocation of human Cdc25C during prophase.
      ), p150Glued (
      • Li H.
      • Liu X.S.
      • Yang X.
      • Song B.
      • Wang Y.
      • Liu X.
      Polo-like kinase 1 phosphorylation of p150Glued facilitates nuclear envelope breakdown during prophase.
      ), Myt1 (
      • Nakajima H.
      • Toyoshima-Morimoto F.
      • Taniguchi E.
      • Nishida E.
      Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate.
      ), and Wee1 (
      • Watanabe N.
      • Arai H.
      • Nishihara Y.
      • Taniguchi M.
      • Watanabe N.
      • Hunter T.
      • Osada H.
      M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP.
      ).
      Similarly, expression of fluorescently-tagged fusion proteins, by transfecting vectors encoding cDNAs (
      • Torres J.Z.
      • Miller J.J.
      • Jackson P.K.
      High-throughput generation of tagged stable cell lines for proteomic analysis.
      ) or bacterial artificial chromosomes containing a gene with its endogenous promoter (
      • Hutchins J.R.
      • Toyoda Y.
      • Hegemann B.
      • Poser I.
      • Hériché J.-K.
      • Sykora M.M.
      • Augsburg M.
      • Hudecz O.
      • Buschhorn B.A.
      • Bulkescher J.
      • Conrad C.
      • Comartin D.
      • Schleiffer A.
      • Sarov M.
      • Pozniakovsky A.
      • et al.
      Systematic analysis of human protein complexes identifies chromosome segregation proteins.
      ), has enabled the identification of novel cell division proteins. The use of a fluorescently-tagged protein allows for an easy visual analysis for whether the protein has a relevant localization, such as at the kinetochores during mitosis, and is particularly useful when an antibody for the protein of interest is unavailable, either because the protein of interest is novel or because commercially available antibodies could not be validated. Combined with other analyses, such as proteomic data, these approaches have been used to identify novel protein complexes and pathways, such as a subunit of the APC/C (
      • Hutchins J.R.
      • Toyoda Y.
      • Hegemann B.
      • Poser I.
      • Hériché J.-K.
      • Sykora M.M.
      • Augsburg M.
      • Hudecz O.
      • Buschhorn B.A.
      • Bulkescher J.
      • Conrad C.
      • Comartin D.
      • Schleiffer A.
      • Sarov M.
      • Pozniakovsky A.
      • et al.
      Systematic analysis of human protein complexes identifies chromosome segregation proteins.
      ), the MOZART family of tubulin-associated proteins (
      • Hutchins J.R.
      • Toyoda Y.
      • Hegemann B.
      • Poser I.
      • Hériché J.-K.
      • Sykora M.M.
      • Augsburg M.
      • Hudecz O.
      • Buschhorn B.A.
      • Bulkescher J.
      • Conrad C.
      • Comartin D.
      • Schleiffer A.
      • Sarov M.
      • Pozniakovsky A.
      • et al.
      Systematic analysis of human protein complexes identifies chromosome segregation proteins.
      ), and the katanin family of microtubule-severing enzymes (
      • Cheung K.
      • Senese S.
      • Kuang J.
      • Bui N.
      • Ongpipattanakul C.
      • Gholkar A.
      • Cohn W.
      • Capri J.
      • Whitelegge J.P.
      • Torres J.Z.
      Proteomic analysis of the mammalian Katanin family of microtubule-severing enzymes defines Katanin p80 subunit B-like 1 (KATNBL1) as a regulator of mammalian Katanin microtubule-severing.
      ).
      Together, these genetic approaches have defined a parts list of the critical factors that are required for proper cell division. Importantly, they have allowed for the dissection of key cell division processes like centrosome homeostasis, early mitotic spindle assembly, spindle assembly checkpoint function, and cytokinesis. These studies have also aided the understanding of human genetic diseases, like developmental disorders and cancers, that have cell division dysregulation at the core of their pathophysiology.

      Proteomic dissection of cell division

      Classical yeast two-hybrid screens have been used to identify novel protein–protein interactions (
      • Jeong A.L.
      • Lee S.
      • Park J.S.
      • Han S.
      • Jang C.-Y.
      • Lim J.-S.
      • Lee M.S.
      • Yang Y.
      Cancerous inhibitor of protein phosphatase 2A (CIP2A) protein is involved in centrosome separation through the regulation of NIMA (never in mitosis gene A)-related kinase 2 (NEK2) protein activity.
      ,
      • Hwang L.H.
      • Lau L.F.
      • Smith D.L.
      • Mistrot C.A.
      • Hardwick K.G.
      • Hwang E.S.
      • Amon A.
      • Murray A.W.
      Budding yeast Cdc20: a target of the spindle checkpoint.
      ) and to define key domains or amino acids necessary for protein–protein interactions (Fig. 1, upper left) (
      • Vidal M.
      • Braun P.
      • Chen E.
      • Boeke J.D.
      • Harlow E.
      Genetic characterization of a mammalian protein–protein interaction domain by using a yeast reverse two-hybrid system.
      ). However, modern proteomic approaches have greatly expanded the identification of novel protein–protein interactions and protein complexes involved in cell division. We outline two main approaches to the proteomic mapping of cell division: first, affinity-based purifications, based on the strength of protein–protein interactions; and second, proximity-based purifications, based on the spatiotemporal localization of the protein of interest. In affinity purifications, a tagged protein is expressed within cells, and the protein complexes are immunoprecipitated via antibodies that target the protein tag and are analyzed by MS (Fig. 1, upper right) (
      • Torres J.Z.
      • Miller J.J.
      • Jackson P.K.
      High-throughput generation of tagged stable cell lines for proteomic analysis.
      ,
      • Bradley M.
      • Ramirez I.
      • Cheung K.
      • Gholkar A.A.
      • Torres J.Z.
      Inducible LAP-tagged stable cell lines for investigating protein function, spatiotemporal localization and protein interaction networks.
      ). We have used this approach to study various protein complexes of the cell division machinery, including enzymes that regulate the length of the mitotic spindle (
      • Xia X.
      • Gholkar A.
      • Senese S.
      • Torres J.Z.
      A LCMT1-PME-1 methylation equilibrium controls mitotic spindle size.
      ), ubiquitylation complexes that regulate cytokinesis (
      • Gholkar A.A.
      • Senese S.
      • Lo Y.-C.
      • Vides E.
      • Contreras E.
      • Hodara E.
      • Capri J.
      • Whitelegge J.P.
      • Torres J.Z.
      The X-linked-intellectual-disability-associated ubiquitin ligase Mid2 interacts with astrin and regulates astrin levels to promote cell division.
      ), novel light chains of the dynein machinery (
      • Gholkar A.A.
      • Senese S.
      • Lo Y.-C.
      • Capri J.
      • Deardorff W.J.
      • Dharmarajan H.
      • Contreras E.
      • Hodara E.
      • Whitelegge J.P.
      • Jackson P.K.
      • Torres J.Z.
      Tctex1d2 associates with short-rib polydactyly syndrome proteins and is required for ciliogenesis.
      ), and a novel kinesin involved in centrosome cohesion (
      • Torres J.Z.
      • Summers M.K.
      • Peterson D.
      • Brauer M.J.
      • Lee J.
      • Senese S.
      • Gholkar A.A.
      • Lo Y.-C.
      • Lei X.
      • Jung K.
      • Anderson D.C.
      • Davis D.P.
      • Belmont L.
      • Jackson P.K.
      The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly.
      ,
      • Senese S.
      • Cheung K.
      • Lo Y.-C.
      • Gholkar A.A.
      • Xia X.
      • Wohlschlegel J.A.
      • Torres J.Z.
      A unique insertion in STARD9's motor domain regulates its stability.
      ,
      • Torres J.Z.
      STARD9/Kif16a is a novel mitotic kinesin and antimitotic target.
      ). In proximity-based purifications, the protein of interest is tagged with a labeling enzyme such as a BirA biotin-ligase mutant called BioID (
      • Roux K.J.
      • Kim D.I.
      • Raida M.
      • Burke B.
      A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.
      ) (or its derivatives BioID2 (
      • Kim D.I.
      • Jensen S.C.
      • Noble K.A.
      • Kc B.
      • Roux K.H.
      • Motamedchaboki K.
      • Roux K.J.
      An improved smaller biotin ligase for BioID proximity labeling.
      ), TurboID, or miniTurboID (
      • Branon T.C.
      • Bosch J.A.
      • Sanchez A.D.
      • Udeshi N.D.
      • Svinkina T.
      • Carr S.A.
      • Feldman J.L.
      • Perrimon N.
      • Ting A.Y.
      Efficient proximity labeling in living cells and organisms with TurboID.
      )) or a peroxide-based enzyme APEX (
      • Rhee H.-W.
      • Zou P.
      • Udeshi N.D.
      • Martell J.D.
      • Mootha V.K.
      • Carr S.A.
      • Ting A.Y.
      Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.
      ). Upon addition of the small ligand biotin to the cell culture media, these labeling enzymes modify proximal proteins with biotin via accessible lysine residues. Following the labeling step, the cells are lysed in denaturing conditions, biotinylated proteins are immunoprecipitated by binding to streptavidin beads, and protein complexes are analyzed by MS. Examples of proximity-based approaches include the identification of CDK1 protein interactors (
      • Schopp I.M.
      • Amaya Ramirez C.C.
      • Debeljak J.
      • Kreibich E.
      • Skribbe M.
      • Wild K.
      • Béthune J.
      Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes.
      ) and the spatial mapping of protein–protein associations within the centrosome (
      • Firat-Karalar E.N.
      • Rauniyar N.
      • Yates 3rd., J.R.
      • Stearns T.
      • Stearns T.
      Proximity interactions among centrosome components identify regulators of centriole duplication.
      ).
      Our group has been interested not only in defining novel components of the cell division machinery but also how these components interact with each other in a spatiotemporal manner. The mapping of cell division protein–protein interactions has been and will continue to be important for understanding how the cell division machinery coordinates to execute cell division with high fidelity. For example, protein interactors of a mitotic protein kinase could represent components of a protein complex, regulators of its activity or localization, and/or substrates for modification. Therefore, cell division protein–protein interaction networks are critical for defining protein function and more broadly how these proteins affect a specific pathway within the cell division program.

      Chemical dissection of cell division

      Natural and synthetic small molecules that target the cell division machinery are useful research tools that can be used in an acute and temporal manner to dissect the mechanisms of cell division. They can also serve as lead molecules for the development of therapeutics for treating proliferative diseases like cancer. However, these compounds have shown limited use in clinical trials, emphasizing the need to discover new or improved compounds and/or more viable biological targets. Moreover, many critical regulators of cell division have no specific inhibitors, hindering research to improve our understanding of their function and their potential as disease drug targets. Therefore, much progress needs to be made in the discovery and development of small molecule inhibitors and modulators of cell division proteins.
      Recently, we developed a novel cell-based high-throughput chemical screening platform for the discovery of cell cycle phase-specific inhibitors that utilize chemical cell cycle profiling (
      • Senese S.
      • Lo Y.C.
      • Huang D.
      • Zangle T.A.
      • Gholkar A.A.
      • Robert L.
      • Homet B.
      • Ribas A.
      • Summers M.K.
      • Teitell M.A.
      • Damoiseaux R.
      • Torres J.Z.
      Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development.
      ,
      • Lo Y.-C.
      • Senese S.
      • France B.
      • Gholkar A.A.
      • Damoiseaux R.
      • Torres J.Z.
      Computational cell cycle profiling of cancer cells for prioritizing FDA-approved drugs with repurposing potential.
      ). Using this approach we analyzed the cell cycle response of cancer cells to each of ∼80,000 drug-like molecules (Fig. 1, lower left) (
      • Senese S.
      • Lo Y.C.
      • Huang D.
      • Zangle T.A.
      • Gholkar A.A.
      • Robert L.
      • Homet B.
      • Ribas A.
      • Summers M.K.
      • Teitell M.A.
      • Damoiseaux R.
      • Torres J.Z.
      Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development.
      ). This screen identified novel inhibitors of each cell cycle phase. Coupled with our computational program CSNAP (Chemical Similarity Network Analysis Pulldown) that relates chemical properties to biological activity (
      • Lo Y.-C.
      • Senese S.
      • Li C.-M.
      • Hu Q.
      • Huang Y.
      • Damoiseaux R.
      • Torres J.Z.
      Large-scale chemical similarity networks for target profiling of compounds identified in cell-based chemical screens.
      ,
      • Lo Y.-C.
      • Senese S.
      • Damoiseaux R.
      • Torres J.Z.
      3D chemical similarity networks for structure-based target prediction and scaffold hopping.
      ), this screen presented 266 compounds that impeded cell division and identified many potential biological targets. As an example of the utility of this method, we demonstrated that the novel compound MI-181 was a microtubule destabilizer like colchicine, bound near the colchicine-binding pocket (
      • McNamara D.E.
      • Senese S.
      • Yeates T.O.
      • Torres J.Z.
      Structures of potent anticancer compounds bound to tubulin.
      ), and had a potency and efficacy similar to taxol (
      • Senese S.
      • Lo Y.C.
      • Huang D.
      • Zangle T.A.
      • Gholkar A.A.
      • Robert L.
      • Homet B.
      • Ribas A.
      • Summers M.K.
      • Teitell M.A.
      • Damoiseaux R.
      • Torres J.Z.
      Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development.
      ). Recently, we screened more than 180,000 chemical compounds and found a small molecule that arrested leukemia cells in G2 and triggered an apoptotic cell death (
      • Xia X.
      • Lo Y.-C.
      • Gholkar A.A.
      • Senese S.
      • Ong J.Y.
      • Velasquez E.F.
      • Damoiseaux R.
      • Torres J.Z.
      Leukemia cell cycle chemical profiling identifies the G2-phase leukemia specific inhibitor leusin-1.
      ). Similarly, chemical screens have also been used to identify APC/C inhibitors (
      • Verma R.
      • Peters N.R.
      • D'Onofrio M.
      • Tochtrop G.P.
      • Sakamoto K.M.
      • Varadan R.
      • Zhang M.
      • Coffino P.
      • Fushman D.
      • Deshaies R.J.
      • King R.W.
      Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.
      ) and mitotic kinase inhibitors (for example, Plk1 (
      • Steegmaier M.
      • Hoffmann M.
      • Baum A.
      • Lénárt P.
      • Petronczki M.
      • Krssák M.
      • Gürtler U.
      • Garin-Chesa P.
      • Lieb S.
      • Quant J.
      • Grauert M.
      • Adolf G.R.
      • Kraut N.
      • Peters J.-M.
      • Rettig W.J.
      BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo.
      ) and Aurora kinases (
      • Ditchfield C.
      • Johnson V.L.
      • Tighe A.
      • Ellston R.
      • Haworth C.
      • Johnson T.
      • Mortlock A.
      • Keen N.
      • Taylor S.S.
      Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores.
      )) that have been used to study their corresponding protein’s functions in spindle assembly and the spindle-assembly checkpoint (Aurora kinase B (
      • Gadea B.B.
      • Ruderman J.V.
      Aurora kinase inhibitor ZM447439 blocks chromosome-induced spindle assembly, the completion of chromosome condensation, and the establishment of the spindle integrity checkpoint in Xenopus egg extracts.
      ), Plk1 (
      • Lénárt P.
      • Petronczki M.
      • Steegmaier M.
      • Di Fiore B.
      • Lipp J.J.
      • Hoffmann M.
      • Rettig W.J.
      • Kraut N.
      • Peters J.-M.
      The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.
      ), and APC/C (
      • Zeng X.
      • Sigoillot F.
      • Gaur S.
      • Choi S.
      • Pfaff K.L.
      • Oh D.-C.
      • Hathaway N.
      • Dimova N.
      • Cuny G.D.
      • King R.W.
      Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage.
      )).
      Although much work has been done to chemically dissect cell division, much work lies ahead to define inhibitors of the cell division machinery. Importantly, most chemical studies have focused on structure-based approaches, which rely on the prior identification of key cell division enzymes through genetic approaches and an understanding of their 3D structure. High-throughput phenotypic chemical profiling of cell division pathways is still lacking. Additionally, new synthetic and natural chemical libraries with broad chemical space continue to become available and represent opportunities for the discovery of molecules that will enable researchers to interrogate cell division. Finally, much effort has been invested in targeting the active site of mitotic enzymes, but the targeting of key protein–protein interactions with small molecules like peptidomimetics has been lagging.

      Structural dissection of cell division

      Studies into the structure of key proteins and protein complexes in cell division have elucidated key mechanisms in the assembly and function of the cell division machinery. Although there have been many important structural studies, we focus on Mad2, one of the key regulators of the spindle assembly checkpoint. Structural studies have been particularly useful in elucidating the role of Mad2 within cell division because Mad2 function depends on its structure. Using NMR studies, it was discovered that Mad2 alternates between two main structural conformations, an open (O-Mad2) and a closed (C-Mad2) state, differing mainly at the C-terminal tail (
      • Luo X.
      • Tang Z.
      • Rizo J.
      • Yu H.
      The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20.
      ,
      • Luo X.
      • Tang Z.
      • Xia G.
      • Wassmann K.
      • Matsumoto T.
      • Rizo J.
      • Yu H.
      The Mad2 spindle checkpoint protein has two distinct natively folded states.
      ). Only C-Mad2 is active and able to bind to Mad1 and Cdc20. Conversion of O-Mad2 to C-Mad2 requires the formation of an O-/C-Mad2 heterodimer (
      • Hara M.
      • Özkan E.
      • Sun H.
      • Yu H.
      • Luo X.
      Structure of an intermediate conformer of the spindle checkpoint protein Mad2.
      ,
      • Yang M.
      • Li B.
      • Liu C.-J.
      • Tomchick D.R.
      • Machius M.
      • Rizo J.
      • Yu H.
      • Luo X.
      Insights into Mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric Mad2 dimer.
      • Hewitt L.
      • Tighe A.
      • Santaguida S.
      • White A.M.
      • Jones C.D.
      • Musacchio A.
      • Green S.
      • Taylor S.S.
      Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1–C-Mad2 core complex.
      ). Crystal structures of Mad2–Mad1 complexes demonstrated a flexible C-terminal tail termed the “safety belt” or “hinge loop” (
      • Dimitrova Y.N.
      • Jenni S.
      • Valverde R.
      • Khin Y.
      • Harrison S.C.
      Structure of the MIND complex defines a regulatory focus for yeast kinetochore assembly.
      ) involved in regulating C-Mad2 binding to Mad1 (
      • Sironi L.
      • Mapelli M.
      • Knapp S.
      • De Antoni A.
      • Jeang K.-T.
      • Musacchio A.
      Crystal structure of the tetrameric Mad1-Mad2 core complex: implications of a “safety belt” binding mechanism for the spindle checkpoint.
      ) and Cdc20 (
      • Chao W.C.
      • Kulkarni K.
      • Zhang Z.
      • Kong E.H.
      • Barford D.
      Structure of the mitotic checkpoint complex.
      ) and prohibiting the metaphase–anaphase transition. These structural studies helped elucidate the means by which Mad2 functions within the mitotic checkpoint complex.
      Increasing developments in cryo-EM have allowed for more complex structures to be elucidated. For example, cryo-EM studies have solved the structure of the APC/C (
      • Chang L.F.
      • Zhang Z.
      • Yang J.
      • McLaughlin S.H.
      • Barford D.
      Molecular architecture and mechanism of the anaphase-promoting complex.
      ,
      • Chang L.
      • Zhang Z.
      • Yang J.
      • McLaughlin S.H.
      • Barford D.
      Atomic structure of the APC/C and its mechanism of protein ubiquitination.
      ), helping to explain the purpose of both APC/C-binding E2 ubiquitin–conjugating enzymes Ube2c and Ube2s (
      • Brown N.G.
      • VanderLinden R.
      • Watson E.R.
      • Weissmann F.
      • Ordureau A.
      • Wu K.-P.
      • Zhang W.
      • Yu S.
      • Mercredi P.Y.
      • Harrison J.S.
      • Davidson I.F.
      • Qiao R.
      • Lu Y.
      • Dube P.
      • Brunner M.R.
      • et al.
      Dual RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by APC/C.
      ) and clarifying the mechanism of Mad2 inhibition of the APC/C (
      • Yamaguchi M.
      • VanderLinden R.
      • Weissmann F.
      • Qiao R.
      • Dube P.
      • Brown N.G.
      • Haselbach D.
      • Zhang W.
      • Sidhu S.S.
      • Peters J.-M.
      • Stark H.
      • Schulman B.A.
      Cryo-EM of mitotic checkpoint complex-bound APC/C reveals reciprocal and conformational regulation of ubiquitin ligation.
      ). Moreover, complex structures like the kinetochores have also been visualized by cryo-EM. Although traditional X-ray crystallography methods have been used to solve the structures of some kinetochore complexes, such as the MIND complex (
      • Dimitrova Y.N.
      • Jenni S.
      • Valverde R.
      • Khin Y.
      • Harrison S.C.
      Structure of the MIND complex defines a regulatory focus for yeast kinetochore assembly.
      ) in Fig. 1 (lower middle), cryo-EM structures of yeast kinetochores and kinetochore-associated proteins in situ (
      • Ng C.T.
      • Deng L.
      • Chen C.
      • Lim H.H.
      • Shi J.
      • Surana U.
      • Gan L.
      Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situ.
      ), purified from yeast (
      • Gonen S.
      • Akiyoshi B.
      • Iadanza M.G.
      • Shi D.
      • Duggan N.
      • Biggins S.
      • Gonen T.
      The structure of purified kinetochores reveals multiple microtubule-attachment sites.
      ), or reassembled in vitro (
      • Hinshaw S.M.
      • Harrison S.C.
      The structure of the Ctf19c/CCAN from budding yeast.
      ) have elucidated the composition, geometry, and assembly and disassembly of eukaryotic kinetochores. Structural information, particularly of large structures like the kinetochores or centrosomes, is important for understanding the protein complexes formed during mitosis and for developing small molecules that can disrupt these interactions.

      Computational dissection of cell division

      Computational and mathematical approaches to study cell division have complemented and informed biochemical and biological techniques. One of the earliest attempts toward rationalizing mitotic entry suggested that, because of feedback loops between Cdc2, its activator Cdc25, and its inhibitor Wee1, Cdc2 activity should oscillate within the cell cycle as a function of cyclin concentration (
      • Novak B.
      • Tyson J.J.
      Numerical analysis of a comprehensive model of M-phase control in Xenopus oocyte extracts and intact embryos.
      ). These models were later confirmed by experiments that revealed Cdc2 exhibited hysteresis and bistability: regulation of Cdc2 prevents premature mitotic exit because a higher concentration of cyclin B is needed to enter mitosis than to maintain a mitotic state (
      • Pomerening J.R.
      • Sontag E.D.
      • Ferrell J.E.
      Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2.
      ,
      • Sha W.
      • Moore J.
      • Chen K.
      • Lassaletta A.D.
      • Yi C.-S.
      • Tyson J.J.
      • Sible J.C.
      Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts.
      ). A mathematical model that assessed cell growth as a function of protein kinase activity (
      • Chen K.C.
      • Calzone L.
      • Csikasz-Nagy A.
      • Cross F.R.
      • Novak B.
      • Tyson J.J.
      Integrative analysis of cell cycle control in budding yeast.
      ) suggested that an unknown phosphatase might regulate Nek1, a phosphatase later identified to be PP2A–Cdc55 (
      • Queralt E.
      • Lehane C.
      • Novak B.
      • Uhlmann F.
      Downregulation of PP2ACdc55 phosphatase by separase initiates mitotic exit in budding yeast.
      ). Other mathematical models have taken similar approaches to assess the roles of the spindle assembly checkpoint components relative to checkpoint function (
      • Henze R.
      • Dittrich P.
      • Ibrahim B.
      A dynamical model for activating and silencing the mitotic checkpoint.
      ,
      • Mistry H.B.
      • MacCallum D.E.
      • Jackson R.C.
      • Chaplain M.A.
      • Davidson F.A.
      Modeling the temporal evolution of the spindle assembly checkpoint and role of Aurora B kinase.
      ) and to model the mitotic spindle as a function of biophysical forces (
      • Civelekoglu-Scholey G.
      • Sharp D.J.
      • Mogilner A.
      • Scholey J.M.
      Model of chromosome motility in Drosophila embryos: adaptation of a general mechanism for rapid mitosis.
      ) and microtubule dynamics and cell size (
      • Lacroix B.
      • Letort G.
      • Pitayu L.
      • Sallé J.
      • Stefanutti M.
      • Maton G.
      • Ladouceur A.-M.
      • Canman J.C.
      • Maddox P.S.
      • Maddox A.S.
      • Minc N.
      • Nédélec F.
      • Dumont J.
      Microtubule dynamics scale with cell size to set spindle length and assembly timing.
      ).
      Computational techniques to glean information from time-lapse imaging of cell division have also been developed. With the advent of advanced imaging software and fluorescently-tagged proteins, researchers have generated spatiotemporal data about protein localization and concentration, resulting in information about protein complex assembly and disassembly (
      • Cai Y.
      • Hossain M.J.
      • Hériché J.-K.
      • Politi A.Z.
      • Walther N.
      • Koch B.
      • Wachsmuth M.
      • Nijmeijer B.
      • Kueblbeck M.
      • Martinic-Kavur M.
      • Ladurner R.
      • Alexander S.
      • Peters J.-M.
      • Ellenberg J.
      Experimental and computational framework for a dynamic protein atlas of human cell division.
      ). Combining datasets from different proteins allowed for the prediction of protein complexes and for the assessment of protein stoichiometry within a complex. Among other results, this imaging technique enabled the quantification of the number of cohesion molecules on DNA during mitosis, confirmed a 1:1 stoichiometry of Aurora kinase B and Borealin, and visualized Aurora kinase B localization to the cytokinetic bridge (
      • Cai Y.
      • Hossain M.J.
      • Hériché J.-K.
      • Politi A.Z.
      • Walther N.
      • Koch B.
      • Wachsmuth M.
      • Nijmeijer B.
      • Kueblbeck M.
      • Martinic-Kavur M.
      • Ladurner R.
      • Alexander S.
      • Peters J.-M.
      • Ellenberg J.
      Experimental and computational framework for a dynamic protein atlas of human cell division.
      ).
      Beyond microscopy, computational approaches have also been used to discover novel substrates of mitotic protein kinases. The basic structure of these algorithms is to use sequence information of known phosphorylation sites to identify a consensus phosphorylation motif and predict novel substrates, as outlined in Fig. 1 (lower right) for Plk1. Many computational tools that expand on this basic approach have been published (
      • Ayati M.
      • Wiredja D.
      • Schlatzer D.
      • Maxwell S.
      • Li M.
      • Koyutürk M.
      • Chance M.R.
      CoPhosK: a method for comprehensive kinase substrate annotation using co-phosphorylation analysis.
      ,
      • Song J.
      • Wang H.
      • Wang J.
      • Leier A.
      • Marquez-Lago T.
      • Yang B.
      • Zhang Z.
      • Akutsu T.
      • Webb G.I.
      • Daly R.J.
      PhosphoPredict: a bioinformatics tool for prediction of human kinase-specific phosphorylation substrates and sites by integrating heterogeneous feature selection.
      ); we highlight a recent study that identified SPICE1 as an Aurora kinase A substrate via a computational algorithm and validated the interaction via biochemistry (
      • Deretic J.
      • Kerr A.
      • Welburn J.P.I.
      A rapid computational approach identifies SPICE1 as an Aurora kinase substrate.
      ).
      Given the wealth of information generated by chemical, proteomic, and genetic screens and cheminformatics and bioinformatics analyses, there is a pressing need to develop computational methods to integrate and analyze these data. In regard to this, our group recently used computational cell cycle profiling for prioritizing Food and Drug Administration–approved drugs with the potential for repurposing as anticancer therapies (
      • Lo Y.-C.
      • Senese S.
      • France B.
      • Gholkar A.A.
      • Damoiseaux R.
      • Torres J.Z.
      Computational cell cycle profiling of cancer cells for prioritizing FDA-approved drugs with repurposing potential.
      ). Methods like this that combine and synthesize data sets from multiple sources into multiparametric analyses will become increasingly critical for developing a comprehensive view of cell division and how best to target it for therapeutic purposes.

      Future perspectives

      Although much has been discovered about the mechanisms that drive cell division, many novel factors that play a role in cell division are still being discovered. For example, endogenous RNA interference (RNAi) has been shown to regulate the expression of cell division proteins like Plk1 (
      • Wang Z.-D.
      • Shen L.-P.
      • Chang C.
      • Zhang X.-Q.
      • Chen Z.-M.
      • Li L.
      • Chen H.
      • Zhou P.-K.
      Long noncoding RNA lnc-RI is a new regulator of mitosis via targeting miRNA-210–3p to release PLK1 mRNA activity.
      ,
      • Shi W.
      • Alajez N.M.
      • Bastianutto C.
      • Hui A.B.
      • Mocanu J.D.
      • Ito E.
      • Busson P.
      • Lo K.-W.
      • Ng R.
      • Waldron J.
      • O'Sullivan B.
      • Liu F.-F.
      Significance of Plk1 regulation by miR-100 in human nasopharyngeal cancer.
      ), Mad1 (
      • Bhattacharjya S.
      • Nath S.
      • Ghose J.
      • Maiti G.P.
      • Biswas N.
      • Bandyopadhyay S.
      • Panda C.K.
      • Bhattacharyya N.P.
      • Roychoudhury S.
      miR-125b promotes cell death by targeting spindle assembly checkpoint gene MAD1 and modulating mitotic progression.
      ), Bub1 (
      • Luo M.
      • Weng Y.
      • Tang J.
      • Hu M.
      • Liu Q.
      • Jiang F.
      • Yang D.
      • Liu C.
      • Zhan X.
      • Song P.
      • Bai H.
      • Li B.
      • Shi Q.
      MicroRNA-450a-3p represses cell proliferation and regulates embryo development by regulating Bub1 expression in mouse.
      ), and Aurora kinase B (
      • Mäki-Jouppila J.H.
      • Pruikkonen S.
      • Tambe M.B.
      • Aure M.R.
      • Halonen T.
      • Salmela A.-L.
      • Laine L.
      • Børresen-Dale A.-L.
      • Kallio M.J.
      MicroRNA let-7b regulates genomic balance by targeting Aurora B kinase.
      ). Many other RNAi have been shown to affect at least one aspect of cell division (
      • Hwang W.-L.
      • Jiang J.-K.
      • Yang S.-H.
      • Huang T.-S.
      • Lan H.-Y.
      • Teng H.-W.
      • Yang C.-Y.
      • Tsai Y.-P.
      • Lin C.-H.
      • Wang H.-W.
      • Yang M.-H.
      MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells.
      • Roy S.
      • Hooiveld G.J.
      • Seehawer M.
      • Caruso S.
      • Heinzmann F.
      • Schneider A.T.
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      • Sonntag R.
      • Luedde M.
      • Trautwein C.
      • Stein I.
      • Pikarsky E.
      • Loosen S.
      • Tacke F.
      • et al.
      microRNA 193a-5p regulates levels of nucleolar- and spindle-associated protein 1 to suppress hepatocarcinogenesis.
      ,
      • Takacs C.M.
      • Giraldez A.J.
      miR-430 regulates oriented cell division during neural tube development in zebrafish.
      ,
      • Pruikkonen S.
      • Kallio M.J.
      Excess of a Rassf1-targeting microRNA, miR-193a-3p, perturbs cell division fidelity.
      • Kriegel A.J.
      • Terhune S.S.
      • Greene A.S.
      • Noon K.R.
      • Pereckas M.S.
      • Liang M.
      Isomer-specific effect of microRNA miR-29b on nuclear morphology.
      ), and some have no identified targets (
      • Stein P.
      • Rozhkov N.V.
      • Li F.
      • Cárdenas F.L.
      • Davydenko O.
      • Vandivier L.E.
      • Gregory B.D.
      • Hannon G.J.
      • Schultz R.M.
      • Schultz R.M.
      Essential role for endogenous siRNAs during meiosis in mouse oocytes.
      ). Given the clinical importance of these RNAi and the therapeutic potential of exogenous RNAi, a systematic understanding of how different forms of RNAi influence the proteins involved in cell division may help uncover novel levels of regulation for cell division.
      In addition to RNAi, small molecules and reactive oxygen species (ROS) have been shown to play critical roles in mitotic progression. For example, folate deficiency leads to replicative stress during DNA replication and consequently to mis-segregation defects during mitosis (
      • Bjerregaard V.A.
      • Garribba L.
      • McMurray C.T.
      • Hickson I.D.
      • Liu Y.
      Folate deficiency drives mitotic missegregation of the human FRAXA locus.
      ). Similarly, the lipid family of phosphoinositides was shown to directly influence mitotic progression through proteins like NuMA (
      • Kotak S.
      • Busso C.
      • Gönczy P.
      NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane.
      ) or phosphatases (
      • Sierra Potchanant E.A.
      • Cerabona D.
      • Sater Z.A.
      • He Y.
      • Sun Z.
      • Gehlhausen J.
      • Nalepa G.
      INPP5E preserves genomic stability through regulation of mitosis.
      ) and by regulating cytoskeletal elements (
      • Zheng P.
      • Baibakov B.
      • Wang X.-H.
      • Dean J.
      PtdIns(3,4,5)P3 is constitutively synthesized and required for spindle translocation during meiosis in mouse oocytes.
      ,
      • Tuncay H.
      • Brinkmann B.F.
      • Steinbacher T.
      • Schürmann A.
      • Gerke V.
      • Iden S.
      • Ebnet K.
      JAM-A regulates cortical dynein localization through Cdc42 to control planar spindle orientation during mitosis.
      ). Sterols have also been shown to play a role in cell division: cells deprived of cholesterol have difficulty undergoing cytokinesis (
      • Fernández C.
      • Lobo
      • Md Mdel V.
      • Gómez-Coronado D.
      • Lasunción M.A.
      Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation.
      ), and the cholesterol derivative pregnenolone localizes to the spindle poles, binds Shugoshin 1, and promotes centriole cohesion (
      • Hamasaki M.
      • Matsumura S.
      • Satou A.
      • Takahashi C.
      • Oda Y.
      • Higashiura C.
      • Ishihama Y.
      • Toyoshima F.
      Pregnenolone functions in centriole cohesion during mitosis.
      ). In S. pombe, intracellular concentrations of glucose affect Wee1 activity and thus cell size at mitotic entry (
      • Allard C.A.H.
      • Opalko H.E.
      • Moseley J.B.
      Stable Pom1 clusters form a glucose-modulated concentration gradient that regulates mitotic entry.
      ). Whether glucose or other metabolites serve roles during mitosis in human cells is largely unexplored. Interestingly, in cancer cells, ROS levels are elevated during mitosis, leading to an increased oxidation of biomolecules, but the functional implications of this oxidation, if any, are unknown (
      • Patterson J.C.
      • Joughin B.A.
      • van de Kooij B.
      • Lim D.C.
      • Lauffenburger D.A.
      • Yaffe M.B.
      ROS and oxidative stress are elevated in mitosis during asynchronous cell cycle progression and are exacerbated by mitotic arrest.
      ). Thus, comprehensive metabolomic, lipidomic, and nucleic acid studies of cell division are likely to yield interesting and previously underappreciated biological aspects of cell division (Fig. 1, upper middle).

      Concluding remarks

      Methods to dissect the mechanisms that govern cell division have progressed rapidly over the last few decades. The strategies discussed here allow for a genome- or proteome-wide assessment of proteins, drugs, and small molecules involved in cell division. In addition, advances in structural biology and computation have aided the study of cell division, particularly with regard to complex structures that are difficult to study with traditional biochemical techniques. Altogether, these approaches have allowed for the discovery and study of the ensemble of proteins and other factors necessary for proper cell division.

      Acknowledgments

      We thank the Journal of Biological Chemistry for artistic help in constructing Fig. 1.

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