CAP Profiles

Bio

Academic Appointments

Administrative Appointments

  • Chairperson, Department of Biochemistry (2019 - Present)
  • Senator, Stanford University Faculty Senate (2019 - Present)
  • Chairperson, Committee on Graduate Admissions and Policy (2017 - 2018)

Honors & Awards

  • Mayent-Rothschild Institute Curie Award, Institute Curie (2019)
  • Stanford Faculty Excellence in Teaching Award, Stanford Medical School (2014)
  • American Cancer Society Research Scholar, American Cancer Society (2011-2014)
  • Gordon Family Scholar of the Damon Runyon Foundation, Damon Runyon Cancer Research Foundation (2005-2007)
  • Damon Runyon Postdoctoral Fellow, Damon Runyon Cancer Research Foundation (1998-2001)
  • Dartmouth College Senior Fellow, Dartmouth College (1989)

Boards, Advisory Committees, Professional Organizations

  • External Scientific Advisor, 4D Nucleome Project - NIH Common Fund (2015 - Present)
  • Fellowship Award Committee, Damon Runyon Cancer Research Foundation (2018 - Present)
  • Scientific Advisory Board, LifeTime Initiative (2018 - Present)

Professional Education

  • BA, Dartmouth College, Biology (1989)
  • Ph.D., University of California at San Francisco, Biochemistry (1998)
  • Post-Doctoral Fellow, Harvard Medical School, Cell Biology (2003)

Contact

Links

Research & Scholarship

Current Research and Scholarly Interests

Our goal is to understand how chromosomes are faithfully transmitted during cell division and how chromosomes are structured and organized in the nucleus. Many cellular processes act on the chromosome to specialize different chromosomal domains for unique functions in the biology of cells. One of the best examples of a specialized chromatin domain is the eukaryotic centromere and kinetochore that forms at a single site on each human chromosome and ensures the proper segregation of the genome during each cell division cycle. The laboratory studies the genetic and epigenetic mechanisms that control the formation of human centromeres and kinetochores. Cells use a variety of different mechanisms to change the function of chromosomes. We have also focused on understanding how RNAs that associate with chromosomes regulate the reorganization of chromatin into silent heterochromatic domains. Our recent efforts have been directed at understanding how long range interactions between chromosomes are used to organize the genome within the nucleus and to control gene expression and chromosome dynamics. We use a combination of digital microscopy to extract quantitative information about the dynamics of chromosomes in living cells, biochemical reconstitution to assemble chromatin and chromosomes in vitro, and genetics to manipulate the chromosome segregation process in order to study how chromosome-distribution systems function in eukaryotes.

Teaching

2019-20 Courses

Stanford Advisees

Graduate and Fellowship Programs

Publications

All Publications

  • Mapping Transcriptome-Wide and Genome-Wide RNA-DNA Contacts with Chromatin-Associated RNA Sequencing (ChAR-seq). Methods in molecular biology (Clifton, N.J.) Limouse, C., Jukam, D., Smith, O. K., Fryer, K. A., Straight, A. F. 2020; 2161: 115–42

    Abstract

    RNAs play key roles in the cell as molecular intermediates for protein synthesis and as regulators of nuclear processes such as splicing, posttranscriptional regulation, or chromatin remodeling. Various classes of non-coding RNAs, including long non-coding RNAs (lncRNAs), can bind chromatin either directly or via interaction with chromatin binding proteins. It has been proposed that lncRNAs regulate cell-state-specific genes by coordinating the locus-dependent activity of chromatin-modifying complexes. Yet, the vast majority of lncRNAs have unknown functions, and we know little about the specific loci they regulate. A key step toward understanding chromatin regulation by RNAs is to map the genomic loci with which every nuclear RNA interacts and, reciprocally, to identify all RNAs that target a given locus. Our ability to generate such data has been limited, until recently, by the lack of methods to probe the genomic localization of more than a few RNAs at a time. Here, we describe a protocol for ChAR-seq, an RNA-DNA proximity ligation method that maps the binding loci for thousands of RNAs at once and without the need for specific RNA or DNA probe sequences. The ChAR-seq approach generates chimeric RNA-DNA molecules in situ and then converts those chimeras to DNA for next-generation sequencing. Using ChAR-seq we detect many types of chromatin-associated RNA, both coding and non-coding. Understanding the RNA-DNA interactome and its changes during differentiation or disease with ChAR-seq will likely provide key insights into chromatin and RNA biology.

    View details for DOI 10.1007/978-1-0716-0680-3_10

    View details for PubMedID 32681510

  • Chromatin-Associated RNA Sequencing (ChAR-seq). Current protocols in molecular biology Jukam, D., Limouse, C., Smith, O. K., Risca, V. I., Bell, J. C., Straight, A. F. 2019: e87

    Abstract

    RNA is a fundamental component of chromatin. Noncoding RNAs (ncRNAs) can associate with chromatin to influence gene expression and chromatin state; many also act at long distances from their transcriptional origin. Yet we know almost nothing about the functions or sites of action for most ncRNAs. Current methods to identify sites of RNA interaction with the genome are limited to the study of a single RNA at a time. Here we describe a protocol for ChAR-seq, a strategy to identify all chromatin-associated RNAs and map their DNA contacts genome-wide. In ChAR-seq, proximity ligation of RNA and DNA to a linker molecule is used to construct a chimeric RNA-DNA molecule that is converted to DNA for sequencing. In a single assay, ChAR-seq can discover de novo chromatin interactions of distinct RNAs, including nascent transcripts, splicing RNAs, and long noncoding RNAs (lncRNAs). Resulting "maps" of genome-bound RNAs should provide new insights into RNA biology. © 2019 by John Wiley & Sons, Inc.

    View details for PubMedID 30786161

  • CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization. The EMBO journal French, B. T., Straight, A. F. 2019

    Abstract

    Chromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. New CENP-A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP-A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP-A assembly is mediated in part by the three-protein Mis18 complex (Mis18alpha, Mis18beta, M18BP1). Here, we show that Xenopus M18BP1 localizes to centromeres during metaphase-prior to CENP-A assembly-by binding to CENP-C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP-C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP-C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP-A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase.

    View details for PubMedID 30606714

  • Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts ELIFE Bell, J. C., Jukam, D., Teran, N. A., Risca, V. I., Smith, O. K., Johnson, W. L., Skotheim, J. M., Greenleaf, W., Straight, A. F. 2018; 7

    Abstract

    RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

    View details for PubMedID 29648534

  • Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS BIOPHYSICAL JOURNAL Limouse, C., Bell, J. C., Fuller, C. J., Straight, A. F., Mabuchi, H. 2018; 114 (7): 1539–50

    Abstract

    Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level (∼100-1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.

    View details for PubMedID 29642025

    View details for PubMedCentralID PMC5954409

  • Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell MOLECULAR BIOLOGY OF THE CELL Cao, S., Zhou, K., Zhang, Z., Luger, K., Straight, A. F. 2018; 29 (6): 751–62

    Abstract

    Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

    View details for PubMedID 29343552

  • Centromere and Kinetochore Assembly in Xenopus laevis Egg Extract. Cold Spring Harbor protocols Flores Servin, J. C., Straight, A. F. 2018

    Abstract

    During cell division, chromosomes must be equally segregated to daughter cells. Centromeres, the primary interaction site between chromosomes and microtubules, mediate faithful chromosome segregation during mitosis. Functional studies of centromere proteins in cells have proven difficult, as mutation or deletion of most centromeric proteins often results in cell lethality. In this protocol, sperm chromatin or reconstituted chromatin arrays, together with Xenopus laevis egg extracts, are used to overcome these limitations and study centromere and kinetochore assembly in vitro. X. laevis egg extract is a powerful tool, as it can be readily cycled in vitro by addition of calcium and easily modified biochemically. Coupled with the addition of customizable reconstituted chromatin arrays or sperm chromatin, X. laevis egg extract provides distinct advantages over cell-based approaches in which similar experiments would not be feasible. Following incubation in egg extract, reconstituted centromeric chromatin arrays and sperm chromatin specifically assemble core centromere and kinetochore components that can be analyzed via immunofluorescence.

    View details for PubMedID 29475995

  • RNA-mediated regulation of heterochromatin CURRENT OPINION IN CELL BIOLOGY Johnson, W. L., Straight, A. F. 2017; 46: 102–9

    Abstract

    The formation of condensed, transcriptionally repressed heterochromatin is essential for controlling gene expression throughout development, silencing parasitic DNA elements, and for genome stability and inheritance. Cells employ diverse mechanisms for controlling heterochromatin states through proteins that modify DNA and histones. An emerging theme is that chromatin-associated RNAs play important roles in regulating heterochromatin proteins by controlling their initial recruitment to chromatin, their stable association with chromatin, their spread along chromatin, or their enzymatic activity. Major challenges for the field include not only identifying regulatory RNAs, but understanding the underlying biochemical mechanisms for how RNAs associate with chromatin, the specificity of interactions between heterochromatin proteins and RNA, and how these binding events manifest in cells to orchestrate RNA-mediated regulation of heterochromatin.

    View details for PubMedID 28614747

    View details for PubMedCentralID PMC5729926

  • Form and function of topologically associating genomic domains in budding yeast PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Eser, U., Chandler-Brown, D., Ay, F., Straight, A. F., Duang, Z., Noble, W. S., Skotheim, J. M. 2017; 114 (15): E3061-E3070

    Abstract

    The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained budding yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the long-known effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the budding yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins.

    View details for DOI 10.1073/pnas.1612256114

    View details for Web of Science ID 000398789800011

    View details for PubMedID 28348222

    View details for PubMedCentralID PMC5393236

  • Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping. Nature Risca, V. I., Denny, S. K., Straight, A. F., Greenleaf, W. J. 2017; 541 (7636): 237-241

    Abstract

    Chromatin structure at the length scale encompassing local nucleosome-nucleosome interactions is thought to play a crucial role in regulating transcription and access to DNA. However, this secondary structure of chromatin remains poorly understood compared with the primary structure of single nucleosomes or the tertiary structure of long-range looping interactions. Here we report the first genome-wide map of chromatin conformation in human cells at the 1-3 nucleosome (50-500 bp) scale, obtained using ionizing radiation-induced spatially correlated cleavage of DNA with sequencing (RICC-seq) to identify DNA-DNA contacts that are spatially proximal. Unbiased analysis of RICC-seq signal reveals regional enrichment of DNA fragments characteristic of alternating rather than adjacent nucleosome interactions in tri-nucleosome units, particularly in H3K9me3-marked heterochromatin. We infer differences in the likelihood of nucleosome-nucleosome contacts among open chromatin, H3K27me3-marked, and H3K9me3-marked repressed chromatin regions. After calibrating RICC-seq signal to three-dimensional distances, we show that compact two-start helical fibre structures with stacked alternating nucleosomes are consistent with RICC-seq fragmentation patterns from H3K9me3-marked chromatin, while non-compact structures and solenoid structures are consistent with open chromatin. Our data support a model of chromatin architecture in intact interphase nuclei consistent with variable longitudinal compaction of two-start helical fibres.

    View details for DOI 10.1038/nature20781

    View details for PubMedID 28024297

  • Editorial overview: The cell nucleus: New discoveries on nuclear structure, dynamics and function. Current opinion in cell biology Foisner, R., Straight, A. F. 2017; 46: iv-vi

    View details for PubMedID 28693757

  • The Power of Xenopus Egg Extract for Reconstitution of Centromere and Kinetochore Function. Progress in molecular and subcellular biology French, B. T., Straight, A. F. 2017; 56: 59–84

    Abstract

    Faithful transmission of genetic information during cell division requires attachment of chromosomes to the mitotic spindle via the kinetochore. In vitro reconstitution studies are beginning to uncover how the kinetochore is assembled upon the underlying centromere, how the kinetochore couples chromosome movement to microtubule dynamics, and how cells ensure the site of kinetochore assembly is maintained from one generation to the next. Here we give special emphasis to advances made in Xenopus egg extract, which provides a unique, biochemically tractable in vitro system that affords the complexity of cytoplasm and nucleoplasm to permit reconstitution of the dynamic, cell cycle-regulated functions of the centromere and kinetochore.

    View details for PubMedID 28840233

  • The Power of Xenopus Egg Extract for Reconstitution of Centromere and Kinetochore Function Centromeres and Kinetochores French, B. T., Straight, A. F. edited by Black, B. E. Springer. 2017: 59–84

    View details for DOI 10.1007/978-3-319-58592-5_3

  • Xenopus laevis M18BP1 Directly Binds Existing CENP-A Nucleosomes to Promote Centromeric Chromatin Assembly. Developmental cell French, B. T., Westhorpe, F. G., Limouse, C., Straight, A. F. 2017; 42 (2): 190–99.e10

    Abstract

    Vertebrate centromeres are epigenetically defined by nucleosomes containing the histone H3 variant, CENP-A. CENP-A nucleosome assembly requires the three-protein Mis18 complex (Mis18α, Mis18β, and M18BP1) that recruits the CENP-A chaperone HJURP to centromeres, but how the Mis18 complex recognizes centromeric chromatin is unknown. Using Xenopus egg extract, we show that direct, cell-cycle-regulated binding of M18BP1 to CENP-A nucleosomes recruits the Mis18 complex to interphase centromeres to promote new CENP-A nucleosome assembly. We demonstrate that Xenopus M18BP1 binds CENP-A nucleosomes using a motif that is widely conserved except in mammals. The M18BP1 motif resembles a CENP-A nucleosome binding motif in CENP-C, and we show that CENP-C competes with M18BP1 for CENP-A nucleosome binding at centromeres. We show that both CENP-C and M18BP1 recruit HJURP to centromeres for new CENP-A assembly. This study defines cellular mechanisms for recruiting CENP-A assembly factors to existing CENP-A nucleosomes for the epigenetic inheritance of centromeres.

    View details for PubMedID 28743005

    View details for PubMedCentralID PMC5544353

  • RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin. eLife Johnson, W. L., Yewdell, W. T., Bell, J. C., McNulty, S. M., Duda, Z., O'Neill, R. J., Sullivan, B. A., Straight, A. F. 2017; 6

    Abstract

    Heterochromatin formed by the SUV39 histone methyltransferases represses transcription from repetitive DNA sequences and ensures genomic stability. How SUV39 enzymes localize to their target genomic loci remains unclear. Here, we demonstrate that chromatin-associated RNA contributes to the stable association of SUV39H1 with constitutive heterochromatin in human cells. We find that RNA associated with mitotic chromosomes is concentrated at pericentric heterochromatin, and is encoded, in part, by repetitive α-satellite sequences, which are retained in cis at their transcription sites. Purified SUV39H1 directly binds nucleic acids through its chromodomain; and in cells, SUV39H1 associates with α-satellite RNA transcripts. Furthermore, nucleic acid binding mutants destabilize the association of SUV39H1 with chromatin in mitotic and interphase cells - effects that can be recapitulated by RNase treatment or RNA polymerase inhibition - and cause defects in heterochromatin function. Collectively, our findings uncover a previously unrealized function for chromatin-associated RNA in regulating constitutive heterochromatin in human cells.

    View details for PubMedID 28760200

    View details for PubMedCentralID PMC5538822

  • Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres. Nature communications Shang, W., Hori, T., Westhorpe, F. G., Godek, K. M., Toyoda, A., Misu, S., Monma, N., Ikeo, K., Carroll, C. W., Takami, Y., Fujiyama, A., Kimura, H., Straight, A. F., Fukagawa, T. 2016; 7: 13465-?

    Abstract

    Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A-H4-HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48-Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A-H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres.

    View details for DOI 10.1038/ncomms13465

    View details for PubMedID 27811920

    View details for PubMedCentralID PMC5097169

  • MIS12/MIND Control at the Kinetochore. Cell Ladurner, R., Straight, A. F. 2016; 167 (4): 889-891

    Abstract

    Kinetochores are complex multiprotein machines that link chromosomes to dynamic microtubules for chromosome segregation. Two studies in Cell reveal the structure of the human MIS12 and budding yeast MIND kinetochore complexes and the regulatory mechanisms that enable them to link chromosomes to microtubules during mitosis.

    View details for DOI 10.1016/j.cell.2016.10.036

    View details for PubMedID 27814517

  • In Vitro Kinetochore Assembly. Methods in molecular biology (Clifton, N.J.) Miell, M. D., Straight, A. F. 2016; 1413: 111-133

    Abstract

    The kinetochore is the primary site of interaction between chromosomes and microtubules of the mitotic spindle during chromosome segregation. Kinetochores are composed of more than 100 proteins that transiently assemble during mitosis at a single epigenetically defined region on each chromosome, known as the centromere. Kinetochore assembly and activity must be tightly regulated to ensure proper microtubule interaction and faithful chromosome segregation. Kinetochore malfunction can result in chromosome segregation defects leading to aneuploidy and cell death. As such, cell free and reconstituted systems to analyze kinetochore formation and function are invaluable in probing the biochemical activities of kinetochores. In vitro approaches to studying kinetochores have enabled the manipulation of kinetochore protein structure, function, interactions, and regulation that are not possible in cells. Here we outline a cell-free approach for the assembly of centromeres and recruitment of functional kinetochores that enables their manipulation and analysis.

    View details for DOI 10.1007/978-1-4939-3542-0_8

    View details for PubMedID 27193846

    View details for PubMedCentralID PMC4956618

  • A cell-free CENP-A assembly system defines the chromatin requirements for centromere maintenance JOURNAL OF CELL BIOLOGY Westhorpe, F. G., Fuller, C. J., Straight, A. F. 2015; 209 (6): 789-801

    Abstract

    Centromeres are defined by the presence of CENP-A nucleosomes in chromatin and are essential for accurate chromosome segregation. Centromeric chromatin epigenetically seeds new CENP-A nucleosome formation, thereby maintaining functional centromeres as cells divide. The features within centromeric chromatin that direct new CENP-A assembly remain unclear. Here, we developed a cell-free CENP-A assembly system that enabled the study of chromatin-bound CENP-A and soluble CENP-A separately. We show that two distinct domains of CENP-A within existing CENP-A nucleosomes are required for new CENP-A assembly and that CENP-A nucleosomes recruit the CENP-A assembly factors CENP-C and M18BP1 independently. Furthermore, we demonstrate that the mechanism of CENP-C recruitment to centromeres is dependent on the density of underlying CENP-A nucleosomes.

    View details for DOI 10.1083/jcb.201503132

    View details for Web of Science ID 000356998200005

    View details for PubMedID 26076692

    View details for PubMedCentralID PMC4477859

  • Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition. Proceedings of the National Academy of Sciences of the United States of America Amodeo, A. A., Jukam, D., Straight, A. F., Skotheim, J. M. 2015; 112 (10): E1086-95

    Abstract

    During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.

    View details for DOI 10.1073/pnas.1413990112

    View details for PubMedID 25713373

    View details for PubMedCentralID PMC4364222

  • Regulating the timing of CENP-A nucleosome assembly by phosphorylation. Developmental cell Miell, M. D., Straight, A. F. 2015; 32 (1): 1-2

    Abstract

    In this issue of Developmental Cell, Yu et al. (2015) demonstrate that CENP-A phosphorylation by CDK1 inhibits its association with the chaperone protein HJURP and that the removal of this modification at mitotic exit is a key regulatory event that controls the timing of new CENP-A nucleosome formation at centromeres.

    View details for DOI 10.1016/j.devcel.2014.12.020

    View details for PubMedID 25584791

  • The centromere: epigenetic control of chromosome segregation during mitosis. Cold Spring Harbor perspectives in biology Westhorpe, F. G., Straight, A. F. 2015; 7 (1)

    View details for DOI 10.1101/cshperspect.a015818

    View details for PubMedID 25414369

  • The centromere: epigenetic control of chromosome segregation during mitosis. Cold Spring Harbor perspectives in biology Westhorpe, F. G., Straight, A. F. 2014; 7 (1)

    Abstract

    A fundamental challenge for the survival of all organisms is maintaining the integrity of the genome in all cells. Cells must therefore segregate their replicated genome equally during each cell division. Eukaryotic organisms package their genome into a number of physically distinct chromosomes, which replicate during S phase and condense during prophase of mitosis to form paired sister chromatids. During mitosis, cells form a physical connection between each sister chromatid and microtubules of the mitotic spindle, which segregate one copy of each chromatid to each new daughter cell. The centromere is the DNA locus on each chromosome that creates the site of this connection. In this review, we present a brief history of centromere research and discuss our current knowledge of centromere establishment, maintenance, composition, structure, and function in mitosis.

    View details for DOI 10.1101/cshperspect.a015818

    View details for PubMedID 25414369

  • Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma NATURE GENETICS Lee, C. S., Bhaduri, A., Mah, A., Johnson, W. L., Ungewickell, A., Aros, C. J., Nguyen, C. B., Rios, E. J., Siprashvili, Z., Straight, A., Kim, J., Aasi, S. Z., Khavari, P. A. 2014; 46 (10): 1060-1062

    Abstract

    Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.

    View details for DOI 10.1038/ng.3091

    View details for Web of Science ID 000342554100007

    View details for PubMedCentralID PMC4324615

  • Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma. Nature genetics Lee, C. S., Bhaduri, A., Mah, A., Johnson, W. L., Ungewickell, A., Aros, C. J., Nguyen, C. B., Rios, E. J., Siprashvili, Z., Straight, A., Kim, J., Aasi, S. Z., Khavari, P. A. 2014; 46 (10): 1060-1062

    Abstract

    Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.

    View details for DOI 10.1038/ng.3091

    View details for PubMedID 25194279

  • Reply to "CENP-A octamers do not confer a reduction in nucleosome height by AFM". Nature structural & molecular biology Miell, M. D., Straight, A. F., Allshire, R. C. 2014; 21 (1): 5-8

    View details for DOI 10.1038/nsmb.2744

    View details for PubMedID 24389543

  • Swapping CENP-A at the centromere. Nature cell biology French, B. T., Straight, A. F. 2013; 15 (9): 1028-1030

    Abstract

    Faithful genome segregation depends on the functions of the eukaryotic centromere, which is characterized by the histone variant CENP-A. Gene replacement in human cells and fission yeast has now been used to show how CENP-A biochemically encodes centromere identity, as well as reveal an unexpected role for CENP-B in centromere function.

    View details for DOI 10.1038/ncb2833

    View details for PubMedID 23999616

  • CENP-A confers a reduction in height on octameric nucleosomes. Nature structural & molecular biology Miell, M. D., Fuller, C. J., Guse, A., Barysz, H. M., Downes, A., Owen-Hughes, T., Rappsilber, J., Straight, A. F., Allshire, R. C. 2013; 20 (6): 763-765

    Abstract

    Nucleosomes with histone H3 replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes from human and Drosophila have been reported to have reduced heights as compared to canonical octameric H3 nucleosomes, thus suggesting a unique tetrameric hemisomal composition. We demonstrate that octameric CENP-A nucleosomes assembled in vitro exhibit reduced heights, indicating that they are physically distinct from H3 nucleosomes and negating the need to invoke the presence of hemisomes.

    View details for DOI 10.1038/nsmb.2574

    View details for PubMedID 23644598

  • Functions of the centromere and kinetochore in chromosome segregation. Current opinion in cell biology Westhorpe, F. G., Straight, A. F. 2013; 25 (3): 334-340

    Abstract

    Centromeres play essential roles in equal chromosome segregation by directing the assembly of the microtubule binding kinetochore and serving as the cohesion site between sister chromatids. Here, we review the significant recent progress in our understanding of centromere protein assembly and how centromere proteins form the foundation of the kinetochore.

    View details for DOI 10.1016/j.ceb.2013.02.001

    View details for PubMedID 23490282

  • A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C. Science Kato, H., Jiang, J., Zhou, B., Rozendaal, M., Feng, H., Ghirlando, R., Xiao, T. S., Straight, A. F., Bai, Y. 2013; 340 (6136): 1110-1113

    Abstract

    Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Kinetochore assembly depends on specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A and H2B. We further found that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through hydrophobic interactions facilitated by nucleosome docking.

    View details for DOI 10.1126/science.1235532

    View details for PubMedID 23723239

  • Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant CHROMOSOME RESEARCH Earnshaw, W. C., Allshire, R. C., Black, B. E., Bloom, K., Brinkley, B. R., BROWN, W., Cheeseman, I. M., Choo, K. H., Copenhaver, G. P., DeLuca, J. G., Desai, A., Diekmann, S., Erhardt, S., Fitzgerald-Hayes, M., Foltz, D., Fukagawa, T., Gassmann, R., Gerlich, D. W., Glover, D. M., Gorbsky, G. J., HARRISON, S. C., Heun, P., Hirota, T., Jansen, L. E., Karpen, G., Kops, G. J., Lampson, M. A., LENS, S. M., Losada, A., Luger, K., Maiato, H., Maddox, P. S., Margolis, R. L., Masumoto, H., McAinsh, A. D., Mellone, B. G., Meraldi, P., Musacchio, A., Oegema, K., O'Neill, R. J., Salmon, E. D., Scott, K. C., Straight, A. F., Stukenberg, P. T., Sullivan, B. A., Sullivan, K. F., SUNKEL, C. E., Swedlow, J. R., Walczak, C. E., Warburton, P. E., Westermann, S., WILLARD, H. F., Wordeman, L., Yanagida, M., Yen, T. J., YODA, K., Cleveland, D. W. 2013; 21 (2): 101-106

    Abstract

    The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

    View details for DOI 10.1007/s10577-013-9347-y

    View details for Web of Science ID 000317688800001

    View details for PubMedID 23580138

  • Fluorescent protein applications in microscopy. Methods in cell biology Johnson, W. L., Straight, A. F. 2013; 114: 99-123

    Abstract

    The use of fluorescent proteins (FPs) in modern cell biology and microscopy has had an extraordinary impact on our ability to investigate dynamic processes in living cells. FPs are unique in that fluorescence is encoded solely by the primary amino acid sequence of the FP and does not require enzymatic modification or cofactors. This genetically encoded fluorescence enables the expression of FPs in diverse cells and organisms and the detection of that fluorescence in living systems. This chapter focuses on microscopy-based applications of FP detection to monitor protein localization, dynamics, interaction, and the cellular environment.

    View details for DOI 10.1016/B978-0-12-407761-4.00005-1

    View details for PubMedID 23931504

  • A cell-free system for functional centromere and kinetochore assembly NATURE PROTOCOLS Guse, A., Fuller, C. J., Straight, A. F. 2012; 7 (10): 1847-1869

    Abstract

    This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell-free system lends itself to use in protein immunodepletion, complementation and drug inhibition as a tool to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification and protein post-translational modification. This system provides a distinct advantage over cell-based investigations in which perturbing centromere and kinetochore function often results in lethality. After incubation in egg extract, reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins, which locally stabilize microtubules and, on microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment takes 3 d.

    View details for DOI 10.1038/nprot.2012.112

    View details for Web of Science ID 000309508200009

    View details for PubMedID 23018190

  • Imaging nanometre-scale structure in cells using in situ aberration correction JOURNAL OF MICROSCOPY Fuller, C. J., Straight, A. F. 2012; 248 (1): 90-101

    Abstract

    Accurate distance measurements of cellular structures on a length scale relevant to single macromolecules or macromolecular complexes present a major challenge for biological microscopy. In addition to the inherent challenges of overcoming the limits imposed by the diffraction of light, cells themselves are a complex and poorly understood optical environment. We present an extension of the high-resolution colocalization method to measure three dimensional distances between diffraction-limited objects using standard widefield fluorescence microscopy. We use this method to demonstrate that in three dimensions, cells intrinsically introduce a large and variable amount of chromatic aberration into optical measurements. We present a means of correcting this aberration in situ [termed 'Colocalization and In-situ Correction of Aberration for Distance Analysis' (CICADA)] by exploiting the fact that there is a linear relationship between the degree of aberration between different wavelengths. By labelling a cellular structure with redundantly multi-colour labelled antibodies, we can create an intracellular fiducial marker for correcting the individual aberrations between two different wavelengths in the same cells. Our observations demonstrate that with suitable corrections, nanometre scale three-dimensional distance measurements can be used to probe the substructure of macromolecular complexes within cells.

    View details for DOI 10.1111/j.1365-2818.2012.03654.x

    View details for Web of Science ID 000308655400010

    View details for PubMedID 22906048

  • The Split Personality of CENP-A Nucleosomes CELL Westhorpe, F. G., Straight, A. F. 2012; 150 (2): 245-247

    Abstract

    The composition and structure of centromeric nucleosomes, which contain the histone H3 variant CENP-A, is intensely debated. Two independent studies in this issue, in yeast and human cells, now suggest that CENP-A nucleosomes adopt different structures depending on the stage of the cell cycle.

    View details for DOI 10.1016/j.cell.2012.07.003

    View details for Web of Science ID 000306595700004

    View details for PubMedID 22817887

  • Dynamics of CENP-N kinetochore binding during the cell cycle JOURNAL OF CELL SCIENCE Hellwig, D., Emmerth, S., Ulbricht, T., Doering, V., Hoischen, C., Martin, R., Samora, C. P., McAinsh, A. D., Carroll, C. W., Straight, A. F., Meraldi, P., Diekmann, S. 2011; 124 (22): 3871-3883

    Abstract

    Accurate chromosome segregation requires the assembly of kinetochores, multiprotein complexes that assemble on the centromere of each sister chromatid. A key step in this process involves binding of the constitutive centromere-associated network (CCAN) to CENP-A, the histone H3 variant that constitutes centromeric nucleosomes. This network is proposed to operate as a persistent structural scaffold for assembly of the outer kinetochore during mitosis. Here, we show by fluorescence resonance energy transfer (FRET) that the N-terminus of CENP-N lies in close proximity to the N-terminus of CENP-A in vivo, consistent with in vitro data showing direct binding of CENP-N to CENP-A. Furthermore, we demonstrate in living cells that CENP-N is bound to kinetochores during S phase and G2, but is largely absent from kinetochores during mitosis and G1. By measuring the dynamics of kinetochore binding, we reveal that CENP-N undergoes rapid exchange in G1 until the middle of S phase when it becomes stably associated with kinetochores. The majority of CENP-N is loaded during S phase and dissociates again during G2. We propose a model in which CENP-N functions as a fidelity factor during centromeric replication and reveal that the CCAN network is considerably more dynamic than previously appreciated.

    View details for DOI 10.1242/jcs.088625

    View details for Web of Science ID 000298145400014

    View details for PubMedID 22100916

  • CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly JOURNAL OF CELL BIOLOGY Moree, B., Meyer, C. B., Fuller, C. J., Straight, A. F. 2011; 194 (6): 855-871

    Abstract

    Eukaryotic chromosomes segregate by attaching to microtubules of the mitotic spindle through a chromosomal microtubule binding site called the kinetochore. Kinetochores assemble on a specialized chromosomal locus termed the centromere, which is characterized by the replacement of histone H3 in centromeric nucleosomes with the essential histone H3 variant CENP-A (centromere protein A). Understanding how CENP-A chromatin is assembled and maintained is central to understanding chromosome segregation mechanisms. CENP-A nucleosome assembly requires the Mis18 complex and the CENP-A chaperone HJURP. These factors localize to centromeres in telophase/G1, when new CENP-A chromatin is assembled. The mechanisms that control their targeting are unknown. In this paper, we identify a mechanism for recruiting the Mis18 complex protein M18BP1 to centromeres. We show that depletion of CENP-C prevents M18BP1 targeting to metaphase centromeres and inhibits CENP-A chromatin assembly. We find that M18BP1 directly binds CENP-C through conserved domains in the CENP-C protein. Thus, CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly.

    View details for DOI 10.1083/jcb.201106079

    View details for Web of Science ID 000295026500007

    View details for PubMedID 21911481

    View details for PubMedCentralID PMC3207292

  • In vitro centromere and kinetochore assembly on defined chromatin templates NATURE Guse, A., Carroll, C. W., Moree, B., Fuller, C. J., Straight, A. F. 2011; 477 (7364): 354-U136

    Abstract

    During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.

    View details for DOI 10.1038/nature10379

    View details for Web of Science ID 000294852400036

    View details for PubMedID 21874020

  • Single-molecule fluorescence experiments with real-time feedback: Tracking-FCS, tracking-FRET and tracking diffusometry 242nd National Meeting of the American-Chemical-Society (ACS) Limouse, C., Fuller, C., Zhang, K., Straight, A., Mabuchi, H. AMER CHEMICAL SOC. 2011

    View details for Web of Science ID 000299378306072

  • Single molecule dynamics of chromatin fibers Annual Meeting of the American-Society-for-Cell-Biology (ASCB) Limouse, C., Fuller, C., Straight, A., Mabuchi, H. AMER SOC CELL BIOLOGY. 2011

    View details for Web of Science ID 000305505502220

  • Local Geometry and Elasticity in Compact Chromatin Structure BIOPHYSICAL JOURNAL Koslover, E. F., Fuller, C. J., Straight, A. F., Spakowitz, A. J. 2010; 99 (12): 3941-3950

    Abstract

    The hierarchical packaging of DNA into chromatin within a eukaryotic nucleus plays a pivotal role in both the accessibility of genomic information and the dynamics of replication. Our work addresses the role of nanoscale physical and geometric properties in determining the structure of chromatin at the mesoscale level. We study the packaging of DNA in chromatin fibers by optimization of regular helical morphologies, considering the elasticity of the linker DNA as well as steric packing of the nucleosomes and linkers. Our model predicts a broad range of preferred helix structures for a fixed linker length of DNA; changing the linker length alters the predicted ensemble. Specifically, we find that the twist registry of the nucleosomes, as set by the internucleosome repeat length, determines the preferred angle between the nucleosomes and the fiber axis. For moderate to long linker lengths, we find a number of energetically comparable configurations with different nucleosome-nucleosome interaction patterns, indicating a potential role for kinetic trapping in chromatin fiber formation. Our results highlight the key role played by DNA elasticity and local geometry in regulating the hierarchical packaging of the genome.

    View details for DOI 10.1016/j.bpj.2010.10.024

    View details for Web of Science ID 000285438900017

    View details for PubMedID 21156136

    View details for PubMedCentralID PMC3000514

  • Dual recognition of CENP-A nucleosomes is required for centromere assembly JOURNAL OF CELL BIOLOGY Carroll, C. W., Milks, K. J., Straight, A. F. 2010; 189 (7): 1143-1155

    Abstract

    Centromeres contain specialized nucleosomes in which histone H3 is replaced by the histone variant centromere protein A (CENP-A). CENP-A nucleosomes are thought to act as an epigenetic mark that specifies centromere identity. We previously identified CENP-N as a CENP-A nucleosome-specific binding protein. Here, we show that CENP-C also binds directly and specifically to CENP-A nucleosomes. Nucleosome binding by CENP-C required the extreme C terminus of CENP-A and did not compete with CENP-N binding, which suggests that CENP-C and CENP-N recognize distinct structural elements of CENP-A nucleosomes. A mutation that disrupted CENP-C binding to CENP-A nucleosomes in vitro caused defects in CENP-C targeting to centromeres. Moreover, depletion of CENP-C with siRNA resulted in the mislocalization of all other nonhistone CENPs examined, including CENP-K, CENP-H, CENP-I, and CENP-T, and led to a partial reduction in centromeric CENP-A. We propose that CENP-C binds directly to CENP-A chromatin and, together with CENP-N, provides the foundation upon which other centromere and kinetochore proteins are assembled.

    View details for DOI 10.1083/jcb.201001013

    View details for Web of Science ID 000279188400012

    View details for PubMedID 20566683

    View details for PubMedCentralID PMC2894454

  • RB's original CIN? GENES & DEVELOPMENT Sage, J., Straight, A. F. 2010; 24 (13): 1329-1333

    Abstract

    The retinoblastoma tumor suppressor RB is the downstream mediator of a cellular pathway that is thought to prevent cancer by controlling the ability of cells to enter or exit the cell cycle in G0/G1. Recently, however, accumulating evidence has suggested that RB, its family members p107 and p130, and their partners, the E2F family of transcription factors, may have important cellular functions beyond the G1/S transition of the cell cycle, including during DNA replication and at the transition into mitosis. In this issue of Genes & Development, three studies demonstrate a critical role for RB in proper chromosome condensation, centromeric function, and chromosome stability in mammalian cells, and link these cellular functions of RB to tumor suppression in mice. Here we discuss how transcriptional and post-transcriptional mechanisms under the control of the RB pathway ensure accurate progression through mitosis, thereby preventing cancer development.

    View details for DOI 10.1101/gad.1948010

    View details for Web of Science ID 000279405000001

    View details for PubMedID 20551167

    View details for PubMedCentralID PMC2895191

  • Image analysis benchmarking methods for high-content screen design JOURNAL OF MICROSCOPY-OXFORD Fuller, C. J., Straight, A. F. 2010; 238 (2): 145-161

    Abstract

    The recent development of complex chemical and small interfering RNA (siRNA) collections has enabled large-scale cell-based phenotypic screening. High-content and high-throughput imaging are widely used methods to record phenotypic data after chemical and small interfering RNA treatment, and numerous image processing and analysis methods have been used to quantify these phenotypes. Currently, there are no standardized methods for evaluating the effectiveness of new and existing image processing and analysis tools for an arbitrary screening problem. We generated a series of benchmarking images that represent commonly encountered variation in high-throughput screening data and used these image standards to evaluate the robustness of five different image analysis methods to changes in signal-to-noise ratio, focal plane, cell density and phenotype strength. The analysis methods that were most reliable, in the presence of experimental variation, required few cells to accurately distinguish phenotypic changes between control and experimental data sets. We conclude that by applying these simple benchmarking principles an a priori estimate of the image acquisition requirements for phenotypic analysis can be made before initiating an image-based screen. Application of this benchmarking methodology provides a mechanism to significantly reduce data acquisition and analysis burdens and to improve data quality and information content.

    View details for DOI 10.1111/j.1365-2818.2009.03337.x

    View details for Web of Science ID 000276793100006

    View details for PubMedID 20529062

  • Dissection of CENP-C-directed Centromere and Kinetochore Assembly MOLECULAR BIOLOGY OF THE CELL Milks, K. J., Moree, B., Straight, A. F. 2009; 20 (19): 4246-4255

    Abstract

    Eukaryotic cells ensure accurate chromosome segregation in mitosis by assembling a microtubule-binding site on each chromosome called the kinetochore that attaches to the mitotic spindle. The kinetochore is assembled specifically during mitosis on a specialized region of each chromosome called the centromere, which is constitutively bound by >15 centromere-specific proteins. These proteins, including centromere proteins A and C (CENP-A and -C), are essential for kinetochore assembly and proper chromosome segregation. How the centromere is assembled and how the centromere promotes mitotic kinetochore formation are poorly understood. We have used Xenopus egg extracts as an in vitro system to study the role of CENP-C in centromere and kinetochore assembly. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at the sperm centromere from the egg cytoplasm. Immunodepletion of CENP-C from metaphase egg extract prevents kinetochore formation on sperm chromatin, and depleted extracts can be complemented with in vitro-translated CENP-C. Using this complementation assay, we have identified CENP-C mutants that localized to centromeres but failed to support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K.

    View details for DOI 10.1091/mbc.E09-05-0378

    View details for Web of Science ID 000270352400012

    View details for PubMedID 19641019

    View details for PubMedCentralID PMC2754938

  • Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N NATURE CELL BIOLOGY Carroll, C. W., Silva, M. C., Godek, K. M., Jansen, L. E., Straight, A. F. 2009; 11 (7): 896-U297

    Abstract

    Centromeres are specialized chromosomal domains that direct kinetochore assembly during mitosis. CENP-A (centromere protein A), a histone H3-variant present exclusively in centromeric nucleosomes, is thought to function as an epigenetic mark that specifies centromere identity. Here we identify the essential centromere protein CENP-N as the first protein to selectively bind CENP-A nucleosomes but not H3 nucleosomes. CENP-N bound CENP-A nucleosomes in a DNA sequence-independent manner, but did not bind soluble CENP-A-H4 tetramers. Mutations in CENP-N that reduced its affinity for CENP-A nucleosomes caused defects in CENP-N localization and had dominant effects on the recruitment of CENP-H, CENP-I and CENP-K to centromeres. Depletion of CENP-N using siRNA (short interfering RNA) led to similar centromere assembly defects and resulted in reduced assembly of nascent CENP-A into centromeric chromatin. These data suggest that CENP-N interprets the information encoded within CENP-A nucleosomes and recruits other proteins to centromeric chromatin that are required for centromere function and propagation.

    View details for DOI 10.1038/ncb1899

    View details for Web of Science ID 000267603100020

    View details for PubMedID 19543270

    View details for PubMedCentralID PMC2704923

  • Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation JOURNAL OF CELL BIOLOGY Erhardt, S., Mellone, B. G., Betts, C. M., Zhang, W., Karpen, G. H., Straight, A. F. 2008; 183 (5): 805-818

    Abstract

    Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.

    View details for DOI 10.1083/jcb.200806038

    View details for Web of Science ID 000261232000008

    View details for PubMedID 19047461

  • Polo-Like Kinase Controls Vertebrate Spindle Elongation and Cytokinesis PLOS ONE Brennan, I. M., Peters, U., Kapoor, T. M., Straight, A. F. 2007; 2 (5)

    Abstract

    During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.

    View details for DOI 10.1371/journal.pone.0000409

    View details for Web of Science ID 000207445700011

    View details for PubMedID 17476331

  • Centromeric chromatin gets loaded JOURNAL OF CELL BIOLOGY Carroll, C. W., Straight, A. F. 2007; 176 (6): 735-736

    Abstract

    Centromeric nucleosomes contain a histone H3 variant called centromere protein A (CENP-A) that is required for kinetochore assembly and chromosome segregation. Two new studies, Jansen et al. (see p. 795 of this issue) and Maddox et al. (see p. 757 of this issue), address when CENP-A is deposited at centromeres during the cell division cycle and identify an evolutionally conserved protein required for CENP-A deposition. Together, these studies advance our understanding of centromeric chromatin assembly and provide a framework for investigating the molecular mechanisms that underlie the centromere-specific loading of CENP-A.

    View details for DOI 10.1083/jcb.200702020

    View details for Web of Science ID 000244863900001

    View details for PubMedID 17339381

    View details for PubMedCentralID PMC2064045

  • Fluorescent protein applications in microscopy DIGITAL MICROSCOPY, 3RD EDITION Straight, A. F. 2007; 81: 93-?

    View details for DOI 10.1016/S0091-679X(06)81006-X

    View details for Web of Science ID 000247882100006

    View details for PubMedID 17519164

  • Centromere formation: from epigenetics to self-assembly TRENDS IN CELL BIOLOGY Carroll, C. W., Straight, A. F. 2006; 16 (2): 70-78

    Abstract

    This review is part of the Chromosome segregation and Aneuploidy series that focuses on the importance of chromosome segregation mechanisms in maintaining genome stability. Centromeres are specialized chromosomal domains that serve as the foundation for the mitotic kinetochore, the interaction site between the chromosome and the mitotic spindle. The chromatin of centromeres is distinguished from other chromosomal loci by the unique incorporation of the centromeric histone H3 variant, centromere protein A. Here, we review the genetic and epigenetic factors that control the formation and maintenance of centromeric chromatin and propose a chromatin self-assembly model for organizing the higher-order structure of the centromere.

    View details for DOI 10.1016/j.tcb.2005.12.008

    View details for Web of Science ID 000236080600003

    View details for PubMedID 16412639

  • Absolute stereochemical assignment and fluorescence tuning of the small molecule tool, (-)-blebbistatin EUROPEAN JOURNAL OF ORGANIC CHEMISTRY Lucas-Lopez, C., Patterson, S., Blum, T., Straight, A. F., TOTH, J., Slawin, A. M., Mitchison, T. J., Sellers, J. R., Westwood, N. J. 2005: 1736-1740

    View details for DOI 10.1002/ejoc.200500103

    View details for Web of Science ID 000228959500004

  • Blebbistatin, a myosin II inhibitor, is photoinactivated by blue light BIOCHEMISTRY Sakamoto, T., Limouze, J., Combs, C. A., Straight, A. F., Sellers, J. R. 2005; 44 (2): 584-588

    Abstract

    Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. Blebbistatin inhibits the actin-activated MgATPase activity and in vitro motility of class II myosins. In cells, it has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. In particular, we have found that exposure to light at wavelengths below 488 nm rapidly inactivates the inhibitory action of blebbistatin using the in vitro motility of myosin as an assay. In addition, the inhibition of cytokinetic ring contraction can be reversed by exposure of the cells to blue light. This property may be useful in locally reversing the action of blebbistatin treatment in a cell. However, caution should be exercised as free radicals may be produced upon irradiation of blebbistatin that could result in cell damage.

    View details for DOI 10.1021/bi0483357

    View details for Web of Science ID 000226348000016

    View details for PubMedID 15641783

  • Anillin binds nonmuscle myosin II and regulates the contractile ring MOLECULAR BIOLOGY OF THE CELL Straight, A. F., Field, C. M., Mitchison, T. J. 2005; 16 (1): 193-201

    Abstract

    We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.

    View details for DOI 10.1091/mbc.E04-08-0758

    View details for Web of Science ID 000225954400020

    View details for PubMedID 15496454

    View details for PubMedCentralID PMC539163

  • Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIB BIOCHEMISTRY Ramamurthy, B., Yengo, C. M., Straight, A. F., Mitchison, T. J., Sweeney, H. L. 2004; 43 (46): 14832-14839

    Abstract

    We examined the effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1). Blebbistatin is a small molecule that affects cell blebbing during the process of cell division, which has been shown to decrease the myosin ATPase activity of a number of myosins [Straight et al. (2003) Science 299, 1743-1747]. The steady-state actin-activated ATPase activity of NMIIB S1 was decreased approximately 90% at 40 microM actin in the presence of blebbistatin. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis. Binding to actin in the presence of ADP (0.57 +/-0.08 microM(-1) s(-1)) was reduced slightly in the presence of blebbistatin (0.38 +/- 0.03 microM(-1) s(-1)), while mantADP dissociation from acto-NMIIB S1 was reduced (approximately 30%). P(i) release was blocked in the presence of blebbistatin. Accordingly, the apparent affinity of NMIIB S1 for actin in the presence of ATP was greatly reduced. Based on the above data, we surmise that blebbistatin inhibits the ATPase activity of NMIIB S1 primarily by blocking entry into the strong binding state; secondarily, it reduces the rate of ADP release. These effects are likely mediated by binding of blebbistatin within the myosin cleft that progressively closes in forming the acto-myosin rigor state.

    View details for DOI 10.1021/bi0490284

    View details for Web of Science ID 000225172800034

    View details for PubMedID 15544354

  • Specificity of blebbistatin, an inhibitor of myosin II JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY Limouze, J., Straight, A. F., Mitchison, T., Sellers, J. E. 2004; 25 (4-5): 337-341

    Abstract

    Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. We have examined the specificity and potency of the drug by assaying its effects on the actin-activated MgATPase assay of diverse members of the myosin superfamily. Blebbistatin potently inhibits several striated muscle myosins as well as vertebrate nonmuscle myosin IIA and IIB with IC50 values ranging from 0.5 to 5 microM. Interestingly, smooth muscle which is highly homologous to vertebrate nonmuscle myosin is only poorly inhibited (IC50=80 microM). The drug potently inhibits Dictyostelium myosin II, but poorly inhibits Acanthamoeba myosin II. Blebbistatin did not inhibit representative myosin superfamily members from classes I, V, and X.

    View details for Web of Science ID 000226517500008

    View details for PubMedID 15548862

  • Determining the position of the cell division plane NATURE Canman, J. C., Cameron, L. A., Maddox, P. S., Straight, A., Tirnauer, J. S., Mitchison, T. J., Fang, G. W., Kapoor, T. M., Salmon, E. D. 2003; 424 (6952): 1074-1078

    Abstract

    Proper positioning of the cell division plane during mitosis is essential for determining the size and position of the two daughter cells--a critical step during development and cell differentiation. A bipolar microtubule array has been proposed to be a minimum requirement for furrow positioning in mammalian cells, with furrows forming at the site of microtubule plus-end overlap between the spindle poles. Observations in other species have suggested, however, that this may not be true. Here we show, by inducing mammalian tissue cells with monopolar spindles to enter anaphase, that furrow formation in cultured mammalian cells does not require a bipolar spindle. Unexpectedly, cytokinesis occurs at high frequency in monopolar cells. Division always occurs at a cortical position distal to the chromosomes. Analysis of microtubules during cytokinesis in cells with monopolar and bipolar spindles shows that a subpopulation of stable microtubules extends past chromosomes and binds to the cell cortex at the site of furrow formation. Our data are consistent with a model in which chromosomes supply microtubules with factors that promote microtubule stability and furrowing.

    View details for DOI 10.1038/nature01860

    View details for Web of Science ID 000184984200048

    View details for PubMedID 12904818

  • Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles: implications for spindle mechanics JOURNAL OF CELL BIOLOGY Maddox, P., Straight, A., Coughlin, P., Mitchison, T. J., Salmon, E. D. 2003; 162 (3): 377-382

    Abstract

    Microtubule plus ends dynamically attach to kinetochores on mitotic chromosomes. We directly imaged this dynamic interface using high resolution fluorescent speckle microscopy and direct labeling of kinetochores in Xenopus extract spindles. During metaphase, kinetochores were stationary and under tension while plus end polymerization and poleward microtubule flux (flux) occurred at velocities varying from 1.5-2.5 micro m/min. Because kinetochore microtubules polymerize at metaphase kinetochores, the primary source of kinetochore tension must be the spindle forces that produce flux and not a kinetochore-based mechanism. We infer that the kinetochore resists translocation of kinetochore microtubules through their attachment sites, and that the polymerization state of the kinetochore acts a "slip-clutch" mechanism that prevents detachment at high tension. At anaphase onset, kinetochores switched to depolymerization of microtubule plus ends, resulting in chromosome-to-pole rates transiently greater than flux. Kinetochores switched from persistent depolymerization to persistent polymerization and back again during anaphase, bistability exhibited by kinetochores in vertebrate tissue cells. These results provide the most complete description of spindle microtubule poleward flux to date, with important implications for the microtubule-kinetochore interface and for how flux regulates kinetochore function.

    View details for DOI 10.1083/jcb.200301088

    View details for Web of Science ID 000184667900003

    View details for PubMedID 12900391

  • Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils CELL Xu, J. S., Wang, F., Van Keymeulen, A., Herzmark, P., Straight, A., Kelly, K., Takuwa, Y., Sugimoto, N., Mitchison, T., Bourne, H. R. 2003; 114 (2): 201-214

    Abstract

    Like neutrophilic leukocytes, differentiated HL-60 cells respond to chemoattractant by adopting a polarized morphology, with F-actin in a protruding pseudopod at the leading edge and contractile actin-myosin complexes at the back and sides. Experiments with pharmacological inhibitors, toxins, and mutant proteins show that this polarity depends on divergent, opposing "frontness" and "backness" signals generated by different receptor-activated trimeric G proteins. Frontness depends upon Gi-mediated production of 3'-phosphoinositol lipids (PI3Ps), the activated form of Rac, a small GTPase, and F-actin. G12 and G13 trigger backness signals, including activation of a second GTPase (Rho), a Rho-dependent kinase, and myosin II. Functional incompatibility causes the two resulting actin assemblies to aggregate into separate domains, making the leading edge more sensitive to attractant than the back. The latter effect explains both the neutrophil's ability to polarize in uniform concentrations of chemoattractant and its response to reversal of an attractant gradient by performing a U-turn.

    View details for Web of Science ID 000184378700010

    View details for PubMedID 12887922

  • Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor SCIENCE Straight, A. F., Cheung, A., Limouze, J., Chen, I., Westwood, N. J., Sellers, J. R., Mitchison, T. J. 2003; 299 (5613): 1743-1747

    Abstract

    Completion of cell division during cytokinesis requires temporally and spatially regulated communication from the microtubule cytoskeleton to the actin cytoskeleton and the cell membrane. We identified a specific inhibitor of nonmuscle myosin II, blebbistatin, that inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly. Using blebbistatin and other drugs, we showed that exit from the cytokinetic phase of the cell cycle depends on ubiquitin-mediated proteolysis. Continuous signals from microtubules are required to maintain the position of the cleavage furrow, and these signals control the localization of myosin II independently of other furrow components.

    View details for Web of Science ID 000181519500049

    View details for PubMedID 12637748

  • Self- and actin-templated assembly of mammalian septins DEVELOPMENTAL CELL Kinoshita, M., Field, C. M., Coughlin, M. L., Straight, A. F., Mitchison, T. J. 2002; 3 (6): 791-802

    Abstract

    Septins are polymerizing GTPases required for cytokinesis and cortical organization. The principles by which they are targeted to, and assemble at, specific cell regions are unknown. We show that septins in mammalian cells switch between a linear organization along actin bundles and cytoplasmic rings, approximately 0.6 microm in diameter. A recombinant septin complex self-assembles into rings resembling those in cells. Linear organization along actin bundles was reconstituted by adding an adaptor protein, anillin. Perturbation of septin organization in cells by expression of a septin-interacting fragment of anillin or by septin depletion via siRNA causes loss of actin bundles. We conclude that septins alone self-assemble into rings, that adaptor proteins recruit septins to actin bundles, and that septins help organize these bundles.

    View details for Web of Science ID 000179756100008

    View details for PubMedID 12479805

  • Anaphase onset does not require the microtubule-dependent depletion of kinetochore and centromere-binding proteins JOURNAL OF CELL SCIENCE Canman, J. C., Sharma, N., Straight, A., Shannon, K. B., Fang, G. W., Salmon, E. D. 2002; 115 (19): 3787-3795

    Abstract

    Spindle checkpoint proteins, such as Mad2 and BubR1, and the motors dynein/dynactin and CENP-E usually leave kinetochores prior to anaphase onset by microtubule-dependent mechanisms. Likewise, 'chromosome passenger proteins' including INCENP are depleted from the centromeres after anaphase onset and then move to the midzone complex, an event that is essential for cytokinesis. Here we test whether the cell cycle changes that occur at anaphase onset require or contribute to the depletion of kinetochore and centromere proteins independent of microtubules. This required the development of a novel non-antibody method to induce precocious anaphase onset in vivo by using a bacterially expressed fragment of the spindle checkpoint protein Mad1 capable of activating the APC/C, called GST-Mad1F10. By injecting PtK1 cells in nocodazole with GST-Mad1F10 and processing the cells for immunofluorescence microscopy after anaphase sister chromatid separation in nocodazole we found that Mad2, BubR1, cytoplasmic dynein, CENP-E and the 3F3/2 phosphoepitope remain on kinetochores. Thus depletion of these proteins (or phosphoepitope) at kinetochores is not required for anaphase onset and anaphase onset does not produce their depletion independent of microtubules. In contrast, both microtubules and anaphase onset are required for depletion of the 'chromosome passenger' protein INCENP from centromeres, as INCENP does not leave the chromosomes prior to anaphase onset in the presence or absence of microtubules, but does leave the centromeres after anaphase onset in the presence of microtubules.

    View details for DOI 10.1242/jcs.00057

    View details for Web of Science ID 000178636600009

    View details for PubMedID 12235289

  • A small-molecule inhibitor of skeletal muscle myosin II NATURE CELL BIOLOGY Cheung, A., Dantzig, J. A., Hollingworth, S., Baylor, S. M., Goldman, Y. E., Mitchison, T. J., Straight, A. F. 2002; 4 (1): 83-88

    Abstract

    We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

    View details for DOI 10.1038/ncb734

    View details for Web of Science ID 000173381500021

    View details for PubMedID 11744924

  • Microtubules, membranes and cytokinesis CURRENT BIOLOGY Straight, A. F., Field, C. M. 2000; 10 (20): R760-R770

    Abstract

    Proper division of the cell requires coordination between chromosome segregation by the mitotic spindle and cleavage of the cell by the cytokinetic apparatus. Interactions between the mitotic spindle, the contractile ring and the plasma membrane ensure that the cleavage furrow is properly placed between the segregating chromosomes and that new membrane compartments are formed to produce two daughter cells. The microtubule midzone is able to stimulate the cortex of the cell to ensure proper ingression and completion of the cleavage furrow. Specialized microtubule structures are responsible for directing membrane vesicles to the site of cell cleavage, and vesicle fusion is required for the proper completion of cytokinesis.

    View details for Web of Science ID 000090034300013

    View details for PubMedID 11069103

  • Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity CELL Straight, A. F., Shou, W. Y., Dowd, G. J., Turck, C. W., Deshaies, R. J., Johnson, A. D., Moazed, D. 1999; 97 (2): 245-256

    Abstract

    The Sir2 protein mediates gene silencing and repression of recombination at the rDNA repeats in budding yeast. Here we show that Sir2 executes these functions as a component of a nucleolar complex designated RENT (regulator of nucleolar silencing and telophase exit). Net1, a core subunit of this complex, preferentially cross-links to the rDNA repeats, but not to silent DNA regions near telomeres or to active genes, and tethers the RENT complex to rDNA. Net1 is furthermore required for rDNA silencing and nucleolar integrity. During interphase, Net1 and Sir2 colocalize to a subdomain within the nucleous, but at the end of mitosis a fraction of Sir2 leaves the nucleolus and disperses as foci throughout the nucleus, suggesting that the structure of rDNA silent chromatin changes during the cell cycle. Our findings suggest that a protein complex shown to regulate exit from mitosis is also involved in gene silencing.

    View details for Web of Science ID 000079779800013

    View details for PubMedID 10219245

  • Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast JOURNAL OF CELL BIOLOGY Straight, A. F., Sedat, J. W., Murray, A. W. 1998; 143 (3): 687-694

    Abstract

    The mitotic spindle is a complex and dynamic structure. Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis. We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein. Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Delta mutants lack the rapid phase of anaphase B, kip1Delta mutants show defects in the slow phase of anaphase B, and kip3Delta mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell. The kip3Delta and kip1Delta mutants affect the duration of anaphase, but cin8Delta does not.

    View details for Web of Science ID 000076894300011

    View details for PubMedID 9813090

  • Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle MOLECULAR BIOLOGY OF THE CELL Nabeshima, K., Nakagawa, T., Straight, A. F., Murray, A., Chikashige, Y., Yamashita, Y. M., Hiraoka, Y., Yanagida, M. 1998; 9 (11): 3211-3225

    Abstract

    In higher eukaryotic cells, the spindle forms along with chromosome condensation in mitotic prophase. In metaphase, chromosomes are aligned on the spindle with sister kinetochores facing toward the opposite poles. In anaphase A, sister chromatids separate from each other without spindle extension, whereas spindle elongation takes place during anaphase B. We have critically examined whether such mitotic stages also occur in a lower eukaryote, Schizosaccharomyces pombe. Using the green fluorescent protein tagging technique, early mitotic to late anaphase events were observed in living fission yeast cells. S. pombe has three phases in spindle dynamics, spindle formation (phase 1), constant spindle length (phase 2), and spindle extension (phase 3). Sister centromere separation (anaphase A) rapidly occurred at the end of phase 2. The centromere showed dynamic movements throughout phase 2 as it moved back and forth and was transiently split in two before its separation, suggesting that the centromere was positioned in a bioriented manner toward the poles at metaphase. Microtubule-associating Dis1 was required for the occurrence of constant spindle length and centromere movement in phase 2. Normal transition from phase 2 to 3 needed DNA topoisomerase II and Cut1 but not Cut14. The duration of each phase was highly dependent on temperature.

    View details for Web of Science ID 000076888800017

    View details for PubMedID 9802907

  • In vivo visualization of chromosomes using lac operator-repressor binding TRENDS IN CELL BIOLOGY Belmont, A. S., Straight, A. F. 1998; 8 (3): 121-124

    Abstract

    This article describes a new technique for direct, in vivo visualization of chromosome dynamics based on lac repressor recognition of direct repeats of the lac operator. The method allows the tagging of specific chromosomal sites and thus in situ localization with minimal perturbation of structure. Detection by light microscopy, using GFP-repressor fusion proteins or immunofluorescence, can be complemented by higher-resolution electron microscopy using immunogold staining. Applications of this method will facilitate the investigation of interphase chromosome dynamics, as well as chromosome segregation during cell division in organisms that lack cytologically condensed chromosomes.

    View details for Web of Science ID 000072430700008

    View details for PubMedID 9695822

  • Interphase chromosomes undergo constrained diffusional motion in living cells CURRENT BIOLOGY Marshall, W. F., Straight, A., Marko, J. F., Swedlow, J., Dernburg, A., Belmont, A., Murray, A. W., Agard, D. A., Sedat, J. W. 1997; 7 (12): 930-939

    Abstract

    Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus.To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture.We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.

    View details for Web of Science ID A1997YL44000025

    View details for PubMedID 9382846

  • Cell cycle: Checkpoint proteins and kinetochores CURRENT BIOLOGY Straight, A. F. 1997; 7 (10): R613-R616

    Abstract

    Vertebrate homologs of yeast spindle assembly checkpoint proteins are localized to kinetochores and may act as a sensor for proper chromosome attachment to the mitotic spindle.

    View details for Web of Science ID A1997YB58300012

    View details for PubMedID 9368739

  • Chromosome and low copy plasmid segregation in E-coli: Visual evidence for distinct mechanisms CELL Gordon, G. S., Sitnikov, D., Webb, C. D., Teleman, A., Straight, A., Losick, R., Murray, A. W., Wright, A. 1997; 90 (6): 1113-1121

    Abstract

    We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.

    View details for Web of Science ID A1997XX76800017

    View details for PubMedID 9323139

  • Mitosis in living budding yeast: Anaphase a but no metaphase plate SCIENCE Straight, A. F., Marshall, W. F., Sedat, J. W., Murray, A. W. 1997; 277 (5325): 574-578

    Abstract

    Chromosome movements and spindle dynamics were visualized in living cells of the budding yeast Saccharomyces cerevisiae. Individual chromosomal loci were detected by expression of a protein fusion between green fluorescent protein (GFP) and the Lac repressor, which bound to an array of Lac operator binding sites integrated into the chromosome. Spindle microtubules were detected by expression of a protein fusion between GFP and Tub1, the major alpha tubulin. Spindle elongation and chromosome separation exhibited biphasic kinetics, and centromeres separated before telomeres. Budding yeast did not exhibit a conventional metaphase chromosome alignment but did show anaphase A, movement of the chromosomes to the poles.

    View details for Web of Science ID A1997XM86700054

    View details for PubMedID 9228009

  • Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA MOLECULAR BIOLOGY OF THE CELL Nunnari, J., Marshall, W. F., Straight, A., Murray, A., Sedat, J. W., Walter, P. 1997; 8 (7): 1233-1242

    Abstract

    To gain insight into the process of mitochondrial transmission in yeast, we directly labeled mitochondrial proteins and mitochondrial DNA (mtDNA) and observed their fate after the fusion of two cells. To this end, mitochondrial proteins in haploid cells of opposite mating type were labeled with different fluorescent dyes and observed by fluorescence microscopy after mating of the cells. Parental mitochondrial protein markers rapidly redistributed and colocalized throughout zygotes, indicating that during mating, parental mitochondria fuse and their protein contents intermix, consistent with results previously obtained with a single parentally derived protein marker. Analysis of the three-dimensional structure and dynamics of mitochondria in living cells with wide-field fluorescence microscopy indicated that mitochondria form a single dynamic network, whose continuity is maintained by a balanced frequency of fission and fusion events. Thus, the complete mixing of mitochondrial proteins can be explained by the formation of one continuous mitochondrial compartment after mating. In marked contrast to the mixing of parental mitochondrial proteins after fusion, mtDNA (labeled with the thymidine analogue 5-bromodeoxyuridine) remained distinctly localized to one half of the zygotic cell. This observation provides a direct explanation for the genetically observed nonrandom patterns of mtDNA transmission. We propose that anchoring of mtDNA within the organelle is linked to an active segregation mechanism that ensures accurate inheritance of mtDNA along with the organelle.

    View details for Web of Science ID A1997XL79800006

    View details for PubMedID 9243504

  • Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B-subtilis CELL Webb, C. D., Teleman, A., GORDON, S., Straight, A., Belmont, A., Lin, D. C., Grossman, A. D., Wright, A., Losick, R. 1997; 88 (5): 667-674

    Abstract

    To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.

    View details for Web of Science ID A1997WM41300012

    View details for PubMedID 9054506

  • The spindle assembly checkpoint in budding yeast CELL CYCLE CONTROL Straight, A. F., Murray, A. W. 1997; 283: 425-440

    View details for Web of Science ID A1997BJ41B00033

    View details for PubMedID 9251039

  • GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion CURRENT BIOLOGY Straight, A. F., Belmont, A. S., Robinett, C. C., Murray, A. W. 1996; 6 (12): 1599-1608

    Abstract

    Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy.We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)-Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP-Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate.We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.

    View details for Web of Science ID A1996VX48200023

    View details for PubMedID 8994824

  • In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition JOURNAL OF CELL BIOLOGY Robinett, C. C., Straight, A., Li, G., WILLHELM, C., Sudlow, G., Murray, A., Belmont, A. S. 1996; 135 (6): 1685-1700

    Abstract

    We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

    View details for Web of Science ID A1996WA87900002

    View details for PubMedID 8991083

  • Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast CURRENT BIOLOGY Minshull, J., Straight, A., Rudner, A. D., Dernburg, A. F., Belmont, A., Murray, A. W. 1996; 6 (12): 1609-1620

    Abstract

    Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55 delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.

    View details for Web of Science ID A1996VX48200024

    View details for PubMedID 8994825

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