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1.  A new piece in the kinetochore jigsaw puzzle 
The Journal of Cell Biology  2014;206(4):457-459.
In eukaryotic cell division, the kinetochore mediates chromosome attachment to spindle microtubules and acts as a scaffold for signaling pathways, ensuring the accuracy of chromosome segregation. The architecture of the kinetochore underlies its function in mitosis. In this issue, Hornung et al. (2014. J. Cell Biol. http://dx.doi.org/201403081) identify an unexpected linkage between the inner and outer regions of the kinetochore in budding yeast that suggests a new model for the construction of this interface.
doi:10.1083/jcb.201407048
PMCID: PMC4137055  PMID: 25135931
2.  Meiotic Double-Strand Breaks Uncover and Protect Against Mitotic Errors in the C. elegans Germline 
Current biology : CB  2013;23(23):10.1016/j.cub.2013.10.015.
SUMMARY
In sexually reproducing multi-cellular organisms, genetic information is propagated via the germline, the specialized tissue that generates haploid gametes. The C. elegans germline generates gametes in an assembly line-like process- mitotic divisions under the control of the stem cell niche produce nuclei that, upon leaving the niche, enter into meiosis and progress through meiotic prophase [1]. Here we characterize the effects of perturbing cell division in the mitotic region of the C. elegans germline. We show that mitotic errors result in a spindle checkpoint-dependent cell cycle delay but defective nuclei are eventually formed and enter meiosis. These defective nuclei are eliminated by programmed cell death during meiotic prophase. The cell death based removal of defective nuclei does not require the spindle checkpoint, but instead depends on the DNA damage checkpoint. Removal of nuclei resulting from errors in mitosis also requires Spo11, the enzyme that creates double-strand breaks to initiate meiotic recombination. Consistent with this, double strand breaks are increased in number and persist longer in germlines with mitotic defects. These findings reveal that the process of initiating meiotic recombination inherently selects against nuclei with abnormal chromosomal content generated by mitotic errors, thereby ensuring the genomic integrity of gametes.
doi:10.1016/j.cub.2013.10.015
PMCID: PMC3885542  PMID: 24239117
C. elegans; germline; mitosis; meiosis; recombination; cell death; DNA damage
3.  A Conserved RhoGAP Limits M-phase Contractility and Coordinates with Microtubule Asters to Restrict Active RhoA to the Cell Equator During Cytokinesis 
Developmental cell  2013;26(5):496-510.
SUMMARY
During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein (GAP)-mediated inactivation (RhoA flux) is proposed to limit RhoA zone dimensions. Testing the significance of RhoA flux has been hampered by the fact that the GAP targeting RhoA is not known. Here, we identify M-phase GAP (MP-GAP) as the primary GAP targeting RhoA during mitosis/cytokinesis. MP-GAP inhibition caused excessive RhoA activation in M-phase leading to the uncontrolled formation of large cortical protrusions and late cytokinesis failure. RhoA zone width was broadened by attenuation of the centrosomal asters but was not affected by MP-GAP inhibition alone. Simultaneous aster attenuation and MP-GAP inhibition led to RhoA accumulation around the entire cell periphery. These results identify the major GAP restraining RhoA during cell division and delineate the relative contributions of RhoA flux and centrosomal asters in controlling RhoA zone dimensions.
doi:10.1016/j.devcel.2013.08.005
PMCID: PMC4239416  PMID: 24012485
Mitosis; RhoA; Cytokinesis; MP-GAP; Ect2
4.  A Bub1–Mad1 interaction targets the Mad1–Mad2 complex to unattached kinetochores to initiate the spindle checkpoint 
The Journal of Cell Biology  2014;204(5):647-657.
A Bub1–Mad1 interaction targets the Mad1–Mad2 complex to unattached kinetochores to initiate the spindle checkpoint.
Recruitment of Mad1–Mad2 complexes to unattached kinetochores is a central event in spindle checkpoint signaling. Despite its importance, the mechanism that recruits Mad1–Mad2 to kinetochores is unclear. In this paper, we show that MAD-1 interacts with BUB-1 in Caenorhabditis elegans. Mutagenesis identified specific residues in a segment of the MAD-1 coiled coil that mediate the BUB-1 interaction. In addition to unattached kinetochores, MAD-1 localized between separating meiotic chromosomes and to the nuclear periphery. Mutations in the MAD-1 coiled coil that selectively disrupt interaction with BUB-1 eliminated MAD-1 localization to unattached kinetochores and between meiotic chromosomes, both of which require BUB-1, and abrogated checkpoint signaling. The identified MAD-1 coiled-coil segment interacted with a C-terminal region of BUB-1 that contains its kinase domain, and mutations in this region prevented MAD-1 kinetochore targeting independently of kinase activity. These results delineate an interaction between BUB-1 and MAD-1 that targets MAD-1–MAD-2 complexes to kinetochores and is essential for spindle checkpoint signaling.
doi:10.1083/jcb.201311015
PMCID: PMC3941058  PMID: 24567362
5.  Crosstalk Between Microtubule Attachment Complexes Ensures Accurate Chromosome Segregation* 
Science (New York, N.Y.)  2013;342(6163):10.1126/science.1246232.
The microtubule-based mitotic spindle segregates chromosomes during cell division. During chromosome segregation, the centromeric regions of chromosomes build kinetochores that establish end-coupled attachments to spindle microtubules. Here, we used the C. elegans embryo as a model system to examine the crosstalk between two kinetochore protein complexes implicated in temporally distinct stages of attachment formation. The kinetochore dynein module, which mediates initial lateral microtubule capture, inhibited microtubule binding by the Ndc80 complex, which ultimately forms the end-coupled attachments that segregate chromosomes. The kinetochore dynein module directly regulated Ndc80, independently of phosphorylation by Aurora B kinase, and this regulation was required for accurate segregation. Thus, the conversion from initial dynein-mediated, lateral attachments to correctly oriented, Ndc80-mediated end-coupled attachments is actively controlled.
doi:10.1126/science.1246232
PMCID: PMC3885540  PMID: 24231804
6.  Direct Binding of SAS-6 to ZYG-1 Recruits SAS-6 to the Mother Centriole for Cartwheel Assembly 
Developmental cell  2013;25(3):284-298.
SUMMARY
Assembly of SAS-6 dimers to form the centriolar cartwheel requires the ZYG-1/Plk4 kinase. Here we show that ZYG-1 recruits SAS-6 to the mother centriole independently of its kinase activity; kinase activity is subsequently required for cartwheel assembly. We identify a direct interaction between ZYG-1 and the SAS-6 coiled-coil that explains its kinase activity-independent function in SAS-6 recruitment. Perturbing this interaction, or the interaction between an adjacent segment of the SAS-6 coiled-coil and SAS-5, prevented SAS-6 recruitment and cartwheel assembly. SAS-6 mutants with alanine substitutions in a previously described ZYG-1 target site or in 37 other residues, either phosphorylated by ZYG-1 in vitro or conserved in closely related nematodes, all supported cartwheel assembly. We propose that ZYG-1 binding to the SAS-6 coiled-coil recruits the SAS-6—SAS-5 complex to the mother centriole, where a ZYG-1 kinase activity-dependent step, whose target is unlikely to be SAS-6, triggers cartwheel assembly.
doi:10.1016/j.devcel.2013.03.011
PMCID: PMC3655416  PMID: 23673331
Centriole; Plk4; Centrosome; Mitosis; Spindle
7.  The midbody ring scaffolds the abscission machinery in the absence of midbody microtubules 
The Journal of Cell Biology  2013;203(3):505-520.
The septins, but not midbody microtubules, are important for daughter cell cytoplasmic isolation and ESCRT-dependent midbody ring release during abscission.
Abscission completes cytokinesis to form the two daughter cells. Although abscission could be organized from the inside out by the microtubule-based midbody or from the outside in by the contractile ring–derived midbody ring, it is assumed that midbody microtubules scaffold the abscission machinery. In this paper, we assess the contribution of midbody microtubules versus the midbody ring in the Caenorhabditis elegans embryo. We show that abscission occurs in two stages. First, the cytoplasm in the daughter cells becomes isolated, coincident with formation of the intercellular bridge; proper progression through this stage required the septins (a midbody ring component) but not the membrane-remodeling endosomal sorting complex required for transport (ESCRT) machinery. Second, the midbody and midbody ring are released into a specific daughter cell during the subsequent cell division; this stage required the septins and the ESCRT machinery. Surprisingly, midbody microtubules were dispensable for both stages. These results delineate distinct steps during abscission and highlight the central role of the midbody ring, rather than midbody microtubules, in their execution.
doi:10.1083/jcb.201306036
PMCID: PMC3824018  PMID: 24217623
8.  Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae 
PLoS Genetics  2014;10(5):e1004327.
In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.
Author Summary
Lynch syndrome (hereditary nonpolyposis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. In this syndrome, predisposition to cancer results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the human mismatch repair genes MLH1, MSH2, MSH6 or PMS2. In addition to these genes, various DNA replication factors and the excision factor EXO1 function in the repair of damaged DNA by the MMR pathway. In Saccharomyces cerevisiae, the MLH2 gene encodes a MutL homolog protein whose role in DNA mismatch repair has been unclear. Here, we used phylogenetic analysis to demonstrate that the S. cerevisiae Mlh2 protein and the mammalian Pms1 protein are homologs. A combination of genetics, biochemistry and imaging studies were used to demonstrate that the Mlh1-Mlh2 complex is recruited to mispair-containing DNA by the Msh2-Msh6 and Msh2-Msh3 mispair recognition complexes where it forms foci that colocalize with Mlh1-Pms1 foci (note that scPms1 is the homolog of hPms2) and augments the function of the Mlh1-Pms1 complex. Thus, this work establishes the Mlh1-Mlh2 complex as a non-essential accessory factor that functions in MMR.
doi:10.1371/journal.pgen.1004327
PMCID: PMC4014439  PMID: 24811092
9.  What the Hec is up with Mouse Oocyte Meiosis? 
Developmental cell  2013;25(1):3-4.
In this issue of Developmental Cell, Gui and Homer (2013) report that the proper execution of meiosis I in mouse oocytes requires the stabilization of cyclin B2 by the kinetochore protein Hec1, revealing unanticipated functions for both proteins.
doi:10.1016/j.devcel.2013.03.018
PMCID: PMC3804012  PMID: 23597481
10.  Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores 
The Journal of Cell Biology  2013;202(5):735-746.
Depletion analyses and nanometer-scale mapping of spindle assembly checkpoint proteins reveal how these proteins are integrated within the substructure of the kinetochore.
Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation.
doi:10.1083/jcb.201304197
PMCID: PMC3760617  PMID: 23979716
11.  Tension Sensing by Aurora B Kinase is Independent of Survivin-Based Centromere Localization 
Nature  2013;497(7447):118-121.
Accurate segregation of the replicated genome requires chromosome biorientation on the spindle. Biorientation is ensured by Aurora B kinase, a member of the 4-subunit chromosomal passenger complex (CPC)1,2. Localization of the CPC to the inner centromere is central to the current model for how tension ensures chromosome biorientation—kinetochore-spindle attachments not under tension remain close to the inner centromere and are destabilized by Aurora B phosphorylation, whereas kinetochores under tension are pulled away from the influence of Aurora B, stabilizing their microtubule attachments3–5. Here we show that an engineered truncation of the INCENP/Sli15 subunit of budding yeast CPC that eliminates association with the inner centromere nevertheless supports proper chromosome segregation during both mitosis and meiosis. Truncated INCENP/Sli15 suppresses the deletion phenotypes of the inner centromere-targeting proteins Survivin/Bir1, Borealin/Nbl1, Bub1 and Sgo16. Unlike wildtype INCENP/Sli15, truncated INCENP/Sli15 localizes to pre-anaphase spindle microtubules. Premature targeting of full-length INCENP/Sli15 to microtubules by preventing Cdk1 phosphorylation also suppresses inviability of Survivin/Bir1 deletion. These results suggest that activation of Aurora B/Ipl1 by clustering either on chromatin or on microtubules is sufficient for chromosome biorientation.
doi:10.1038/nature12057
PMCID: PMC3644022  PMID: 23604256
12.  Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway 
PLoS Genetics  2013;9(10):e1003869.
Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.
Author Summary
Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. In addition to these genes, various DNA replication factors and the excision factor EXO1 function in the repair of damaged DNA by the MMR pathway. Although EXO1 is considered to be the major repair nuclease functioning in mismatch repair, the relatively low mutation rates caused by an exo1 deletion suggest otherwise. Here we used genetics, microscopy and protein biochemistry to analyze the model organism Saccharomyces cerevisiae to further characterize a poorly understood mismatch repair pathway that functions in the absence of EXO1 that is highly dependent on the Mlh1-Pms1 complex. Surprisingly, we found that the highly conserved metal binding site that is critical for the endonuclease activity of the Mlh1-Pms1 heterodimer is required for MMR in the absence of Exo1 to a much greater extent than in the presence of Exo1. Thus, this work establishes that there are at least two different polynucleotide excision pathways that function in MMR.
doi:10.1371/journal.pgen.1003869
PMCID: PMC3814310  PMID: 24204293
13.  Acentrosomal Spindle Assembly & Chromosome Segregation During Oocyte Meiosis 
Trends in Cell Biology  2012;22(5):241-249.
The ability to reproduce relies in most eukaryotes on specialized cells called gametes. Gametes are formed by the process of meiosis in which, after a single round of replication, two successive cell divisions reduce the ploidy of the genome. Fusion of gametes at fertilization reconstitutes diploidy. In most animal species, chromosome segregation during female meiosis occurs on spindles assembled in the absence of the major microtubule-organizing center, the centrosome. In mammals, oocyte meiosis is error-prone and underlies the majority of birth aneuploidies. Here, we review recent work on acentrosomal spindle formation and chromosome alignment/separation during oocyte meiosis in different animal models.
doi:10.1016/j.tcb.2012.02.007
PMCID: PMC3348331  PMID: 22480579
14.  An inverse relationship to germline transcription defines centromeric chromatin in C. elegans 
Nature  2012;484(7395):534-537.
Centromeres are chromosomal loci that direct segregation of the genome during cell division. The histone H3 variant CENP-A (also known as CenH3) defines centromeres in monocentric organisms, which confine centromere activity to a discrete chromosomal region, and holocentric organisms, which distribute centromere activity along the chromosome length1–3. Because the highly repetitive DNA found at most centromeres is neither necessary nor sufficient for centromere function, stable inheritance of CENP-A nucleosomal chromatin is postulated to epigenetically propagate centromere identity4. Here, we show that in the holocentric nematode Caenorhabditis elegans pre-existing CENP-A nucleosomes are not necessary to guide recruitment of new CENP-A nucleosomes. This is indicated by lack of CENP-A transmission by sperm during fertilization and by removal and subsequent reloading of CENP-A during oogenic meiotic prophase. Genome-wide mapping of CENP-A location in embryos and quantification of CENP-A molecules in nuclei revealed that CENP-A is incorporated at low density in domains that cumulatively encompass half the genome. Embryonic CENP-A domains are established in a pattern inverse to regions that are transcribed in the germline and early embryo, and ectopic transcription of genes in a mutant germline altered the pattern of CENP-A incorporation in embryos. Furthermore, regions transcribed in the germline but not embryos fail to incorporate CENP-A throughout embryogenesis. We propose that germline transcription defines genomic regions that exclude CENP-A incorporation in progeny, and that zygotic transcription during early embryogenesis remodels and reinforces this basal pattern. These findings link centromere identity to transcription and shed light on the evolutionary plasticity of centromeres.
doi:10.1038/nature10973
PMCID: PMC3538161  PMID: 22495302
15.  Structure, Assembly and Reading of Centromeric Chromatin 
Centromeres are epigenetically defined chromatin domains marked by the presence of the histone H3 variant CENP-A. Here we review recent structural and biochemical work on CENP-A, and advances in understanding the mechanisms that propagate and read centromeric chromatin domains.
doi:10.1016/j.gde.2011.11.005
PMCID: PMC3319190  PMID: 22178421
16.  Hierarchical assembly of the eggshell and permeability barrier in C. elegans 
The Journal of Cell Biology  2012;198(4):731-748.
Assembly of the trilaminar eggshell and development of the permeability barrier after fertilization in C. elegans are distinct in their timing and mechanism.
In metazoans, fertilization triggers the assembly of an extracellular coat that constitutes the interface between the embryo and its environment. In nematodes, this coat is the eggshell, which provides mechanical rigidity, prevents polyspermy, and is impermeable to small molecules. Using immunoelectron microscopy, we found that the Caenorhabditis elegans eggshell was composed of an outer vitelline layer, a middle chitin layer, and an inner layer containing chondroitin proteoglycans. The switch between the chitin and proteoglycan layers was achieved by internalization of chitin synthase coincident with exocytosis of proteoglycan-containing cortical granules. Inner layer assembly did not make the zygote impermeable as previously proposed. Instead, correlative light and electron microscopy demonstrated that the permeability barrier was a distinct envelope that formed in a separate step that required fatty acid synthesis, the sugar-modifying enzyme PERM-1, and the acyl chain transfer enzyme DGTR-1. These findings delineate the hierarchy of eggshell assembly and define key molecular mechanisms at each step.
doi:10.1083/jcb.201206008
PMCID: PMC3514041  PMID: 22908315
17.  Visualization of Eukaryotic DNA Mismatch Repair Reveals Distinct Recognition and Repair Intermediates 
Cell  2011;147(5):1040-1053.
Summary
DNA Mismatch Repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae mispairs are primarily detected by the Msh2-Msh6 complex and corrected following subsequent recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. Msh2-Msh6 formed foci in S-phase that colocalized with replication factories; this localized pool accounted for 10–15% of MMR in wild-type cells but was essential for MMR in the absence of the exonuclease Exo1. Mlh1-Pms1 also formed foci that, while requiring Msh2-Msh6 for their formation, rarely colocalized with Msh2-Msh6. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These results suggest that (I) mispair recognition can occur via either a replication factory-targeted or a second distinct pool of Msh2-Msh6, and (II) superstoichiometric Mlh1-Pms1 assembly triggered by mispair-bound Msh2-Msh6 defines sites of active MMR.
doi:10.1016/j.cell.2011.10.025
PMCID: PMC3478091  PMID: 22118461
18.  Rapid De Novo Centromere Formation Occurs Independently of Heterochromatin Protein 1 in C. elegans Embryos 
Current biology : CB  2011;21(21):1800-1807.
Summary
DNA injected into the C. elegans germline forms extrachromosomal arrays that segregate during cell division [1, 2]. The mechanisms underlying array formation and segregation are not known. Here, we show that extrachromosomal arrays form de novo centromeres at high frequency, providing unique access to a process that occurs with extremely low frequency in other systems [3–8]. De novo centromerized arrays recruit centromeric chromatin and kinetochore proteins and autonomously segregate on the spindle. Live imaging following DNA injection revealed that arrays form after oocyte fertilization via homologous recombination and non-homologous end joining. Individual arrays gradually transition from passive inheritance to active segregation during the early embryonic divisions. The Heterochromatin Protein 1 (HP1) family proteins HPL-1 and HPL-2 are dispensable for de novo centromerization even though arrays become strongly enriched for the heterochromatin-associated H3K9me3 modification over time. Partial inhibition of HP1 family proteins accelerates the acquisition of segregation competence. In addition to reporting the first direct visualization of new centromere formation in living cells, these findings reveal that naked DNA rapidly builds de novo centromeres in C. elegans embryos in an HP1-independent manner, and suggest that, rather than being a prerequisite, HP1-dependent heterochromatin antagonizes de novo centromerization.
doi:10.1016/j.cub.2011.09.016
PMCID: PMC3249440  PMID: 22018540
mitosis; centromere; kinetochore; heterochromatin; microtubule; tubulin; cell division; spindle; CenH3; CENP-A; histone variant; HP1
19.  Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore 
The Journal of Cell Biology  2012;196(4):469-482.
A microtubule-binding site in the extreme N terminus of KNL-1 is dispensable for load-bearing attachments but participates in checkpoint silencing at the kinetochore.
Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.
doi:10.1083/jcb.201111107
PMCID: PMC3284002  PMID: 22331849
20.  A High-Resolution C. elegans Essential Gene Network Based on Phenotypic Profiling of a Complex Tissue 
Cell  2011;145(3):470-482.
SUMMARY
High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach—profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes—pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.
doi:10.1016/j.cell.2011.03.037
PMCID: PMC3086541  PMID: 21529718
21.  Affinity Purification of Protein Complexes in C. elegans 
Methods in Cell Biology  2011;106:289-322.
C. elegans is a powerful metazoan model system to address fundamental questions in cell and developmental biology. Research in C. elegans has traditionally focused on genetic, physiological, and cell biological approaches. However, C. elegans is also a facile system for biochemistry: worms are easy to grow in large quantities, the functionality of tagged fusion proteins can be assessed using mutants or RNAi, and the relevance of putative interaction partners can be rapidly tested in vivo. Combining biochemistry with function-based genetic and RNA interference screens can rapidly accelerate the delineation of protein networks and pathways in diverse contexts. In this chapter, we focus on two strategies to identify protein–protein interactions: single-step immunoprecipitation and tandem affinity purification. We describe methods for growth of worms in large-scale liquid culture, preparation of worm and embryo extracts, immunoprecipitation, and tandem affinity purification. In addition, we describe methods to test specificity of antibodies, strategies for optimizing starting material, and approaches to distinguish specific from non-specific interactions.
doi:10.1016/B978-0-12-544172-8.00011-6
PMCID: PMC3319706  PMID: 22118282
22.  A PSHaver for Centromeric Histones 
Molecular cell  2010;40(3):351-352.
doi:10.1016/j.molcel.2010.10.031
PMCID: PMC3003300  PMID: 21070962
23.  The chromosomal passenger complex and centralspindlin independently contribute to contractile ring assembly 
The Journal of Cell Biology  2011;193(1):155-169.
In contrast to their sequential roles in midzone assembly, the CPC and centralspindlin act through independent mechanisms to regulate contractile ring assembly.
The chromosomal passenger complex (CPC) and centralspindlin are conserved cytokinesis regulators that localize to the spindle midzone, which forms between the separating chromosomes. Previous work placed the CPC and centralspindlin in a linear pathway that governs midzone formation. Using Caenorhabditis elegans embryos, we test whether there is a similar linear relationship between centralspindlin and the CPC in contractile ring constriction during cytokinesis. We show that simultaneous inhibition of the CPC kinase Aurora BAIR-2 and the centralspindlin component MKLP1ZEN-4 causes an additive constriction defect. Consistent with distinct roles for the proteins, inhibition of filamentous septin guanosine triphosphatases alleviates constriction defects in Aurora BAIR-2–inhibited embryos, whereas inhibition of Rac does so in MKLP1ZEN-4-inhibited embryos. Centralspindlin and the CPC are not required to enrich ring proteins at the cell equator but instead regulate formation of a compact mature ring. Therefore, in contrast to the linear midzone assembly pathway, centralspindlin and the CPC make independent contributions to control transformation of the sheet-like equatorial band into a ribbon-like contractile ring at the furrow tip.
doi:10.1083/jcb.201008138
PMCID: PMC3082186  PMID: 21464231
24.  Acute Drug Treatment in the Early C. elegans Embryo 
PLoS ONE  2011;6(9):e24656.
Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.
doi:10.1371/journal.pone.0024656
PMCID: PMC3173474  PMID: 21935434
25.  Uncoordinated Loss of Chromatid Cohesion Is a Common Outcome of Extended Metaphase Arrest 
PLoS ONE  2011;6(8):e22969.
Chromosome segregation requires coordinated separation of sister chromatids following biorientation of all chromosomes on the mitotic spindle. Chromatid separation at the metaphase-to-anaphase transition is accomplished by cleavage of the cohesin complex that holds chromatids together. Here we show using live-cell imaging that extending the metaphase bioriented state using five independent perturbations (expression of non-degradable Cyclin B, expression of a Spindly point mutant that prevents spindle checkpoint silencing, depletion of the anaphase inducer Cdc20, treatment with a proteasome inhibitor, or treatment with an inhibitor of the mitotic kinesin CENP-E) leads to eventual scattering of chromosomes on the spindle. This scattering phenotype is characterized by uncoordinated loss of cohesion between some, but not all sister chromatids and subsequent spindle defects that include centriole separation. Cells with scattered chromosomes persist long-term in a mitotic state and eventually die or exit. Partial cohesion loss-associated scattering is observed in both transformed cells and in karyotypically normal human cells, albeit at lower penetrance. Suppressing microtubule dynamics reduces scattering, suggesting that cohesion at centromeres is unable to resist dynamic microtubule-dependent pulling forces on the kinetochores. Consistent with this view, strengthening cohesion by inhibiting the two pathways responsible for its removal significantly inhibits scattering. These results establish that chromosome scattering due to uncoordinated partial loss of chromatid cohesion is a common outcome following extended arrest with bioriented chromosomes in human cells. These findings have important implications for analysis of mitotic phenotypes in human cells and for development of anti-mitotic chemotherapeutic approaches in the treatment of cancer.
doi:10.1371/journal.pone.0022969
PMCID: PMC3149067  PMID: 21829677

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