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Kerry Bloom

Publications and source records attributed to Kerry Bloom.

At least 19 recordsLinked to original sources
PubMed2006

Mps1 phosphorylation of Dam1 couples kinetochores to microtubule plus ends at metaphase.

BACKGROUND: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends. RESULTS: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells. CONCLUSIONS: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.

Cell Cycle Proteins↗
PubMed2006

Measuring nanometer scale gradients in spindle microtubule dynamics using model convolution microscopy.

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at approximately 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A beta-tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length approximately 14%, and decreases tension at kinetochores. The beta-tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.

Chromosome Segregation↗
PubMed2006

NoCut: cytokinesis in check.

In this issue of Cell, Norden et al. (2006) describe a new pathway, NoCut, that links the completion of chromosome segregation to the onset of cytokinesis in budding yeast. In NoCut, Aurora kinase (Ipl1) at the spindle midzone negatively regulates cytokinesis through two proteins previously identified to be involved in cell polarity, Boi1 and Boi2.

Aurora Kinases↗
PubMed2005

Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae.

Nuclear movement before karyogamy in eukaryotes is known as pronuclear migration or as nuclear congression in Saccharomyces cerevisiae. In this study, S. cerevisiae is used as a model system to study microtubule (MT)-dependent nuclear movements during mating. We find that nuclear congression occurs through the interaction of MT plus ends rather than sliding and extensive MT overlap. Furthermore, the orientation and attachment of MTs to the shmoo tip before cell wall breakdown is not required for nuclear congression. The MT plus end-binding proteins Kar3p, a class 14 COOH-terminal kinesin, and Bik1p, the CLIP-170 orthologue, localize to plus ends in the shmoo tip and initiate MT interactions and depolymerization after cell wall breakdown. These data support a model in which nuclear congression in budding yeast occurs by plus end MT capture and depolymerization, generating forces sufficient to move nuclei through the cytoplasm. This is the first evidence that MT plus end interactions from oppositely oriented organizing centers can provide the force for organelle transport in vivo.

Cell Cycle Proteins↗
PubMed2005

Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex.

Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3-Exo70 and Rho1-Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.

Biomarkers↗
PubMed2005

Chromosome segregation: seeing is believing.

For chromosome segregation in mitosis, each centromere directs assembly of a complex, proteinaceous structure - the kinetochore, which connects the chromosome to microtubules of the mitotic spindle. A recent study has provided important new insights into the mechanism by which kinetochores capture spindle microtubules.

Chromosome Segregation↗
PubMed2005

Tension-dependent regulation of microtubule dynamics at kinetochores can explain metaphase congression in yeast.

During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.

Cell Cycle Proteins↗
PubMed2005

Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis.

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.

Amino Acid Sequence↗
PubMed2005

The role of centromere-binding factor 3 (CBF3) in spindle stability, cytokinesis, and kinetochore attachment.

The spindle midzone is critical for spindle stability and cytokinesis. Chromosomal passenger proteins relocalize from chromosomes to the spindle midzone after anaphase onset. The recent localization of the inner-kinetochore, centromere-binding factor 3 (CBF3) complex to the spindle midzone in budding yeast has led to the discovery of novel functions for this complex in addition to its essential role at kinetochores. In G1/S cells, CBF3 components are detected along dynamic microtubules, where they can "search-and-capture" newly replicated centromeres. During anaphase, CBF3 is transported to the microtubule plus-ends of the spindle midzone. Consistent with this localization, cells containing a mutation in the CBF3 subunit Ndc10p show defects in spindle stability during anaphase. In addition, ndc10-1 cells show defects during cytokinesis, resulting in a defect in cell abscission. These results highlight the importance of midzone-targeted proteins in coordinating mitosis with cell division. Here we discuss these findings and explore the significance of CBF3 transport to microtubule plus-ends at the spindle midzone.

Cell Cycle↗
PubMed2004

Chromosome fragmentation after induction of a double-strand break is an active process prevented by the RMX repair complex.

Chromosome aberrations are common outcomes of exposure to DNA-damaging agents or altered replication events and are associated with various diseases and a variety of carcinomas, including leukemias, lymphomas, sarcomas, and epithelial tumors. The incidence of aberrations can be greatly increased as a result of defects in DNA repair pathways. Although there is considerable information about the molecular events associated with the induction and repair of a double-strand break (DSB), little is known about the events that ultimately lead to translocations or deletions through the formation of chromosome breaks or the dissociation of broken ends. We describe a system for visualizing DNA ends at the site of a DSB in living cells. After induction of the break, DNA ends flanking the DSB site in wild-type cells remained adjacent. Loss of a functional RMX complex (Rad50/Mre11/Xrs2) or a mutation in the Rad50 Zn-hook structure resulted in DNA ends being dispersed in approximately 10%-20% of cells. Thus, the RMX complex holds broken ends together and counteracts mitotic spindle forces that can be destructive to damaged chromosomes.

Blotting, Southern↗
PubMed2004

Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase.

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.

Centromere↗
PubMed2004

The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit.

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.

Anaphase↗
PubMed2003

The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast.

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.

Binding Sites↗
PubMed2003

Yeast kinetochores do not stabilize Stu2p-dependent spindle microtubule dynamics.

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule-kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.

Benomyl↗
PubMed2003

Microtubule cytoskeleton: navigating the intracellular landscape.

Recent studies have significantly advanced our understanding of how a dividing cell asymmetrically positions the mitotic spindle--a key process in metazoan development--while maintaining a dynamic spindle state that can respond and reorient when necessary.

Animals↗