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E D Salmon

Publications and source records attributed to E D Salmon.

At least 37 records · Page 2Linked to original sources
PubMed2004

Microtubule plus-end dynamics in Xenopus egg extract spindles.

Microtubule dynamics underlie spindle assembly, yet we do not know how the spindle environment affects these dynamics. We developed methods for measuring two key parameters of microtubule plus-end dynamic instability in Xenopus egg extract spindles. To measure plus-end polymerization rates and localize growing plus ends, we used fluorescence confocal imaging of EB1. This revealed plus-end polymerization throughout the spindle at approximately 11 microm/min, similar to astral microtubules, suggesting polymerization velocity is not regionally regulated by the spindle. The ratio of EB1 to microtubule fluorescence revealed an enrichment of polymerizing ends near the spindle middle, indicating enhanced nucleation or rescue there. We measured depolymerization rates by creating a front of synchronized depolymerization in spindles severed with microneedles. This front could be tracked by polarization and fluorescence microscopy as it advanced from each cut edge toward the associated pole. Both imaging modalities revealed rapid depolymerization ( approximately 30 microm/min) superimposed on a subset of microtubules stable to depolymerization. Larger spindle fragments contained a higher percentage of stable microtubules, which we believe were oriented with their minus ends facing the cut. Depolymerization was blocked by the potent microtubule stabilizing agent hexylene glycol, but was unaffected by alpha-MCAK antibody and AMPPNP, which block catastrophe and kinesin motility, respectively. These measurements move us closer to understanding the complete life history of a spindle microtubule.

Adenylyl Imidodiphosphate↗
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

Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores.

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.

Calcium-Binding Proteins↗
PubMed2003

Merotelic kinetochore orientation occurs frequently during early mitosis in mammalian tissue cells and error correction is achieved by two different mechanisms.

Merotelic kinetochore orientation is an error that occurs when a single kinetochore becomes attached to microtubules from two spindle poles rather than just to one pole. We obtained the first evidence that merotelic kinetochore orientation occurs very frequently during early mitosis in mammalian tissue cells and that two different correction mechanisms are critical for accurate chromosome segregation in cells possessing bipolar spindles and unperturbed chromosomes. Our data show that about 30% of prometaphase PtK1 cells possess one or more merotelically oriented kinetochores. This frequency is increased to over 90% in cells recovering from a nocodazole-induced mitotic block. A delay in establishing spindle bipolarity is responsible for the high frequency of merotelic orientations seen in cells recovering from nocodazole, but not in untreated cells. The frequency of anaphase cells with merotelically oriented lagging chromosomes is 1% in untreated cells and 18% in cells recovering from nocodazole. Prolonging metaphase by 2 hours reduced the frequency of anaphase cells with lagging chromosomes both for untreated and for nocodazole-treated cells. Surprisingly, anaphase lagging chromosomes represented a very small fraction of merotelic kinetochore orientations present in late metaphase. Our data indicate that two correction mechanisms operate to prevent chromosome missegregation due to merotelic kinetochore orientation. The first, a pre-anaphase correction mechanism increases the ratio of kinetochore microtubules attached to the correct versus incorrect pole and might eventually result in kinetochore reorientation before anaphase onset. The increase in microtubule ratio to opposite poles is the groundwork for a second mechanism, active in anaphase, that promotes the segregation of merotelically oriented chromosomes to the correct pole.

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

Determining the position of the cell division plane.

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.

Anaphase↗
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

PAR proteins regulate microtubule dynamics at the cell cortex in C. elegans.

BACKGROUND: The PAR proteins are known to be localized asymmetrically in polarized C. elegans, Drosophila, and human cells and to participate in several cellular processes, including asymmetric cell division and spindle orientation. Although astral microtubules are known to play roles in these processes, their behavior during these events remains poorly understood. RESULTS: We have developed a method that makes it possible to examine the residence time of individual astral microtubules at the cell cortex of developing embryos. Using this method, we found that microtubules are more dynamic at the posterior cortex of the C. elegans embryo compared to the anterior cortex during spindle displacement. We further observed that this asymmetry depends on the PAR-3 protein and heterotrimeric G protein signaling, and that the PAR-2 protein affects microtubule dynamics by restricting PAR-3 activity to the anterior of the embryo. CONCLUSIONS: These results indicate that PAR proteins function to regulate microtubule dynamics at the cortex during microtubule-dependent cellular processes.

Animals↗
PubMed2003

Recovery, visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study.

Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system.

Actins↗
PubMed2003

Mechanisms of microtubule-based kinetochore positioning in the yeast metaphase spindle.

It has been hypothesized that spatial gradients in kMT dynamic instability facilitate mitotic spindle formation and chromosome movement. To test this hypothesis requires the analysis of kMT dynamics, which have not been resolved at the single kMT level in living cells. The budding yeast spindle offers an attractive system in which to study kMT dynamics because, in contrast to animal cells, there is only one kMT per kinetochore. To visualize metaphase kMT plus-end dynamics in yeast, a strain containing a green fluorescent protein fusion to the kinetochore protein, Cse4, was imaged by fluorescence microscopy. Although individual kinetochores were not resolvable, we found that models of kMT dynamics could be evaluated by simulating the stochastic kMT dynamics and then simulating the fluorescence imaging of kMT plus-end-associated kinetochores. Statistical comparison of model-predicted images to experimentally observed images demonstrated that a pure dynamic instability model for kMT dynamics in the yeast metaphase spindle was unacceptable. However, when a temporally stable spatial gradient in the catastrophe or rescue frequency was added to the model, there was reasonable agreement between the model and the experiment. These results provide the first evidence of temporally stable spatial gradients of kMT catastrophe and/or rescue frequency in living cells.

Cell Movement↗
PubMed2002

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells.

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.

Calcium-Binding Proteins↗
PubMed2002

Poleward microtubule flux is a major component of spindle dynamics and anaphase a in mitotic Drosophila embryos.

During cell division, eukaryotic cells assemble dynamic microtubule-based spindles to segregate replicated chromosomes. Rapid spindle microtubule turnover, likely derived from dynamic instability, has been documented in yeasts, plants and vertebrates. Less studied is concerted spindle microtubule poleward translocation (flux) coupled to depolymerization at spindle poles. Microtubule flux has been observed only in vertebrates, although there is indirect evidence for it in insect spermatocytes and higher plants. Here we use fluorescent speckle microscopy (FSM) to demonstrate that mitotic spindles of syncytial Drosophila embryos exhibit poleward microtubule flux, indicating that flux is a widely conserved property of spindles. By simultaneously imaging chromosomes (or kinetochores) and flux, we provide evidence that flux is the dominant mechanism driving chromosome-to-pole movement (anaphase A) in these spindles. At 18 degrees C and 24 degrees C, separated sister chromatids moved poleward at average rates (3.6 and 6.6 microm/min, respectively) slightly greater than the mean rates of poleward flux (3.2 and 5.2 microm/min, respectively). However, at 24 degrees C the rate of kinetochore-to-pole movement varied from slower than to twice the mean rate of flux, suggesting that although flux is the dominant mechanism, kinetochore-associated microtubule depolymerization contributes to anaphase A.

Anaphase↗
PubMed2002

Anaphase onset does not require the microtubule-dependent depletion of kinetochore and centromere-binding proteins.

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.

Anaphase↗
PubMed2002

Chromosome dynamics: new light on Aurora B kinase function.

Aurora B family kinases play an essential role in chromosome segregation and cytokinesis. Recent work suggests that the kinase activity is required for bipolar chromosome orientation, kinetochore assembly, spindle checkpoint and microtubule dynamics. Aurora B also has additional functions in chromosome condensation and cohesion.

Animals↗
PubMed2002

Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching.

FtsZ, the major cytoskeletal component of the bacterial cell-division machine, assembles into a ring (the Z-ring) that contracts at septation. FtsZ is a bacterial homolog of tubulin, with similar tertiary structure, GTP hydrolysis, and in vitro assembly. We used green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching to show that the E. coli Z-ring is extremely dynamic, continually remodeling itself with a half-time of 30 s. ZipA, a membrane protein involved in cell division that colocalizes with FtsZ, was equally dynamic. The Z-ring of the mutant ftsZ84, which has 1/10 the guanosine triphosphatase activity of wild-type FtsZ in vitro, showed a 9-fold slower turnover in vivo. This finding implies that assembly dynamics are determined primarily by GTP hydrolysis. Despite the greatly reduced assembly dynamics, the ftsZ84 cells divide with a normal cell-cycle time.

Bacterial Proteins↗