Biomedical subjects

K Bloom

Publications and source records attributed to K Bloom.

At least 91 records · Page 5Linked to original sources

PubMed2001

Control of spindle polarity and orientation in Saccharomyces cerevisiae.

Control of mitotic spindle orientation represents a major strategy for the generation of cell diversity during development of metazoans. Studies in the budding yeast Saccharomyces cerevisiae have contributed towards our present understanding of the general principles underlying the regulation of spindle positioning in an asymmetrically dividing cell. In S. cerevisiae, the mitotic spindle must orient along the cell polarity axis, defined by the site of bud emergence, to ensure correct nuclear division between the mother and daughter cells. Establishment of spindle polarity dictates this process and relies on the concerted control of spindle pole function and a precise program of cues originating from the cell cortex that directs cytoplasmic microtubule attachments during spindle morphogenesis. These cues cross talk with the machinery responsible for bud-site selection, indicating that orientation of the spindle in yeast cells is mechanistically coupled to the definition of a polarity axis and the division plane. Here, we propose a model integrating the inherently asymmetric properties of the spindle pathway with the program of positional information contributing towards orienting the spindle in budding yeast. Because the basic machinery orienting the spindle in higher-eukaryotic cells appears to be conserved, it might be expected that similar principles govern centrosome asymmetry in the course of metazoan development.

Chromosome Segregation↗

PubMed2001

Phosphorylation of gamma-tubulin regulates microtubule organization in budding yeast.

gamma-Tubulin is essential for microtubule nucleation in yeast and other organisms; whether this protein is regulated in vivo has not been explored. We show that the budding yeast gamma-tubulin (Tub4p) is phosphorylated in vivo. Hyperphosphorylated Tub4p isoforms are restricted to G1. A conserved tyrosine near the carboxy terminus (Tyr445) is required for phosphorylation in vivo. A point mutation, Tyr445 to Asp, causes cells to arrest prior to anaphase. The frequency of new microtubules appearing in the SPB region and the number of microtubules are increased in tub4-Y445D cells, suggesting this mutation promotes microtubule assembly. These data suggest that modification of gamma-tubulin is important for controlling microtubule number, thereby influencing microtubule organization and function during the yeast cell cycle.

Alleles↗

PubMed2001

Comparison of in-patient and out-patient penile prosthesis surgery.

Between December 1996 and December 1998, 79 inflatable penile implant insertions have been performed at our institution by a single surgeon. The objective of this analysis was to compare our in-patient and out-patient experience with penile prosthesis insertion with respect to ease of performance and complication profiles. Data was collected in a prospective manner for both groups (in-patient, n = 33 and out-patient, n = 46). The two groups were compared with respect to intra-operative blood loss, operative time, time lost from work, narcotic use and complication rates. Both groups of patients experienced similar operative blood loss, essentially identical operative times, time lost from work and narcotic use. Most importantly, overall complication rates were 6% for the in-patient group and 4% for the out-patient group. Inflatable penile implant surgery is feasible in an ambulatory surgical setting. There is no difference in complication rates, loss of time from work, or intra-operative and post-operative course. Furthermore, there is a significant saving at our institution by performing the procedure in an out-patient fashion. In-patient prosthetic surgery is reserved for secondary procedures following a prior implant infection or primary implants in men with significant co-morbidities that require in-patient postoperative monitoring.

Aged↗

PubMed2001

ASH1 mRNA localization in three acts.

Novel green fluorescent protein (GFP) labeling techniques targeting specific mRNA transcripts reveal discrete phases of mRNA localization in yeast: packaging, transport, and docking. In budding yeast, ASH1 mRNA is translocated via actin and myosin to the tip of growing cells. A GFP-decorated reporter transcript containing the ASH1 3' untranslated region gRNA(ASH1) forms spots of fluorescence localized to a cortical domain at the bud tip, relocates to the mother-bud neck before cell separation, and finally migrates to the incipient bud site before the next budding cycle. The correct positioning of the mRNA requires at least six proteins: She1p-5p and Bud6p/Aip3p. gRNA(ASH1) localization in mutant strains identified three functional categories for the She proteins: mRNA particle formation (She2p and She4p), mRNA transport into the bud (She1p/Myo4p and She3p), and mRNA tethering at the bud tip (She5p/Bni1p and Bud6p/Aip3p). Because localization of the mRNA within the bud does not a priori restrict the translated protein, we examine the distribution of a mother-specific protein (Yta6p) translated from a mRNA directed into the bud. Yta6p remains associated with the mother cortex despite localization of the mRNA to the bud. This video essay traces the life history of a localized mRNA transcript, describes the roles of proteins required to polarize and anchor the mRNA, and demonstrates at least one instance where mRNA localization does not effect protein localization.

Biological Transport↗

PubMed2001

Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.

We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

Anaphase↗

PubMed2001

Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast.

Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing alpha-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.

Animals↗

PubMed2000

The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast.

BACKGROUND: Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions. RESULTS: Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration. CONCLUSIONS: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration.

Actins↗

PubMed2000

Measurement of J/psi and psi(2S) polarization in pp collisions at sqrt[s] = 1.8 TeV.

We have measured the polarization of J/psi and psi(2S) mesons produced in pp collisions at sqrt[s] = 1.8 TeV, using data collected at the Collider Detector at Fermilab during 1992-1995. The polarization of promptly produced J/psi [psi(2S)] mesons is isolated from those produced in B-hadron decay, and measured over the kinematic range 4 [5.5]<P(T)<20 GeV/c and |y|<0.6. For P(T) greater than or approximately equal 12 GeV/c we do not observe significant polarization in the prompt component.

Journal Article↗

PubMed2000

Search for scalar top and scalar bottom quarks in pp collisions at square root s=1.8 TeV.

We have searched for direct pair production of scalar top and scalar bottom quarks in 88 pb-1 of pp collisions at sqrt[s]=1.8 TeV with the CDF detector. We looked for events with a pair of heavy flavor jets and missing energy, consistent with scalar top (bottom) quark decays to a charm (bottom) quark and a neutralino. The numbers of events that pass our selections show no significant deviation from standard model expectations. We compare our results to the next-to-leading order scalar quark production cross sections to exclude regions in scalar quark-neutralino mass parameter space.

Journal Article↗

PubMed2000

Search for a W' boson via the decay mode W'-->munumu in 1.8 TeV pp collisions.

We report the results of a search for a W' boson produced in pp collisions at a center-of-mass energy of 1.8 TeV using a 107 pb-1 data sample recorded by the Collider Detector at Fermilab. We consider the decay channel W'-->&munumu and search for anomalous production of high transverse mass munumu lepton pairs. We observe no excess of events above background and set limits on the rate of W' boson production and decay relative to standard model W boson production and decay using a fit of the transverse mass distribution observed. If we assume standard model strength couplings of the W' boson to quark and lepton pairs, we exclude a W' boson with invariant mass less than 660 GeV/c2 at 95% confidence level.

Journal Article↗

PubMed2000

Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast.

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

CDC28 Protein Kinase, S cerevisiae↗

PubMed2000

It's a kar9ochore to capture microtubules.

Microtubule orientation to cortical spatial cues is essential for the fidelity of asymmetric cellular processes. A cortical microtubule-capture site, composed of Bim1 and Kar9, has now been identified in yeast. Bim1 is the yeast homologue of EB1, a binding partner of the adenomatous polyposis coli (APC), indicating that important features of this complex may be highly conserved.

Cell Cycle Proteins↗

PubMed2000

Bud6 directs sequential microtubule interactions with the bud tip and bud neck during spindle morphogenesis in Saccharomyces cerevisiae.

In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule-cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule-neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Delta mutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule-cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity.

CDC28 Protein Kinase, S cerevisiae↗

PubMed2000

Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants.

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.

Anaphase↗

PubMed2000

Intrathecal baclofen for management of spastic cerebral palsy: multicenter trial.

Intrathecal baclofen infusion has demonstrated effectiveness in decreasing spasticity of spinal origin. Oral antispasticity medication is minimally effective or not well tolerated in cerebral palsy. This study assessed the effectiveness of intrathecal baclofen in reducing spasticity in cerebral palsy. Candidates were screened by randomized, double-blind, intrathecal injections of baclofen and placebo. Responders were defined as those who experienced an average reduction of 1.0 in the lower extremities on the Ashworth Scale for spasticity. Responders received intrathecal baclofen via the SynchroMed System and were followed for up to 43 months. Fifty-one patients completed screening and 44 entered open-label trials. Lower-extremity spasticity decreased from an average baseline score of 3.64 to 1.90 at 39 months. A decrease in upper extremity spasticity was evidenced over the same study period. Forty-two patients reported adverse events. Most common reports were hypotonia, seizures (no new onset), somnolence, and nausea or vomiting. Fifty-nine percent of the patients experienced procedural or system-related events. Spasticity in patients with cerebral palsy can be treated effectively by continuous intrathecal baclofen. Adverse events, although common, were manageable.

Adolescent↗

PubMed1999

The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle.

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

Actins↗

PubMed1999

Localization and anchoring of mRNA in budding yeast.

BACKGROUND: Eukaryotic cells localize selected mRNAs to a region of the cell as a means to sequester proteins. Signals within the 3' untranslated region (3' UTR) facilitate mRNA localization by both actin and microtubule cytoskeletal systems. Recently, an mRNA in the yeast Saccharomyces cerevisiae, ASH1, was shown to coalesce into a discrete particle that is maintained at the bud tip. Mutations in five genes, SHE1-SHE5, cause defects in particle formation and/or localization of the ASH1 transcript. Factors at the destination of the mRNA transport remain to be identified. RESULTS: We have developed a system to label mRNA in living yeast with green fluorescent protein (GFP) and follow the dynamics of mRNA movement and localization. Constitutively expressing an ASH1 mRNA containing the bacteriophage MS2 coat-protein binding site adjacent to the ASH1 3' UTR allowed us to visualize ASH1 mRNA with an MS2-coat-protein-GFP fusion protein (together denoted 'gRNAASH1'). The gRNAASH1 was restricted to the bud tip in small to large budded cells, migrated to the bud neck prior to cell separation and then rapidly relocalized to the incipient site of bud growth. It also localized to regions of polarized growth during mating. In cells lacking Bud6p/Aip3p or Bnilp/She5p, which are involved in polarity establishment and actin organization, gRNAASH1 migrated to the bud but failed to remain at the bud tip. These studies reveal discrete transport and anchoring steps in mRNA localization. CONCLUSIONS: The ASH1 mRNA was maintained at sites of polarized growth throughout the vegetative and mating cell cycles. Bud6p/Aip3p and Bni1p/She5p are required to maintain the transcript at the cortical bud cap.

DNA-Binding Proteins↗

PubMed1999

Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae.

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.

Cell Fusion↗