Biomedical subjects
Publications and source records attributed to K Bloom.
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.
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.
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.
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.
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.
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.
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.
Techniques to label mRNA with green fluorescent protein (GFP) have provided the first real-time images of RNA motility in live yeast cells. Genetic screens for factors responsible for mRNA asymmetry (e. g. SHE genes) in yeast identified type V myosin among other proteins. Analysis of mRNA movement in various she mutants revealed the role of motor proteins in long-range transport, factors for particle formation, and cortical anchors for docking the mRNA.
Patients with Klippel-Trenaunay (KT) syndrome have a complex constellation of anomalies that includes cutaneous capillary malformation (usually on an affected limb), abnormal development of the deep and superficial veins, and limb asymmetry, usually enlargement. Mixed vascular malformations may be present and include capillary, venous, arterial, and lymphatic systems. The records of 79 patients referred for vascular anomalies were reviewed and 49 were found to have the three "cardinal" anomalies of KT syndrome. Twenty-six females and 23 males had 46 affected legs (27 right legs), 23 affected arms (15 right), 21 affected trunks, and 10 affected heads. Thirty-six had only one affected quadrant, 8 had two, and 5 had three or more. Although 40 patients had increased limb girth, measurable length discrepancy was noted in only 17 individuals. Patients were evaluated using a noninvasive imaging strategy including color duplex ultrasonography, MRI, lymphoscintigraphy, and plain radiographs. Treatment included compression, pulsed-dye laser treatment, reduction of arteriovenous malformations, and orthopedic procedures for overgrowth. All KT cases in this series occurred sporadically. We speculate that KT syndromes may be due to a somatic mutation for a factor critical to vasculogenesis and angiogenesis in embryonic development.
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Localization of dynein-green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at approximately 0.3-1.5 micron/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.
Tagging expressed proteins with the green fluorescent protein (GFP) from Aequorea victoria [1] is a highly specific and sensitive technique for studying the intracellular dynamics of proteins and organelles. We have developed, as a probe, a fusion protein of the carboxyl terminus of dynein and GFP (dynein-GFP), which fluorescently labels the astral microtubules of the budding yeast Saccharomyces cerevisiae. This paper describes the modifications to our multimode microscope imaging system [2,3], the acquisition of three-dimensional (3-D) data sets and the computer processing methods we have developed to obtain time-lapse recordings of fluorescent astral microtubule dynamics and nuclear movements over the complete duration of the 90-120 minute yeast cell cycle. This required low excitation light intensity to prevent GFP photobleaching and phototoxicity, efficient light collection by the microscope optics, a cooled charge-coupled device (CCD) camera with high quantum efficiency, and image reconstruction from serial optical sections through the 6 micron-wide yeast cell to see most or all of the astral molecules. Methods are also described for combining fluorescent images of the microtubules labeled with dynein-GFP with high resolution differential interference contrast (DIC) images of nuclear and cellular morphology [4], and fluorescent images of the chromosomes stained with 4,6-diamidino-2-phenylindole (DAPI) [5].
Activation of a facultative, dicentric chromosome provides a unique opportunity to introduce a double strand DNA break into a chromosome at mitosis. Time lapse video enhanced-differential interference contrast analysis of the cellular response upon dicentric activation reveals that the majority of cells initiates anaphase B, characterized by pole-pole separation, and pauses in mid-anaphase for 30-120 min with spindles spanning the neck of the bud before completing spindle elongation and cytokinesis. The length of the spindle at the delay point (3-4 microm) is not dependent on the physical distance between the two centromeres, indicating that the arrest represents surveillance of a dicentric induced aberration. No mid-anaphase delay is observed in the absence of the RAD9 checkpoint gene, which prevents cell cycle progression in the presence of damaged DNA. These observations reveal RAD9-dependent events well past the G2/M boundary and have considerable implications in understanding how chromosome integrity and the position and state of the mitotic spindle are monitored before cytokinesis.
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