A switch in microtubule dynamics at the onset of anaphase B in the mitotic spindle of Schizosaccharomyces pombe.
Microtubule dynamics have key roles in mitotic spindle assembly and chromosome movement . Fast turnover of spindle microtubules at metaphase and polewards flux of microtubules (polewards movement of the microtubule lattice with depolymerization at the poles) at both metaphase and anaphase have been observed in mammalian cells . Imaging spindle dynamics in genetically tractable yeasts is now possible using green fluorescent protein (GFP)-tagging of tubulin and sites on chromosomes      . We used photobleaching of GFP-labeled tubulin to observe microtubule dynamics in the fission yeast Schizosaccharomyces pombe. Photobleaching did not perturb progress through mitosis. Bleached marks made on the spindle during metaphase recovered their fluorescence rapidly, indicating fast microtubule turnover. Recovery was spatially non-uniform, but we found no evidence for polewards flux. Marks made during anaphase B did not recover fluorescence, and were observed to slide away from each other at the same rate as spindle elongation. Fast microtubule turnover at metaphase and a switch to stable microtubules at anaphase suggest the existence of a cell-cycle-regulated molecular switch that controls microtubule dynamics and that may be conserved in evolution. Unlike the situation for vertebrate spindles, microtubule depolymerization at poles and polewards flux may not occur in S. pombe mitosis. We conclude that GFP-tubulin photobleaching in conjunction with mutant cells should aid research on molecular mechanisms causing and regulating dynamics. (+info)
Direct visualization of the movement of the monomeric axonal transport motor UNC-104 along neuronal processes in living Caenorhabditis elegans.
The formation and function of axons depends on the microtubule-based transport of cellular components from their sites of synthesis in the neuronal cell body to their sites of utilization at the axon terminus. To directly visualize this axonal transport in a living organism, we constructed transgenic lines of Caenorhabditis elegans that express green fluorescent protein fused to the monomeric synaptic vesicle transport motor, UNC-104. This UNC-104:: GFP construct rescued the Unc-104 mutant phenotype and was expressed throughout the nervous system. Using time-lapse confocal fluorescence microscopy, we were able to visualize fluorescent motor proteins moving in both directions along neuronal processes, some of which were identified definitely as axons and others as dendrites. Using kymograph analysis, we followed the movement of >900 particles. Most of them moved in one direction, but not necessarily at the same velocity. Ten percent of the observed particles reversed direction of movement during the period of observation, and 10% exhibited periods of movement interspersed with pauses. During episodes of persistent movement, particles moved at an average velocity of 1.02 microm/sec, which is close to the in vitro velocity of microtubule gliding driven by purified monomeric kinesin at high motor density. To our knowledge, this is the first direct visualization and analysis of the movement of specifically labeled microtubule motor proteins along axons in vivo. (+info)
Motility in Echinosphaerium nucleofilum. I. An analysis of particle motions in the axopodia and a direct test of the involvement of the axoneme.
The motion of particles in the axopodia of Echinosphaerium nucleofilum is saltatory. In the present study, photokymograph records of 123 motions from six axopodia have been analyzed. Particles followed rectilinear paths of from 1 to 15 mum while in continuous motion at an average velocity of 0.66 plus or minus 0.32 mum/s. The velocity of the particles was variable in 36% of the cases measured. Some motions were punctuated by pauses either before continuing in the same direction or reversing. Frequently, several particles were moving at the same velocity, but neighboring particles showed no motion or moved in the opposite direction. Two particles occasionally contacted one another and travelled as a unit for varying lengths of time but subsequently moved independently. These motions reflect the underlying mechanism of motive force production. Furthermore, a glass microneedle can be substituted for the microtubular axoneme in the axopodia. In these artificial axopodia, bidirectional particle motions occurred which were similar to those in normal axopodia. Colchicine, at the threshold dose for axonemal dissolution, had no affect on these particel motions. It is concluded that the microtubular axoneme is not responsible for particle motions and also that individual microtubules are unlikely candidates for motive force production in this system. (+info)
Experimental device for detecting laryngeal movement during swallowing.
It has been reported that swallowing is a rhythmic movement, in which the onset of the oro-pharyngeal stage of swallowing starts from the mylohyoid muscle, followed by movement of the oral and pharyngeal muscles, and reaching the superior esophageal sphincter muscle. This is defined as the oro-pharyngeal stage of swallowing. It has also been reported that along with this movement, the larynx elevates in an antero-superior direction. To investigate the swallowing movement, it would be useful to be able to detect the start of swallowing movements from the body surface. Such a device was designed in this study to investigate the relationships between the onset of laryngeal movement and the EMG initiation of the anterior digastric muscle. Although experimental conditions must be further examined, we were able to record the reproducible movement and the position of larynx using our device provides another tool for studying the swallowing movement. (+info)
C. elegans PAR proteins function by mobilizing and stabilizing asymmetrically localized protein complexes.
BACKGROUND: The PAR proteins are part of an ancient and widely conserved machinery for polarizing cells during animal development. Here we use a combination of genetics and live imaging methods in the model organism Caenorhabditis elegans to dissect the cellular mechanisms by which PAR proteins polarize cells. RESULTS: We demonstrate two distinct mechanisms by which PAR proteins polarize the C. elegans zygote. First, we show that several components of the PAR pathway function in intracellular motility, producing a polarized movement of the cell cortex. We present evidence that this cortical motility may drive the movement of cellular components that must become asymmetrically distributed, including both germline-specific ribonucleoprotein complexes and cortical domains containing the PAR proteins themselves. Second, PAR-1 functions to refine the asymmetric localization of germline ribonucleoprotein complexes by selectively stabilizing only those complexes that reach the PAR-1-enriched posterior cell cortex during the period of cortical motility. CONCLUSIONS: These results identify two cellular mechanisms by which the PAR proteins polarize the C. elegans zygote, and they suggest mechanisms by which PAR proteins may polarize cells in diverse animal systems. (+info)
Laser microsurgery in fission yeast; role of the mitotic spindle midzone in anaphase B.
INTRODUCTION: During anaphase B in mitosis, polymerization and sliding of overlapping spindle microtubules (MTs) contribute to the outward movement the spindle pole bodies (SPBs). To probe the mechanism of spindle elongation, we combine fluorescence microscopy, photobleaching, and laser microsurgery in the fission yeast Schizosaccharomyces pombe. RESULTS: We demonstrate that a green laser cuts intracellular structures in yeast cells with high spatial specificity. By using laser microsurgery, we cut mitotic spindles labeled with GFP-tubulin at various stages of anaphase B. Although cutting generally caused early anaphase spindles to disassemble, midanaphase spindle fragments continued to elongate. In particular, when the spindle was cut near a SPB, the larger spindle fragment continued to elongate in the direction of the cut. Photobleach marks showed that sliding of overlapping midzone MTs was responsible for the elongation of the spindle fragment. Spindle midzone fragments not connected to either of the two spindle poles also elongated. Equatorial microtubule organizing center (eMTOC) activity was not affected in cells with one detached pole but was delayed or absent in cells with two detached poles. CONCLUSIONS: These studies reveal that the spindle midzone is necessary and sufficient for the stabilization of MT ends and for spindle elongation. By contrast, SPBs are not required for elongation, but they contribute to the attachment of the nuclear envelope and chromosomes to the spindle, and to cell cycle progression. Laser microsurgery provides a means by which to dissect the mechanics of the spindle in yeast. (+info)
Cortactin promotes cell motility by enhancing lamellipodial persistence.
BACKGROUND: Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration. RESULTS: Cells deficient in cortactin have impaired cell migration and invasion. Kymography analyses of live-cell imaging studies demonstrate that cortactin-knockdown cells have a selective defect in the persistence of lamellipodial protrusions. The motility and protrusion defects are fully rescued by cortactin molecules, provided both the Arp2/3 complex and F-actin binding sites are intact. Consistent with this requirement for simultaneous contacts with Arp2/3 and F-actin, cortactin is recruited by Arp2/3 complex to lamellipodia and binds with a higher affinity to ATP/ADP-Pi-F-actin than to ADP-F-actin. In situ labeling of lamellipodia revealed that the relative levels of free barbed ends of actin filaments are reduced by over 30% in the cortactin-knockdown cells; however, there is no change in Arp2/3-complex localization to lamellipodia. Cortactin-knockdown cells also have a selective defect in the assembly of new adhesions in protrusions, as assessed by analysis of GFP-paxillin dynamics in living cells. CONCLUSIONS: Cortactin enhances lamellipodial persistence, at least in part through regulation of Arp2/3 complex. The presence of cortactin also enhances the rate of new adhesion formation in lamellipodia. In vivo, these functions may be important during directed cell motility. (+info)
Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella.
Intraflagellar transport (IFT) of particles along flagellar microtubules is required for the assembly and maintenance of eukaryotic flagella and cilia. In Chlamydomonas, anterograde and retrograde particles viewed by light microscopy average 0.12-microm and 0.06-microm diameter, respectively. Examination of IFT particle structure in growing flagella by electron microscopy revealed similar size aggregates composed of small particles linked to each other and to the membrane and microtubules. To determine the relationship between the number of particles and flagellar length, the rate and frequency of IFT particle movement was measured in nongrowing, growing, and shortening flagella. In all flagella, anterograde and retrograde IFT averaged 1.9 microm/s and 2.7 microm/s, respectively, but retrograde IFT was significantly slower in flagella shorter than 4 mum. The number of flagellar IFT particles was not fixed, but depended on flagellar length. Pauses in IFT particle entry into flagella suggest the presence of a periodic "gate" that permits up to 4 particles/s to enter a flagellum. (+info)