Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast.
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ABSTRACT:
SUBMITTER: Omer S
PROVIDER: S-EPMC7578358 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Dynein is a huge multisubunit microtubule (MT)-based motor, whose motor domain resides in the heavy chain. The heavy chain comprises a ring of six AAA (ATPases associated with diverse cellular activities) modules with two slender protruding domains, the tail and stalk. It has been proposed that during the ATP hydrolysis cycle, this tail domain swings against the AAA ring as a lever arm to generate the power stroke. However, there is currently no direct evidence to support the model that the tail swing is tightly linked to dynein motility. To address the question of whether the power stroke of the tail drives MT sliding, we devised an in vitro motility assay using genetically biotinylated cytoplasmic dyneins anchored on a glass surface in the desired orientation with a biotin-streptavidin linkage. Assays on the dyneins with the site-directed biotin tag at eight different locations provided evidence that robust MT sliding is driven by the power stroke of the tail. Furthermore, the assays revealed slow MT sliding independent of dynein orientation on the glass surface, which is mechanically distinct from the sliding driven by the power stroke of the tail.
| S-EPMC1634414 | biostudies-literature
Project description:The ring-shaped cohesin complex is thought to topologically hold sister chromatids together from their synthesis in S phase until chromosome segregation in mitosis. How cohesin stably binds to chromosomes for extended periods, without impeding other chromosomal processes that also require access to the DNA, is poorly understood. Budding yeast cohesin is loaded onto DNA by the Scc2-Scc4 cohesin loader at centromeres and promoters of active genes, from where cohesin translocates to more permanent places of residence at transcription termination sites. Here we show that, at the GAL2 and MET17 loci, pre-existing cohesin is pushed downstream along the DNA in response to transcriptional gene activation, apparently without need for intermittent dissociation or reloading. We observe translocation intermediates and find that the distribution of most chromosomal cohesin is shaped by transcription. Our observations support a model in which cohesin is able to slide laterally along chromosomes while maintaining topological contact with DNA. In this way, stable cohesin binding to DNA and enduring sister chromatid cohesion become compatible with simultaneous underlying chromosomal activities, including but maybe not limited to transcription.
| S-EPMC4929932 | biostudies-literature
Project description:The ring-shaped cohesin complex is thought to topologically hold sister chromatids together from their synthesis in S-phase until chromosome segregation in mitosis. How cohesin stably binds to chromosomes for extended periods, without impeding other chromosomal processes that also require access to the DNA, is poorly understood. Budding yeast cohesin is loaded onto DNA by the Scc2-Scc4 cohesin loader at centromeres and promoters of active genes, from where cohesin translocates to more permanent places of residence at transcription termination sites. Here we show that, at the GAL2 and MET17 loci, pre-existing cohesin is pushed downstream along the DNA in response to transcriptional gene activation, apparently without need for intermittent dissociation or reloading. We observe translocation intermediates and find that the distribution of most chromosomal cohesin is shaped by transcription. Our observations support a model in which cohesin is able to slide laterally along chromosomes while maintaining topological contact with DNA. In this way, stable cohesin binding to DNA and enduring sister chromatid cohesion become compatible with simultaneous underlying chromosomal activities, including but maybe not limited to transcription.
2016-04-21 | GSE80464 | GEO
Project description:Microtubules (MTs) are important for cellular structure, transport of cargoes and segregation of chromosomes and organelles during mitosis. The stochastic growth and shrinkage of MTs, known as dynamic instability, is necessary for these functions. Previous studies to determine how individual MT-associated proteins (MAPs) affect MT dynamics have been performed either through in vivo studies, which provide limited opportunity for observation of individual MTs or manipulation of conditions, or in vitro studies, which focus either on purified proteins, and therefore lack cellular complexity, or on cell extracts made from genetically intractable organisms. In order to investigate the ensemble activities of all MAPs on MT dynamics using lysates made from a genetically tractable organism, we developed a cell-free assay for budding yeast lysates using total internal reflection fluorescence (TIRF) microscopy. Lysates were prepared from yeast strains expressing GFP-tubulin. MT polymerization from pre-assembled MT seeds adhered to a coverslip was observed in real time. Through use of cell division cycle (cdc) and MT depolymerase mutants, we found that MT polymerization and dynamic instability are dependent on the cell cycle state and the activities of specific MAPs.
| S-EPMC6398484 | biostudies-literature
Project description:Coupling between ATPase and track binding sites is essential for molecular motors to move along cytoskeletal tracks. In dynein, these sites are separated by a long coiled coil stalk that must mediate communication between them, but the underlying mechanism remains unclear. Here we show that changes in registration between the two helices of the coiled coil can perform this function. We locked the coiled coil at three specific registrations using oxidation to disulfides of paired cysteine residues introduced into the two helices. These trapped ATPase activity either in a microtubule-independent high or low state, and microtubule binding activity either in an ATP-insensitive strong or weak state, depending on the registry of the coiled coil. Our results provide direct evidence that dynein uses sliding between the two helices of the stalk to couple ATPase and microtubule binding activities during its mechanochemical cycle.
| S-EPMC2757048 | biostudies-literature
Project description:During mitosis, cells must spatiotemporally regulate gene expression programs to ensure accurate cellular division. Failures to properly regulate mitotic progression result in aneuploidy, a hallmark of cancer. Entry and exit from mitosis is largely controlled by waves of cyclin-dependent kinase (CDK) activity coupled to targeted protein degradation. The correct timing of CDK-based mitotic regulation is coordinated with the structure and function of microtubules. To determine whether mitotic gene expression is also regulated by the integrity of microtubules, we performed ribosome profiling and mRNA-sequencing in the presence and absence of microtubules in the budding yeast Saccharomyces cerevisiae. We discovered a coordinated translational and transcriptional repression of genes involved in cell wall biology processes when microtubules are disrupted. The genes targeted for repression in the absence of microtubules are enriched for downstream targets of a feed-forward pathway that controls cytokinesis and septum degradation and is regulated by the Cbk1 kinase, the Regulation of Ace2 Morphogenesis (RAM) pathway. We demonstrate that microtubule disruption leads to aberrant subcellular localization of Cbk1 in a manner that partially depends on the spindle position checkpoint. Furthermore, constitutive activation of the RAM pathway in the absence of microtubules leads to growth defects. Taken together, these results uncover a previously unknown link between microtubule function and the proper execution of mitotic gene expression programs to ensure that cell division does not occur prematurely.
| S-EPMC9511886 | biostudies-literature
Project description:Recent experimental studies of the role of microtubule sliding in neurite outgrowth suggested a qualitative model, according to which kinesin-1 motors push the minus-end-out microtubules against the cell membrane and generate the early cell processes. At the later stage, dynein takes over the sliding, expels the minus-end-out microtubules from the neurites, and pulls in the plus-end-out microtubules that continue to elongate the nascent axon. This model leaves unanswered a number of questions: why is dynein unable to generate the processes alone, whereas kinesin-1 can? What is the role of microtubule dynamics in process initiation and growth? Can the model correctly predict the rates of process growth in control and dynein-inhibited cases? What triggers the transition from kinesin-driven to dynein-driven sliding? To answer these questions, we combine computational modeling of a network of elastic dynamic microtubules and kinesin-1 and dynein motors with measurements of the process growth kinetics and pharmacological perturbations in Drosophila S2 cells. The results verify quantitatively the qualitative model of the microtubule polarity sorting and suggest that dynein-powered elongation is effective only when the processes are longer than a threshold length, which explains why kinesin-1 alone, but not dynein, is sufficient for the process growth. Furthermore, we show that the mechanism of process elongation depends critically on microtubule dynamic instability. Both modeling and experimental measurements show, surprisingly, that dynein inhibition accelerates the process extension. We discuss implications of the model for the general problems of cell polarization, cytoskeletal polarity emergence, and cell process protrusion.
| S-EPMC6260207 | biostudies-other
Project description:LIS1 is a critical regulator of dynein function during mitosis and organelle transport. Here, we investigated how Pac1, the budding yeast LIS1 homologue, regulates dynein targeting and activity during nuclear migration. We show that Pac1 and Dyn1 (dynein heavy chain) are dependent upon each other and upon Bik1 (budding yeast CLIP-170 homologue) for plus end localization, whereas Bik1 is independent of either. Dyn1, Pac1 and Bik1 interact in vivo at the plus ends, where an excess amount of Bik1 recruits approximately equal amounts of Pac1 and Dyn1. Overexpression of Pac1 enhanced plus end targeting of Dyn1 and vice versa, while affinity-purification of Dyn1 revealed that it exists in a complex with Pac1 in the absence of Bik1, leading us to conclude that the Pac1-Dyn1 complex preassembles in the cytoplasm prior to loading onto Bik1-decorated plus ends. Strikingly, we found that Pac1-overexpression augments cortical dynein activity through a mechanism distinct from loss of She1, a negative regulator of dynein-dynactin association. While Pac1-overexpression enhances the frequency of cortical targeting for dynein and dynactin, the stoichiometry of these complexes remains relatively unchanged at the plus ends compared to that in wild-type cells (?3 dynein to 1 dynactin). Loss of She1, however, enhances dynein-dynactin association at the plus ends and the cell cortex, resulting in an apparent 1:1 stoichiometry. Our results reveal differential regulation of cortical dynein activity by She1 and Pac1, and provide a potentially new regulatory step in the off-loading model for dynein function.
| S-EPMC3053442 | biostudies-literature
Project description:Cytoplasmic dynein-1 is a minus end-directed microtubule motor that transports numerous cargoes in cell types throughout the evolutionary spectrum. Dynein is regulated by various motor-intrinsic and motor-extrinsic factors that enhance its processivity, recruit it to various cellular sites, or otherwise promote or restrict its activity. Studying dynein activity in higher eukaryotes is complicated by various factors, including the myriad functions in which this motor participates, and the consequential pleotropic effects associated with disrupting its activity. Budding yeast has long been a powerful model system for understanding this enormous motor protein complex, which is highly conserved between yeast and humans at the primary sequence and structural levels. Studies in budding yeast are simplified by the fact that dynein only performs one known function in this organism: to position the mitotic spindle at the site of cell division. Monitoring dynein-mediated spindle movements in budding yeast provides a powerful tool for the quantitative measurements of various motility parameters, and a system with which to assess the consequence of mutations in dynein or its regulators. Here, we provide detailed protocols to perform quantitative measurements of dynein activity in live cells using a combination of fluorescence microscopy and computational methods to track and quantitate dynein-mediated spindle movements. These methods are broadly applicable to anyone that wishes to perform fluorescence microscopy on budding yeast.
| S-EPMC10132083 | biostudies-literature
Project description:Cytoplasmic dynein is a microtubule motor that powers minus-end-directed motility in a variety of biological settings. The budding yeast, Saccharomyces cerevisiae, has been a useful system for the study of dynein, due to its molecular genetics and cell biology capabilities, coupled with the conservation of dynein-pathway proteins. In this review we discuss how budding yeast use dynein to manipulate the position of the mitotic spindle and the nucleus during cell division, using cytoplasmic microtubules, and we describe our current understanding of the genes required for dynein function. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
| S-EPMC2746759 | biostudies-literature