In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.
13 14 Background: Cells are powered by a large set of macromolecular complexes, which 15 work together in a crowded environment. The in situ mechanisms of these complexes 16 are unclear because their 3-D distribution, organization, and interactions are largely 17 unknown. Electron cryotomography (cryo-ET) is a key tool to address these knowledge 18 gaps because it produces cryotomograms --3-D images that reveal biological structure 19 at approximately 4-nm resolution. Cryo-ET does not involve any fixation, dehydration, 20 staining, or plastic embedment, meaning that cellular features are visualized in a life-21 like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we have 22 Conclusions: Cellular cryo-ET data can be mined to obtain new cell-biological, 36 structural, and 3-D statistical insights in situ. Because these data capture cells in a life-37 like state, they contain some structures that are either absent or not visible in traditional 38 EM data. Template matching and subtomogram averaging of known macromolecular 39 complexes can reveal their 3-D distributions and low-resolution structures. Furthermore, 40 these data can serve as testbeds for high-throughput image-analysis pipelines, as 41 training sets for feature-recognition software, for feasibility analysis when planning new 42 structural cell-biology projects, and as practice data for students who are learning 43 cellular cryo-ET. 44 45
Electron cryotomography (cryo-ET) is an increasingly popular technique to study cellular structures and macromolecules in situ. Due to poor penetration of electrons through thick biological samples, the vitreously frozen samples for cryo-ET need to be thin. For frozen-hydrated cells, such samples can be produced either by cryomicrotomy or cryo-FIB-milling. As a result, a tomogram of such a sample contains information of a small fraction of the entire cell volume, making it challenging to image rare structures in the cell or to determine the distribution of scattered structures. Here, we describe the tools and workflow that we designed to facilitate serial cryomicrotomy, which makes possible the exploration of a larger volume of individual cells at molecular resolution. We successfully used serial cryomicrotomy to locate and image the Dam1/DASH complex located at microtubule plus ends inside mitotic Saccharomyces cerevisiae cells.
Background Cells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms—3D images that reveal biological structure at ∼4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET. Findings Here we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiaecollected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB. Conclusions Cellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students.
Abbreviations 16 MT: microtubule 17 kMT: kinetochore microtubule 18 cryo-ET: electron cryotomography / cryo-electron tomography 19 20 Running title: Cryo-ET of the yeast mitotic machinery in vivo 21 1 ABSTRACT 22 In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at 23 their kinetochores. While kinetochore subcomplexes have been studied extensively in 24 563 Yong for advice on chromatography, the Jensen lab for computer access, and members 564 of the Gan group, Jack Johnson, Steve Harrison and Paul Matsudaira for feedback. 565 CTN, CC, LD, and LG were funded by NUS startups R-154-000-515-133, R-154-000-566 524-651, and D-E12-303-154-217, an MOE T2 R-154-000-624-112, with equipment 567 support from NUS YIA R-154-000-558-133. HHL and US were funded by the 568 Biomedical Research Council of A*STAR (Agency of Science Technology and 569 Research), Singapore. 570 571 Contributions 572 CTN -experiments, project design, writing, LD -experiments, CC -project design, 573 experiments, HHL -experiments, JS -training, US -project design, writing, LG -574 experiments, project design, writing.575
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