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.

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

In this study, the complete mitochondrial genome sequence of the Tibetan Mastiff was reported. The total length of the mitogenome is 16,729 bp. It contains the typical structure, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region is in line with other canine animals. We further identified genome-wide variations among different canine mitochondrial genomes and indicated that the D-loop region harbors the most sequence variation, which will provide sequence variation information for the protection and utilization of the Tibetan Mastiff germplasm resource.