Supplementary Components1. the spindle. Kinesin-14, which includes HSET, XCTK2, NCD, Kar3, and Pkl1, is a MT minus end-directed motor localized to the spindle poles, Mouse monoclonal to IHOG able to crosslink parallel MTs to focus the spindle pole during mitosis and meiosis, and to antagonize kinesin-5, a MT plus end-directed motor localized at the spindle midzone, in a force-balance equilibrium to maintain proper spindle length architecture and function 6, 7. Loss of kinesin-14 generally results in chromosome segregation defects 8C13, or aneuploidy. However, how the loss of kinesin-14 leads to aneuploidy has not been determined. We show in fission yeast that loss of kinesin-14 Pkl1 leads to aberrant spindle pole MT protrusions, resulting from kinesin-5 Cut7 sliding the unfocused pole MTs. Long MT protrusions can subsequently push the post-anaphase segregated chromosomes to the site of cell division, resulting in chromosome cut at cytokinesis, thus producing aneuploid cells. Results Pkl1 acts similarly to the metazoan kinesin-14. It is a diffusive MT minus end-directed motor 14, localizing at the spindle pole body (SPB) during mitosis 15C17. Deletion of (cells exhibited aberrant spindle MT protrusions 19. To understand the nature of these protrusions, we performed live-cell imaging of wild-type and cells expressing mCherry-Atb2 (tubulin) and Benzyl chloroformate Sid4-GFP (SPB marker 20). We observed spindle MT protrusions in 85% of cells, compared to none in the wildtype cells (Fig. 1a, 1c). The protrusions were to the spindle long-axis parallel, made an appearance during prophase-metaphase, emanated from each one (58% of cells) or both (27% of cells) spindle poles, and generally had been taken care of throughout anaphase (Fig. 1a, 1c). Significantly, protrusions originated from in the nucleus. Using Cut11-GFP (nuclear membrane marker 21), we noticed protrusions pressing out the nuclear envelope, and puncturing the envelope once the protrusions had been lengthy (Fig. 1b, supplementary Fig also. 3b), indicating power exertion through the protrusions. Open up in another window Shape 1 Pkl1 maintains spindle pole body (SPB) integritya) Time-lapse pictures of wild-type Benzyl chloroformate (cell expressing mCherry-Atb2 (tubulin) and Sid4-GFP (SPB marker) through metaphase and anaphase. The cell has no MT protrusions emanating from the SPB. In contrast, the cell has MT protrusions, which are parallel to the spindle long-axis, emanating from one or both SPB (yellow arrow head). The MT protrusion can be long, reaching the cell tip cortex and buckle (time 28min). Scale bar, 5m. b) Time-lapse images of and cell expressing mCherry-Atb2 and Cut11-GFP (nuclear membrane marker) through anaphase. The cell has a MT protrusion from inside the nucleus pushing out the nuclear membrane (yellow arrow head, time 0min). When the MT protrusion reaches a relatively long length, it punctures through the nuclear membrane (red arrow head, time 8min). Scale bar, 5m. c) Comparative plot of frequency of different MT protrusions in (n=20) and (n=30) cells. cells have no aberrant MT protrusions. Bars represent mean s.d. for multiple experiments. d) Time-lapse images of a mitotic spindle of a cell expressing mCherry-Atb2 and Mal3-GFP (MT plus end tracking protein EB1). Mal3-GFP is present all along the spindle. Distinct dot of Mal3-GFP tracks the short growing MT (red arrow head, time 10C30s), and disappears when the MT depolymerizes (time 40s). The long MT has no Mal3-GFP at its tip (yellow arrow head). Scale Benzyl chloroformate bar, 2m. e) Plot of MT protrusion length and polarity frequency in.