Abstract
The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to move chromosomes. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals the preservation of local architecture in the spindle-center over seconds. Sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers pivot around poles but not chromosomes, retaining their orientation within 3 mm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement in the spindle center. This could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales.
| Original language | English |
|---|---|
| Article number | e53807 |
| Journal | eLife |
| Volume | 9 |
| DOIs | |
| State | Published - Mar 2020 |
Bibliographical note
Publisher Copyright:© Suresh et al.
Funding
We thank Alexey Khodjakov for PtK2 GFP-a-tubulin cells and Timothy Mitchison for FCPT. We are grateful to Le Paliulis for technical advice and key disussions, to Maya Anjur-Deitrich, Charles Asbury, Justin Biel, the Fred Chang Lab, Daniel Fletcher, Jesse Gatlin, Wallace Marshall, Dick McIntosh, Dan Needleman, Adair Oesterle, Yuta Shimamoto, Radhika Subramanian, Iva Tolic and Orion Weiner for helpful discussions, Miquel Rosas Salvans for technical help, Deepak Krishnamurthy and Jason Town for image analysis discussions, the Verkman Lab for the microforge, and Arthur Molines and members of the Dumont Lab for discussions and critical reading of the manuscript. This work was supported by NIH DP2GM119177, NIH 1R01GM134132, NSF CAREER 1554139, NSF 1548297 Center for Cellular Construction, the Rita Allen Foundation and Searle Scholars’ Program (SD), NSF Graduate Research Fellowships (PS and AFL) and a UCSF Kozloff Fellowship (AFL). We thank Alexey Khodjakov for PtK2 GFP-a-tubulin cells and Timothy Mitchison for FCPT. We are grateful to Le Paliulis for technical advice and key disussions, to Maya Anjur-Deitrich, Charles Asbury, Justin Biel, the Fred Chang Lab, Daniel Fletcher, Jesse Gatlin, Wallace Marshall, Dick McIntosh, Dan Needleman, Adair Oesterle, Yuta Shimamoto, Radhika Subramanian, Iva Tolic and Orion Weiner for helpful discussions, Miquel Rosas Salvans for technical help, Deepak Krishnamurthy and Jason Town for image analysis discussions, the Verkman Lab for the microforge, and Arthur Molines and members of the Dumont Lab for discussions and critical reading of the manuscript. This work was supported by NIH DP2GM119177, NIH 1R01GM134132, NSF CAREER 1554139, NSF 1548297 Center for Cellular Construction, the Rita Allen Foundation and Searle Scholars’ Program (SD), NSF Graduate Research Fellowships (PS and AFL) and a UCSF Kozloff Fellowship (AFL). National Institute of General Medical Sciences DP2GM119177 Sophie Dumont National Institute of General Medical Sciences 1R01GM134132 Sophie Dumont National Science Foundation 1554139 CAREER Sophie Dumont National Science Foundation 1548297 Center for Cellular Construction Sophie Dumont Rita Allen Foundation Sophie Dumont Chicago Community Trust Searle Scholars’ Program Sophie Dumont National Science Foundation Graduate Research Fellowship Pooja Suresh Alexandra F Long University of California, San Francisco UCSF Kozloff Fellowship Alexandra F Long The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
| Funders | Funder number |
|---|---|
| Arthur Molines | |
| Center for Cellular Construction Sophie Dumont Rita Allen Foundation Sophie Dumont Chicago Community Trust Searle Scholars’ Program Sophie Dumont National Science Foundation Graduate Research Fellowship Pooja Suresh Alexandra F Long University of California, San Francisco UCSF | |
| National Institute of General Medical Sciences DP2GM119177 Sophie Dumont National Institute of General Medical Sciences | 1548297 |
| Timothy Mitchison for FCPT | |
| U.S. Department of Energy Chinese Academy of Sciences Guangzhou Municipal Science and Technology Project Oak Ridge National Laboratory Extreme Science and Engineering Discovery Environment National Science Foundation National Energy Research Scientific Computing Center National Natural Science Foundation of China | 1554139 |
| National Institutes of Health (NIH) | DP2GM119177 |
| National Institute of General Medical Sciences | R01GM134132 |
| Rita Allen Foundation | |
| University of California San Francisco |
ASJC Scopus subject areas
- General Neuroscience
- General Biochemistry, Genetics and Molecular Biology
- General Immunology and Microbiology