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Abstract
During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here, we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.
Highlights
Pre-anaphase chromosome movements culminate with chromosome alignment at the spindle equator, a distinctive feature of mitosis important for the synchronous anaphase poleward movement of chromatids and proper telophase nuclear reformation (Fonseca et al, 2019; Maiato et al, 2017)
To study the role of protein regulator of cytokinesis 1 (PRC1) and the forces arising from coupling of bridging and k-fibers in chromosome alignment, we developed an optogenetic tool for fast and reversible removal of PRC1 from the spindle to the cell membrane, based on the previously designed improved light inducible dimer system (Guntas et al, 2015)
Addition of opto-PRC1 did not change the duration of metaphase, as inferred from the fraction of cells that entered anaphase during image acquisition, which was similar in cells with endogenous PRC1 and cells treated with PRC1 siRNA and containing opto-PRC1 (79 ± 6%, N = 37, and 71 ± 5%, N = 72, respectively; p = 0.4, Pearson’s Chi-squared test; Figure 1—figure supplement 1C)
Summary
Pre-anaphase chromosome movements culminate with chromosome alignment at the spindle equator, a distinctive feature of mitosis important for the synchronous anaphase poleward movement of chromatids and proper telophase nuclear reformation (Fonseca et al, 2019; Maiato et al, 2017). Pulling forces exerted by k-fiber tips on kinetochores are thought to be regulated by kinesin-8 motor proteins, which are needed for proper kinetochore alignment in various organisms from yeast to humans (Garcia et al, 2002; Stumpff et al, 2008; Wargacki et al, 2010) These motors can ‘measure’ microtubule length because they bind to microtubules along their length and move toward the plus end, leading to more kinesin-8 accumulated at the plus end of longer microtubules, where they promote microtubule catastrophe, that is, a switch from growth to shrinkage (Tischer et al, 2009; Varga et al, 2006). This mechanism can explain kinetochore alignment at the spindle equator
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