Sister Kinetochore Recapture in Fission Yeast Occurs by Two Distinct Mechanisms, Both Requiring Dam1 and Klp2

Abstract

In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. We have studied chromosome recapture in the fission yeast Schizosaccharomyces pombe. We show by live cell analysis that lost kinetochores interact laterally with intranuclear microtubules (INMs) and that both microtubule depolymerization (end-on pulling) and minus-end-directed movement (microtubule sliding) contribute to chromosome retrieval to the spindle pole body (SPB). We find that the minus-end-directed motor Klp2 colocalizes with the kinetochore during its transport to the SPB and contributes to the effectiveness of retrieval by affecting both end-on pulling and lateral sliding. Furthermore, we provide in vivo evidence that Dam1, a component of the DASH complex, also colocalizes with the kinetochore during its transport and is essential for its retrieval by either of these mechanisms. Finally, we find that the position of the unattached kinetochore correlates with the size and orientation of the INMs, suggesting that chromosome recapture may not be a random process.

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

“…By using strains that have a single Dam1 subunit tagged with GFP and expressed from the endogenous promoter (here, called mono-GFP-tagged Dam1), SpDam1 complex was previously detected to localize to kinetochores, the spindle, and intranuclear MTs in mitotic cells (20)(21)(22). In addition to these sites, by careful microscopic examination, we also detected weak, discrete spots of Dam1 assemblies on the interphase cytoplasmic MTs (Fig.…”

“…Alternatively, Dam1 may be biochemically modified in a cell cycle stage-specific manner so that its effect on MT dynamics is drastically altered when cells enter mitosis. Supporting this scenario, intranuclear MTs without association with kinetochores also exhibit reduced depolymerization rate, demonstrating that Dam1 directly modulates spindle MT dynamics outside the context of kinetochore (22).…”

“…It is possible that the absence of Dam1 may reduce the attachment between kinetochores and MTs, which in turn may trigger a to-be-identified regulatory mechanism that halts kMT depolymerization until attachment is secured. Consistently, intranuclear MTs, upon recapturing stray kinetochores in mitosis, virtually cease depolymerization, or stand still, in the absence of Dam1 (22). Alternatively, Dam1 may be biochemically modified in a cell cycle stage-specific manner so that its effect on MT dynamics is drastically altered when cells enter mitosis.…”

“…20). Similar to ScDam1, SpDam1 is localized at kinetochores as well as on the spindle and the intranuclear MT (20)(21)(22), and functions in coupling kinetochores to MTs (22)(23)(24). Using quantitative fluorescence microscopy, it was determined that on average only seven to eight copies of SpDam1 per kinetochore were found (25).…”

The Cik1/Kar3 Motor Complex Is Required for the Proper Kinetochore–Microtubule Interaction After Stressful DNA Replication

“…In fission yeast, the homologs of Kar3 and Dam1 are required for chromosome transport through lateral sliding or the end-on pulling mechanism (Gachet et al 2008), suggesting that the mechanism is conserved. Here we characterized the role of the Cik1/Kar3 motor complex in response to stressful DNA replication, but this function is likely specific to budding yeast because only budding yeast cells establish KT-MT interaction during S-phase.…”