Cell cycle control of spindle pole body duplication and splitting by Sfi1 and Cdc31 in fission yeast

Abstract

Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote the biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in Schizosaccharomyces pombe, from anaphase to early septation and throughout G2 phase. We found that the function of Sfi1-Cdc31 in SPB duplication is accomplished before septation ends and G2 accumulation starts. Thus, Sfi1 early accumulation at mitotic exit might correspond to half-bridge duplication. We further show that Cdc31 phosphorylation on serine 15 in a Cdk1 (encoded by cdc2) consensus site is required for the dissociation of a significant pool of Sfi1 from the bridge and timely segregation of SPBs at mitotic onset. This suggests that the Cdc31 N-terminus modulates the stability of Sfi1-Cdc31 arrays in fission yeast, and impacts on the timing of establishment of spindle bipolarity.

Duplication of the Yeast Spindle Pole Body Once per Cell Cycle

“…However, immunoelectron microscopy localized fission yeast Cdc31 to the bridge of side-by-side SPBs or to a site next to SPBs in an anaphase cell (48). S. pombe Sfi1 (SpSfi1) perfectly colocalizes with Cdc31 at the SPB half-bridge/bridge in high-resolution fluorescence microscopy, and both proteins are essential for SPB duplication (48)(49)(50). The interdependency of the SpSfi1-Cdc31 SPB localization is consistent with an interaction of the two proteins (49).…”

“…S. pombe Sfi1 (SpSfi1) perfectly colocalizes with Cdc31 at the SPB half-bridge/bridge in high-resolution fluorescence microscopy, and both proteins are essential for SPB duplication (48)(49)(50). The interdependency of the SpSfi1-Cdc31 SPB localization is consistent with an interaction of the two proteins (49). Interestingly, electron micrographs reveal a much thicker bridge in fission yeast than in its budding yeast counterpart (12,51).…”

“…While these experiments document incorporation of SpSfi1 into the SPB, they do not resolve the issue of when in the cell cycle the conversion from half-bridge to bridge takes place. However, elegant execution point experiments showed that it is SpSfi1-Cdc31 that becomes incorporated during early anaphase that is critical for SPB duplication in the next cell cycle (49). Therefore, most likely, the half-bridge expands into the bridge during this cell cycle phase in a manner similar to that seen with budding yeast SPBs (13).…”

“…However, light microscopy revealed an interesting biphasic nature with respect to the incorporation of SpSfi1 into the mother SPB after SPB separation with mitotic entry (49,50). The first phase, during which SpSfi1 levels at the SPB increase 1.5-fold, stretches from early anaphase until mid-septation.…”

Live Cell Imaging in Fission Yeast

“…Such shifts are easily controlled with the heated objective collar/slide system of Bioptechs, or the CellASIC or Cherry Biotech microfluidic systems (Velve-Casquillas et al 2010;Bouhlel et al 2015). However, it is important to remember that rapid temperature shifts invoke a stress response that will change cell physiology within 2 min (Toone and Jones 2004), making it essential that appropriate controls are used.…”