Loss of kinesin-8 improves the robustness of the self-assembled spindle in Schizosaccharomyces pombe

Abstract

Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles, the existence of which implies that microtubule spindles self-assemble without the participation of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we recently reported the formation of self-assembled microtubule arrays, which were able to segregate chromosomes, in fission yeast mutants where the contribution of the spindle pole body (SPB, the centrosome equivalent in yeast) was specifically blocked during meiosis. Here, we demonstrate that this unexpected microtubule formation represents a bonafide type of acentrosomal spindle. Moreover, a comparative analysis of these self-assembled spindles and the canonical SPB-dependent spindle reveals similarities and differences: for example, both spindles have a similar polarity, but the location of the γ-tubulin complex differs. We also show that the robustness of self-assembled spindles can be reinforced by eliminating kinesin-8 family members, whereas kinesin-8 mutants have an adverse impact on SPB-dependent spindles. Hence, we consider that reinforced self-assembled spindles in yeast will help to clarify the molecular mechanisms behind acentrosomal meiosis, a crucial step towards better understanding gametogenesis.

Analyzing self-assembled spindle dynamics in fission yeast meiosis using in vivo fluorescence imaging

“…For example, we have used the auxin-induced degron system to deplete specific proteins and latrunculin A to disrupt the F-actin network in this context, adding the drug just before starting the timelapse, with no incubation periods. 2 Initiate the timelapse acquisition. Table 1 Acquisition settings in Spinning Disk confocal system (Olympus/Yokogawa) Fluorescence protein Excitation/Emission Laser power Exposure time z-stack Time interval Objective CFP 445/452-45 10% 200 ms 14 sections 0.4 μm apart 3 min 100×/1.4 NA Oil Plan Apo GFP 491/525-45 10% 200 ms 14 sections 0.4 μm apart 3 min 100×/1.4 NA Oil Plan Apo m-Cherry 561/605-64 60% 200 ms 14 sections 0.4 μm apart 3 min 100×/1.4 NA Oil Plan Apo Table 2 Acquisition settings in Dragonfly 200 confocal system (Olympus/Andor) Fluorescence protein Excitation/Emission Laser power Exposure time z-stack Time interval Objective GFP 488/521-38 30% 50 ms 14 sections 0.4 μm apart 3 min 100×/1.45 NA Oil Plan Apo m-Cherry 561/594-43 10% 50 ms 14 sections 0.4 μm apart 3 min 100×/1.45 NA Oil Plan Apo Table 3 Acquisition settings in Personal DeltaVision system Fluorescence protein Excitation/Emission % Transmittance Exposure time z-stack Time interval Objective CFP 436/470-30 32...…”

“…For example, we have used the auxin-induced degron system to deplete specific proteins and latrunculin A to disrupt the F-actin network in this context, adding the drug just before starting the timelapse, with no incubation periods. 2 …”

“… 1 Remarkably, this spindle is able to segregate chromosomes and operates independently of the SPBs. 1 , 2

Figure 1 The difference between SPB-dependent ( wt ) and self-assembled spindles ( bqt1Δ sad1.2 ) in fission yeast meiosis Left: the normal ( wt ) process involves SPB insertion into the nuclear membrane and the formation of an SPB-dependent spindle at the beginning of fission yeast meiosis. Right: in the bqt1Δ sad1.2 mutant, the SPB insertion into the nuclear envelope (NE) and SPB-dependent spindle formation are abolished, resulting in the formation of a self-assembled spindle in meiosis.

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“…The published articles, Pineda-Santaella and Fernández-Álvarez 1 and Pineda-Santaella et al. 2 include all datasets analyzed during this study.…”

Beyond tradition: exploring the non-canonical functions of telomeres in meiosis

“…As described above, loss of the telomere bouquet in fission yeast causes severe defects in local NE disassembly and, consequently, in the SPB insertion into the NE and spindle formation (Tomita and Cooper, 2007;Pineda-Santaella and Fernández-Álvarez, 2019;Pineda-Santaella et al, 2021). However, approximately 50% of bouquet-mutant cells can form normal spindles by using centromeres in prophase to create a bouquet-like structure…”