Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi. Copyright © 2013 John Wiley & Sons, Ltd.

An important question in cell biology is how cellular organelles partition during cell division. In organisms undergoing closed mitosis, the elongation of an intranuclear spindle drives nuclear division, generating two identically sized nuclei [1, 2]. However, how the site of nuclear division is determined and the underlying mechanism driving nuclear envelope (NE) fission remain largely unknown. Here, using the fission yeast, we show that the microtubule bundler Ase1/PRC1 at the spindle midzone is required for the local concentration of nuclear pore complexes (NPCs) in the region of the NE in contact with the central spindle. As the spindle elongates during anaphase B, components of these NPCs are sequentially eliminated, and this is accompanied by the local remodeling of the NE. These two events lead to the eventual removal of NPCs and nuclear division. In the absence of importin α, NPCs remain stable in this region and no event of NE remodeling is observed. Consequently, cells fail to undergo nuclear division. Thus, our results highlight a new role of the central spindle as a spatial cue that determines the site of nuclear division and point to NPC removal as the triggering event.

Kinetochores (KTs) are large multiprotein complexes that constitute the interface between centromeric chromatin and the mitotic spindle during chromosome segregation. In spite of their essential role, little is known about how centromeres and KTs are assembled and how their precise stoichiometry is regulated. In this study, we show that the nuclear pore basket component Alm1 is required to maintain both the proteasome and its anchor, Cut8, at the nuclear envelope, which in turn regulates proteostasis of certain inner KT components. Consistently, alm1-deleted cells show increased levels of KT proteins, including CENP-CCnp3, spindle assembly checkpoint activation, and chromosome segregation defects. Our data demonstrate a novel function of the nucleoporin Alm1 in proteasome localization required for KT homeostasis.

In eukaryotic cells, the organization of the genome within the nucleus requires the nuclear envelope (NE) and its associated proteins. The nucleus is subjected to mechanical forces produced by the cytoskeleton. The physical properties of the NE and the linkage of chromatin in compacted conformation at sites of cytoskeleton contacts seem to be key for withstanding nuclear mechanical stress. Mechanical perturbations of the nucleus normally occur during nuclear positioning and migration. In addition, cell contraction or expansion occurring for instance during cell migration or upon changes in osmotic conditions also result innuclear mechanical stress. Recent studies in Schizosaccharomyces pombe (fission yeast) have revealed unexpected functions of cytoplasmic microtubules in nuclear architecture and chromosome behavior, and have pointed to NE-chromatin tethers as protective elements during nuclear mechanics. Here, we review and discuss how fission yeast cells can be used to understand principles underlying the dynamic interplay between genome organization and function and the effect of forces applied to the nucleus by the microtubule cytoskeleton.

The formation of biomolecular condensates contributes to intracellular compartmentalization and plays an important role in many cellular processes. The characterization of condensates is however challenging, requiring advanced biophysical or biochemical methods that are often less suitable for in vivo studies. A particular need for easily accessible yet thorough methods that enable the characterization of condensates across different experimental systems thus remains. To address this, we present PhaseMetrics, a semi-automated FIJI-based image analysis pipeline tailored for quantifying particle properties from microscopy data. Tested using the FG-domain of yeast nucleoporin Nup100, PhaseMetrics accurately assesses particle properties across diverse experimental setups, including particles formed in vitro in chemically defined buffers or Xenopus egg extracts and cellular systems. Comparing the results with biochemical assays, we conclude that PhaseMetrics reliably detects changes induced by various conditions, including the presence of polyethylene glycol, 1,6-hexanediol, or a salt gradient, as well as the activity of the molecular chaperone DNAJB6b and the protein disaggregase Hsp104. Given the flexibility in its analysis parameters, the pipeline should also apply to other condensate-forming systems, and we show its application in detecting TDP-43 particles. By enabling the accurate representation of the variability within the population and the detection of subtle changes at the single-condensate level, the method complements conventional biochemical assays. Combined, PhaseMetrics is an easily accessible, customizable pipeline that enables imaging-based quantitative assessment of biomolecular condensates in vitro and in cells, providing a valuable addition to the current toolbox.