Quantitative analysis of nuclear pore complex organization in .

The number, distribution, and composition of nuclear pore complexes (NPCs) in the nuclear envelope varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization are controlled, we analyzed the NPC number and distribution in the fission yeast using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi, and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions.

Redistribution of centrosomal proteins by centromeres and Polo kinase controls partial nuclear envelope breakdown in fission yeast.

Proper mitotic progression in requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB-yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy, we show that the conserved Sad1-UNC-84 homology-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation.

Molecular model of fission yeast centrosome assembly determined by superresolution imaging.

Microtubule-organizing centers (MTOCs), known as centrosomes in animals and spindle pole bodies (SPBs) in fungi, are important for the faithful distribution of chromosomes between daughter cells during mitosis as well as for other cellular functions. The cytoplasmic duplication cycle and regulation of the SPB is analogous to centrosomes, making it an ideal model to study MTOC assembly. Here, we use superresolution structured illumination microscopy with single-particle averaging to localize 14 SPB components and regulators, determining both the relationship of proteins to each other within the SPB and how each protein is assembled into a new structure during SPB duplication.

Profilin regulates F-actin network homeostasis by favoring formin over Arp2/3 complex.

Fission yeast cells use Arp2/3 complex and formin to assemble diverse filamentous actin (F-actin) networks within a common cytoplasm for endocytosis, division, and polarization. Although these homeostatic F-actin networks are usually investigated separately, competition for a limited pool of actin monomers (G-actin) helps to regulate their size and density. However, the mechanism by which G-actin is correctly distributed between rival F-actin networks is not clear.

Fission yeast profilin is tailored to facilitate actin assembly by the cytokinesis formin Cdc12.

The evolutionarily conserved small actin-monomer binding protein profilin is believed to be a housekeeping factor that maintains a general pool of unassembled actin. However, despite similar primary sequences, structural folds, and affinities for G-actin and poly-L-proline, budding yeast profilin ScPFY fails to complement fission yeast profilin SpPRF temperature-sensitive mutant cdc3-124 cells. To identify profilin's essential properties, we built a combinatorial library of ScPFY variants containing either WT or SpPRF residues at multiple positions and carried out a genetic selection to isolate variants that support life in fission yeast.

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast.

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring.