Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

Transient septin sumoylation steers a Fir1-Skt5 protein complex between the split septin ring.

Ubiquitylation and phosphorylation control composition and architecture of the cell separation machinery in yeast and other eukaryotes. The significance of septin sumoylation on cell separation remained an enigma. Septins form an hourglass structure at the bud neck of yeast cells that transforms into a split septin double ring during mitosis. We discovered that sumoylated septins recruit the cytokinesis checkpoint protein Fir1 to the peripheral side of the septin hourglass just before its transformation into the double-ring configuration. As this transition occurs, Fir1 is released from the septins and seamlessly relocates between the split septin rings through synchronized binding to the scaffold Spa2. Fir1 binds and carries the membrane-bound Skt5 on its route to the division plane where the Fir1-Skt5 complex serves as receptor for chitin synthase III.

The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

The structure of a tetrameric septin complex reveals a hydrophobic element essential for NC-interface integrity.

The septins of the yeast Saccharomyces cerevisiae assemble into hetero-octameric rods by alternating interactions between neighboring G-domains or N- and C-termini, respectively. These rods polymerize end to end into apolar filaments, forming a ring beneath the prospective new bud that expands during the cell cycle into an hourglass structure. The hourglass finally splits during cytokinesis into a double ring. Understanding these transitions as well as the plasticity of the higher order assemblies requires a detailed knowledge of the underlying structures. Here we present the first X-ray crystal structure of a tetrameric Shs1-Cdc12-Cdc3-Cdc10 complex at a resolution of 3.2 Å. Close inspection of the NC-interfaces of this and other septin structures reveals a conserved contact motif that is essential for NC-interface integrity of yeast and human septins in vivo and in vitro. Using the tetrameric structure in combination with AlphaFold-Multimer allowed us to propose a model of the octameric septin rod.