An Expanded View of the Eukaryotic Cytoskeleton

A rich and ongoing history of cell biology research has defined the major polymer systems of the eukaryotic cytoskeleton. Recent studies have identified additional proteins that form filamentous structures in cells and can self-assemble into linear polymers when purified. This suggests that the eukaryotic cytoskeleton is an even more complex system than previously considered. In this essay, I examine the case for an expanded definition of the eukaryotic cytoskeleton and present a series of challenges for future work in this area

Eisosomes Provide Membrane Reservoirs for Rapid Expansion of the Yeast Plasma Membrane

Cell surface area rapidly increases during mechanical and hypoosmotic stresses. Such expansion of the plasma membrane requires \u27membrane reservoirs\u27 that provide surface area and buffer membrane tension, but the sources of this membrane remain poorly understood. In principle, the flattening of invaginations and buds within the plasma membrane could provide this additional surface area, as recently shown for caveolae in animal cells. Here, we used microfluidics to study the rapid expansion of the yeast plasma membrane in protoplasts, which lack the rigid cell wall. To survive hypoosmotic stress, yeast cell protoplasts required eisosomes, protein-based structures that generate long invaginations at the plasma membrane. Both budding yeast and fission yeast protoplasts lacking eisosomes were unable to expand like wild-type protoplasts during hypoosmotic stress, and subsequently lysed. By performing quantitative fluorescence microscopy on single protoplasts, we also found that eisosomes disassembled as surface area increased. During this process, invaginations generated by eisosomes at the plasma membrane became flattened, as visualized by scanning electron microscopy. We propose that eisosomes serve as tension-dependent membrane reservoirs for expansion of yeast cells in an analogous manner to caveolae in animal cells

Phosphatases Generate Signal Specificity Downstream of Ssp1 Kinase in Fission Yeast

AMPK-related protein kinases (ARKs) coordinate cell growth, proliferation, and migration with environmental status. It is unclear how specific ARKs are activated at specific times. In the fission yeast Schizosaccharomyces pombe, the CaMKK-like protein kinase Ssp1 promotes cell cycle progression by activating the ARK Cdr2 according to cell growth signals. Here, we demonstrate that Ssp1 activates a second ARK, Ssp2/AMPKα, for cell proliferation in low environmental glucose. Ssp1 activates these two related targets by the same biochemical mechanism: direct phosphorylation of a conserved residue in the activation loop (Cdr2-T166 and Ssp2-T189). Despite a shared upstream kinase and similar phosphorylation sites, Cdr2 and Ssp2 have distinct regulatory input cues and distinct functional outputs. We investigated this specificity and found that distinct protein phosphatases counteract Ssp1 activity toward its different substrates. We identified the PP6 family phosphatase Ppe1 as the primary phosphatase for Ssp2-T189 dephosphorylation. The phosphatase inhibitor Sds23 acts upstream of PP6 to regulate Ssp2-T189 phosphorylation in a manner that depends on energy but not on the intact AMPK heterotrimer. In contrast, Cdr2-T166 phosphorylation is regulated by protein phosphatase 2A but not by the Sds23-PP6 pathway. Thus, our study provides a phosphatase-driven mechanism to induce specific physiological responses downstream of a master protein kinase

Seg1 Controls Eisosome Assembly and Shape

Eisosomes are stable domains at the plasma membrane of the budding yeast Saccharomyces cerevisiae and have been proposed to function in endocytosis. Eisosomes are composed of two main cytoplasmic proteins, Pil1 and Lsp1, that form a scaffold around furrow-like plasma membrane invaginations. We show here that the poorly characterized eisosome protein Seg1/Ymr086w is important for eisosome biogenesis and architecture. Seg1 was required for efficient incorporation of Pil1 into eisosomes and the generation of normal plasma membrane furrows. Seg1 preceded Pil1 during eisosome formation and established a platform for the assembly of other eisosome components. This platform was further shaped and stabilized upon the arrival of Pil1 and Lsp1. Moreover, Seg1 abundance controlled the shape of eisosomes by determining their length. Similarly, the Schizosaccharomyces pombe Seg1-like protein Sle1 was necessary to generate the filamentous eisosomes present in fission yeast. The function of Seg1 in the stepwise biogenesis of eisosomes reveals striking architectural similarities between eisosomes in yeast and caveolae in mammals

Megadalton-Node Assembly by Binding of Skb1 to the Membrane Anchor Slf1

The plasma membrane contains both dynamic and static microdomains. Given the growing appreciation of cortical microdomains in cell biology, it is important to determine the organizational principles that underlie assembly of compartmentalized structures at the plasma membrane. The fission yeast plasma membrane is highly compartmentalized by distinct sets of cortical nodes, which control signaling for cell cycle progression and cytokinesis. The mitotic inhibitor Skb1 localizes to a set of cortical nodes that provide spatial control over signaling for entry into mitosis. However, it has been unclear whether these nodes contain other proteins and how they might be organized and tethered to the plasma membrane. Here we show that Skb1 forms nodes by interacting with the novel protein Slf1, which is a limiting factor for node formation in cells. Using quantitative fluorescence microscopy and in vitro assays, we demonstrate that Skb1-Slf1 nodes are megadalton structures that are anchored to the membrane by a lipid-binding region in the Slf1 C-terminus. We propose a mechanism for higher-order node formation by Skb1 and Slf1, with implications for macromolecular assemblies in diverse cell types

Blt1 and Mid1 Provide Overlapping Membrane Anchors To Position the Division Plane in Fission Yeast

Spatial control of cytokinesis is essential for proper cell division. The molecular mechanisms that anchor the dynamic assembly and constriction of the cytokinetic ring at the plasma membrane remain unclear. In the fission yeast Schizosaccharomyces pombe, the cytokinetic ring is assembled in the cell middle from cortical node precursors that are positioned by the anillin-like protein Mid1. During mitotic entry, cortical nodes mature and then compact into a contractile ring positioned in the cell middle. The molecular link between Mid1 and medial cortical nodes remains poorly defined. Here we show that Blt1, a previously enig- matic cortical node protein, promotes the robust association of Mid1 with cortical nodes. Blt1 interacts with Mid1 through the RhoGEF Gef2 to stabilize nodes at the cell cortex during the early stages of contractile ring assembly. The Blt1 N terminus is re- quired for localization and function, while the Blt1 C terminus promotes cortical localization by interacting with phospholipids. In cells lacking membrane binding by both Mid1 and Blt1, nodes detach from the cell cortex and generate aberrant cytokinetic rings. We conclude that Blt1 acts as a scaffolding protein for precursors of the cytokinetic ring and that Blt1 and Mid1 provide overlapping membrane anchors for proper division plane positioning

Advances in Gene Ontology Utilization Improve Statistical Power of Annotation Enrichment

Gene-annotation enrichment is a common method for utilizing ontology-based annotations in gene and gene-product centric knowledgebases. Effective utilization of these annotations requires inferring semantic linkages by tracing paths through edges in the ontological graph, referred to as relations. However, some relations are semantically problematic with respect to scope, necessitating their omission or modification lest erroneous term mappings occur. To address these issues, we created the Gene Ontology Categorization Suite, or GOcats—a novel tool that organizes the Gene Ontology into subgraphs representing user-defined concepts, while ensuring that all appropriate relations are congruent with respect to scoping semantics. Here, we demonstrate the improvements in annotation enrichment by re-interpreting edges that would otherwise be omitted by traditional ancestor path-tracing methods. Specifically, we show that GOcats’ unique handling of relations improves enrichment over conventional methods in the analysis of two different gene-expression datasets: a breast cancer microarray dataset and several horse cartilage development RNAseq datasets. With the breast cancer microarray dataset, we observed significant improvement (one-sided binomial test p-value = 1.86E-25) in 182 of 217 significantly enriched GO terms identified from the conventional path traversal method when GOcats’ path traversal was used. We also found new significantly enriched terms using GOcats, whose biological relevancy has been experimentally demonstrated elsewhere. Likewise, on the horse RNAseq datasets, we observed a significant improvement in GO term enrichment when using GOcat’s path traversal: one-sided binomial test p-values range from 1.32E-03 to 2.58E-44