Biology subjects

Willet, A. H.

Publications and source records attributed to Willet, A. H..

6 recordsLinked to original sources

bioRxiv2026

A multivalent docking platform and Rcn1-mediated inhibition control the extent of calcineurin recruitment to the cell division site for the dephosphorylation of multiple cytokinetic proteins

Cytokinesis requires coordinated signaling to ensure the accurate physical separation of daughter cells. Calcineurin (CN), a conserved Ca2+/calmodulin-dependent phosphatase, is required for cytokinesis in organisms ranging from yeast to humans, yet how CN is regulated at the division site and the substrates through which it promotes cell division remain poorly understood. Here we use the fission yeast Schizosaccharomyces pombe, which display striking cell division defects in the absence of CN, to define how CN is anchored at the cell division site. We show that CN recruitment to the cytokinetic ring (CR) requires its PxIxIT- and LxVP-binding surfaces and is mediated by multivalent interactions with the CR components paxillin-like Pxl1 and the F-BAR protein Cdc15. Disrupting these interactions nearly eliminates CN from the CR and causes gross cytokinetic defects similar to complete loss of CN function. Cell cycle stage-specific quantitative phosphoproteomics combined with proximity labeling-based proteomics were used to identify candidate CN substrates involved in cytokinesis. Validation of a cohort of these proteins localizing to the CR, including the F-BAR protein Rga7, the actin regulator Aim21 and three protein kinases, revealed that CN targets a broad network of structural and signaling components involved in cell division. We also identify the conserved CN inhibitor Rcn1 as a CN substrate and show that Rcn1 restricts CN accumulation at the CR to provide an additional layer of spatial regulation. Thus, spatial control of CN enables proper protein dephosphorylation for successful cytokinesis.

cell biology↗

bioRxiv2026

A Glucan Synthase-Remodeler Module Organizes Branched Glucan Assembly in the Fungal Cell Wall

The fungal cell wall is an essential extracellular matrix that underpins growth, morphogenesis, and pathogenesis. Cell wall construction requires numerous enzymes that synthesize and remodel extracellular polymers, yet the principles governing their spatial and functional organization remain unclear. In the fission yeast Schizosaccharomyces pombe, we identify Ghs2, a predicted glycoside hydrolase 16 (GH16) domain-containing transmembrane protein, as an obligate binding partner of the {beta}-1,3-glucan synthase Bgs3. Ghs2 and Bgs3 co-localize at sites of polarized growth and physically associate in vivo. Structure-guided modeling positions the Ghs2 GH16 domain proximal to the predicted Bgs3 glucan extrusion pore, suggesting coordinated polymer synthesis and remodeling. Solid-state NMR analyses demonstrate that both Ghs2 and Bgs3 are required for the proper accumulation of branched {beta}-1,3-glucan. Together with genetic and cell biological evidence, these findings support a model in which Bgs3 synthesizes linear {beta}-1,3-glucan and Ghs2 subsequently introduces {beta}-1,6-linked branch points onto the nascent polymer. More broadly, we propose that synthase-modifier pairs may act together to shape polymer architecture during cell wall assembly.

cell biology↗

bioRxiv2025

Solid-State NMR Analysis of Schizosaccharomyces pombe Reveals Role of α-Amylase Family Enzymes in Cell Wall Structure and Function

The fission yeast Schizosaccharomyces pombe is a widely employed model organism for studying the eukaryotic cell cycle. Like plants and bacteria, S. pombe must build a cell wall in concert with its cell cycle, but how cell wall-synthesizing and remodeling enzymes mediate this process remains unclear. Here we characterize the functions of Aah1 and Aah3, two related S. pombe -amylases that are putative members of this evolutionarily conserved family of cell wall-modifying proteins. We found that unlike rod-shaped wildtype S. pombe cells, aah1{Delta} aah3{Delta} cells are nearly spherical, grow slowly, have thickened cell walls, and have severe defects in cell separation following cytokinesis. Solid-state NMR spectroscopy analyses of intact wildtype and aah1{Delta} aah3{Delta} cells revealed that aah1{Delta} aah3{Delta} cell walls are rigidified with a significant reduction in the -glucan matrix, characterized by reduced amounts of the major -1,3-glucan and the minor -1,4-glucan within the rigid and mobile phases; this reduction was compensated for by a two-fold increase in {beta}-glucan content. Indeed, viability of aah1{Delta} aah3{Delta} cells depended on {beta}-glucan upregulation and the cell wall integrity pathway that mediates it. While aah1{Delta} aah3{Delta} cells resemble cells with impaired function of the transglycosylation domain of -glucan synthase 1 (Ags1), increased expression of Aah3 does not compensate for impaired Ags1 function or vice-versa. Overall, our data suggest that Aah1 and Aah3 are required in addition to Ags1, likely downstream, for the transglycosylation of -glucan chains to generate fibers of appropriate dimensions to support proper cell morphology, growth, and division. Significance StatementThis study utilized a range of imaging techniques and high-resolution solid-state NMR spectroscopy of intact S. pombe cells to refine our understanding of S. pombe cell wall composition. This study also determined that two related GPI-anchored -amylase family proteins, Aah1 and Aah3, likely act as transglycosylases non-redundantly with an -glucan synthase in the synthesis of -glucan chains of appropriate content and size to support polarized growth and cell division. Our results also highlight the anti-fungal therapeutic potential of GPI-anchored enzymes acting in concert with glucan synthases.

microbiology↗

bioRxiv2024

A non-muscle α-actinin is an intrinsic component of the cardiac Z-disc and regulates sarcomere turnover, contractility, and heart remodeling

Cardiac sarcomeres generate the fundamental forces behind each heartbeat and are thought to contain only muscle-specific cytoskeletal proteins. We show that a widely expressed actin cross-linking protein, -actinin 4 (ACTN4), is a sarcomere component of the human and zebrafish heart in vivo and in human iPSC-derived cardiac myocytes (CMs) in vitro. A confluence of biochemical experiments, immunofluorescence, and AI modeling suggest ACTN4 forms a heterodimeric complex with muscle-specific ACTN2 at the cardiac Z-disc, the cardiac sarcomere border. ACTN4 depletion from human iPSC-CMs stabilizes canonical sarcomere proteins and drives contractility-dependent cellular hypertrophy while ACTN4 overexpression destabilizes sarcomeres. ACTN4 depletion from zebrafish embryos specifically increases ventricular contractility which drives atrial enlargement, suggesting biomechanically driven atrial remodeling. ACTN4-associated phenotypes in both model systems lack hallmarks of cardiac disease models and an ACTN4 variant in humans is associated with reduced risk for disease. Our findings suggest a "non-muscle" actinin regulates heart contractility and influences clinical outcomes related to heart failure.

cell biology↗

bioRxiv2023

Characterization of Pik1 function in fission yeast reveals its conserved role in lipid synthesis and not cytokinesis

Phosphatidylinositol (PI)-4-phosphate (PI4P) is a lipid found at the plasma membrane (PM) and Golgi in cells from yeast to humans. PI4P is generated from PI by PI4-kinases and can be converted to PI-4,5-bisphosphate [PI(4,5)P2]. Schizosaccharomyces pombe have 2 essential PI4-kinases: Stt4 and Pik1. Stt4 localizes to the PM and its loss from the PM results in a decrease of PM PI4P and PI(4,5)P2. As a result, cells divide non-medially due to disrupted cytokinetic ring-PM anchoring. However, the localization and function of S. pombe Pik1 has not been thoroughly examined. Here, we found that Pik1 localizes exclusively to the trans-Golgi and is required for Golgi PI4P production. We determined that Ncs1 regulates Pik1, but unlike in other organisms, it is not required for Pik1 Golgi localization. When Pik1 function was disrupted, PM PI4P but not PI(4,5)P2 levels were reduced, a major difference with Stt4. We conclude that Stt4 is the chief enzyme responsible for producing the PI4P that generates PI(4,5)P2. Also, that cells with disrupted Pik1 do not divide asymmetrically highlights the specific importance of PM PI(4,5)P2 for cytokinetic ring-PM anchoring. Summary statementFission yeast Pik1 localizes exclusively to the trans-Golgi independently of Ncs1, where it contributes to PI4P but not PI(4,5)P2 synthesis. Pik1 does not affect cytokinesis.

cell biology↗

bioRxiv2023

Membrane binding of endocytic myosin-1s is inhibited by a class of ankyrin repeat proteins

Myosin-1s are monomeric actin-based motors that function at membranes. Myo1 is the single myosin-1 isoform in Schizosaccharomyces pombe that works redundantly with Wsp1-Vrp1 to activate the Arp2/3 complex for endocytosis. Here, we identified Ank1 as an uncharacterized cytoplasmic Myo1 binding partner. We found that in ank1{Delta} cells, Myo1 dramatically redistributed from endocytic patches to decorate the entire plasma membrane and endocytosis was defective. Biochemical analysis and structural predictions suggested that the Ank1 ankyrin repeats bind the Myo1 lever arm and the Ank1 acidic tail binds the Myo1 TH1 domain to prevent TH1-dependent Myo1 membrane binding. Indeed, Ank1 over-expression precluded Myo1 membrane localization and recombinant Ank1 blocked purified Myo1 liposome binding in vitro. Based on biochemical and cell biology analyses, we propose budding yeast Ank1 and human OSTF1 are functional Ank1 orthologs and that cytoplasmic sequestration by small ankyrin repeat proteins is a conserved mechanism regulating myosin-1s in endocytosis. SummaryFission yeast long-tailed myosin-1 binds Ank1. Ank1 ankyrin repeats associate with the Myo1 lever arm and Ank1 acidic tail binds the Myo1 TH1 domain to inhibit Myo1 membrane binding. Ank1 orthologs exists in budding yeast (Ank1) and humans (OSTF1).

cell biology↗