Biology subjects

Prosser, D. C.

Publications and source records attributed to Prosser, D. C..

5 recordsLinked to original sources

bioRxiv2026

A Sac7-Rho1 axis at the plasma membrane controls clathrin-independent endocytosis

In eukaryotes, our understanding of clathrin-independent endocytosis (CIE) lags far behind that of clathrin-mediated endocytosis (CME). CIE plays key roles in internalizing receptors, viruses, bacterial toxins, and pathogens; thus, deeper mechanistic insights are critical for understanding cellular strategies for plasma membrane regulation. Yeast CIE requires a signal relay between the stress sensor Mid2, the guanine nucleotide exchange factor (GEF) Rom1, the Rho1 GTPase, and the formin Bni1. While GEFs promote GTPase activity, GTPase-activating proteins (GAPs) conversely stimulate nucleotide hydrolysis and GTPase inactivation. Here, we provide new insight into CIE, adding the RhoGAP Sac7 as a regulator. SAC7 deletion in CME-deficient cells improved cargo internalization, and Sac7 localizes primarily to the mother cortex. Cells lacking SAC7 accumulate active Rho1 and retain Bni1 at the plasma membrane, where Bni1 retention may subsequently enhance actin assembly needed for CIE. Our results thus demonstrate that Sac7 negatively regulates CIE by restricting cortical Rho1 activity.

cell biology↗

bioRxiv2026

The yeast DENN domain protein Avl9 contributes to recycling and sorting of endosomal cargos

In yeast and humans, the conserved DENN-domain (Differentially Expressed in Normal and Neoplastic tissue) protein Avl9 is thought to play roles in membrane traffic and secretion, but its precise function remains poorly defined. Since DENN-containing proteins are associated with Rab GTPase function, we sought to understand Avl9 function in the context of Rab regulation. Here, we show that Avl9 localizes to peripheral punctae that are consistent with secretory vesicles. Moreover, we demonstrate genetic interactions and co-localization between Avl9 and numerous Rabs in the secretory and endosomal pathways, suggesting a potential function at the interface of secretion and recycling. Consistent with this role, avl9{Delta} results in defective recycling of the endosomal cargo Snc1 but does not alter plasma membrane delivery of an endocytosis-defective Snc1EN- mutant, suggesting that Avl9 is not directly involved in secretory traffic from the TGN to the plasma membrane. The avl9{Delta} recycling defect is exacerbated by the additional loss of RCY1 or SNX4, but not VPS35. Each of these three genes contributes to a distinct endosomal recycling pathway, indicating that Avl9 acts in conjunction with multiple recycling pathways. Summary StatementIn this study, Rioux et al. describe a role for the DENN domain protein Avl9, previously thought to regulate secretion, as a novel factor involved in recycling of cargos from endosomal compartments.

cell biology↗

bioRxiv2025

Stress granules and protein aggregates reveal intracellular resource competition

Stress granules are biomolecular condensates that form in response to environmental stress and disassemble once normal conditions are restored. However, when disassembly fails, stress granules can persist and solidify. While stress granule solidification has been well documented, the cellular mechanisms underlying the transition from reversible to persistent stress granules remain unclear. Persistent stress granules can seed the formation of pathological aggregates, such as TDP-43 in amyotrophic lateral sclerosis1, 2. Although amyloid and tau aggregates are hallmarks of Alzheimers disease, a subset of patients also develop TDP-43 deposits, suggesting a possible role for stress granule solidification in Alzheimers disease progression3-5. Despite theoretical models explaining why persistence and ensuing solidification occurs, strong in vivo evidence is lacking6. Here we show that competition for limited chaperone resources drive stress granule persistence. In the presence of TDP-43 aggregates or yeast amyloid proteins called prions, stress granule disassembly is slowed or halted disassembly. Using yeast prions as a model, we show that the addition of chaperones, specifically the AAA+ ATPase molecular chaperone, Hsp104, resulted in resumption of stress granule disassembly. Our results demonstrate that the competition for shared resources, such as molecular chaperones, can limit stress granule disassembly. We suspect that the presence of pathological aggregates results in resource competition within the aging brain, contributing to the persistence of stress granules and their subsequent solidification and aggregation.

cell biology↗

bioRxiv2024

A new series of fluorogen-activating proteins for quantitative protein trafficking and co-localization studies in S. cerevisiae

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae. SIGNIFICANCE STATEMENT- Quantitative endocytic assays are required to characterize factors that regulate both ligand-dependent and constitutive endocytosis. - We optimize fluorogen-activating proteins (FAPs) technology for use as a live cell imaging probe in yeast that fluoresces in the far-red range for quantitative endocytosis assays. - The FAP tools and approaches generated will facilitate quantitative endocytic and protein recycling assays for yeast cell biologists.

cell biology↗

bioRxiv2023

Actin- and microtubule-based motors contribute to clathrin-independent endocytosis in yeast

Most eukaryotic cells utilize clathrin-mediated endocytosis as well as multiple clathrin-independent pathways to internalize proteins and membranes. Although clathrin-mediated endocytosis has been studied extensively and many machinery proteins have been identified, clathrin-independent pathways remain poorly characterized by comparison. We previously identified the first known yeast clathrin-independent endocytic pathway, which relies on the actin-modulating GTPase Rho1, the formin Bni1 and unbranched actin filaments, but does not require the clathrin coat or core clathrin machinery proteins. In this study, we sought to better understand clathrin-independent endocytosis in yeast by exploring the role of myosins as actin-based motors, since actin is required for endocytosis in yeast. We find that Myo2, which transports secretory vesicles, organelles and microtubules along actin cables to sites of polarized growth, participates in clathrin-independent endocytosis. Unexpectedly, the ability of Myo2 to transport microtubule plus ends to the cell cortex appears to be required for its role in clathrin-independent endocytosis. In addition, dynein, dynactin and proteins involved in cortical microtubule capture are also required. Thus, our results suggest that interplay between actin and microtubules contributes to clathrin-independent internalization in yeast. Summary StatementClathrin-independent endocytosis is a poorly-understood but conserved process. Here, we provide evidence of a role for myosin and dynein as motor proteins involved the yeast clathrin-independent pathway.

cell biology↗