Mroh1, a lysosomal regulator localized by WASH-generated actin

The process of constitutive exocytosis remains poorly characterized. While some roles of the WASH complex in exocytosis are known, the mechanisms governing late-stage exocytosis are unclear. Prior research has shown that the WASH complex is essential for actin nucleation in membrane trafficking. However, no prior work had resolved how lysosomal maturation coordinates with exocytosis. This gap motivated the use of a FACS-based screening approach to identify novel regulators. The study builds on established knowledge of exocytosis in model organisms like Dictyostelium. It was already known that fluorescent dextran uptake can track exocytosis defects. The research introduces a new method to detect exocytosis disruptions. This paper expands on prior findings by focusing on lysosomal regulators.

The aim of this study is to identify proteins involved in late-stage exocytosis. Researchers focused on a poorly understood phase of exocytosis, specifically after lysosome neutralization. The study seeks to uncover new regulators of exocytosis using a FACS-based screening method. The researchers hypothesized that fluorescent dextran retention could reveal exocytosis defects. The motivation stems from gaps in understanding how lysosomes transition to postlysosomes. The study also explores the role of the WASH complex in this process. The goal is to determine whether Mroh1 influences exocytosis timing. The work addresses a specific problem in exocytosis regulation that had not been fully explored.

The researchers developed a FACS-based method to identify exocytosis mutants. They used fluorescent dextran uptake to track exocytosis defects in Dictyostelium. A random mutant pool was screened to detect disruptions in exocytosis. The method involved selecting cells that retained fluorescent dextran. The WASH complex mutants were expected to show exocytosis defects. The study also examined Mroh1 mutants to assess lysosomal maturation. Researchers observed lysosomal dynamics using fluorescent markers. The role of actin in Mroh1 localization was tested using latrunculin treatment.

Screening identified Mroh1 mutants alongside WASH complex mutants. Mroh1 mutants showed normal endosome development but delayed exocytosis. Lysosomes in Mroh1 mutants failed to transition to postlysosomes efficiently. The WASH complex was found to be recycled inefficiently in Mroh1 mutants. Mroh1 localizes to lysosomes in both mammalian and Dictyostelium cells. The protein accumulates on lysosomes during maturation and is removed before exocytosis. WASH-generated actin is required for proper Mroh1 localization. Latrunculin treatment caused Mroh1 to relocalize to small vesicles.

The findings suggest that Mroh1 is involved in a late exocytosis step dependent on actin. The study shows that Mroh1 localization is regulated by the WASH complex. The researchers propose that Mroh1 functions after lysosome neutralization. The results indicate that exocytosis is delayed in Mroh1 mutants. The study supports a role for Mroh1 in coordinating lysosomal maturation with exocytosis. The data suggest that actin dynamics influence Mroh1 localization. The authors conclude that Mroh1 is part of a previously undefined exocytosis mechanism. The findings highlight the importance of actin in exocytosis regulation.