Mroh1, a lysosomal regulator localized by WASH-generated actin

Abstract

The steps leading to constitutive exocytosis are poorly understood. In Dictyostelium WASH complex mutants, exocytosis is blocked, so cells that take up fluorescent dextran from the medium retain it and remain fluorescent. Here, we establish a FACS-based method to select cells that retain fluorescent dextran, allowing identification of mutants with disrupted exocytosis. Screening a pool of random mutants identified members of the WASH complex, as expected, and multiple mutants in the conserved HEAT-repeat-containing protein Mroh1. In mroh1 mutants, endosomes develop normally until the stage where lysosomes neutralize to postlysosomes, but thereafter the WASH complex is recycled inefficiently, and subsequent exocytosis is substantially delayed. Mroh1 protein localizes to lysosomes in mammalian and Dictyostelium cells. In Dictyostelium, it accumulates on lysosomes as they mature and is removed, together with the WASH complex, shortly before the postlysosomes are exocytosed. WASH-generated F-actin is required for correct subcellular localization; in WASH complex mutants, and immediately after latrunculin treatment, Mroh1 relocalizes from the cytoplasm to small vesicles. Thus, Mroh1 is involved in a late and hitherto undefined actin-dependent step in exocytosis.

Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers

“…Consequently, mature pigmented melanosomes become neutral (Raposo et al, 2001) and competent for secretion. Interestingly in Dictyostellium discoideum , lysosomes destined for exocytosis alkalinize their lumen through a WASH- and Arp2/3-dependent retrieval of cargoes, including the vacuolar ATPase (Carnell et al, 2011; King et al, 2013; Thomason et al, 2017). The export of vacuolar ATPase, which associates with melanosomes (Tabata et al, 2008), might serve to raise the pH of melanosomes and promote their secretion.…”

WASP family proteins and formins compete in pseudopod- and bleb-based migration

“…Images were acquired every 2 s. Photobleaching was performed using an Andor mosaic FRAPPA unit. Macropinocytosis was measured exactly as in Thomason et al (2017) .…”

High-throughput Measurement of <em>Dictyostelium discoideum</em> Macropinocytosis by Flow Cytometry

“…Using 60 min as the standard time point when comparing macropinocytosis in different mutants/conditions therefore allows a good signal to be achieved, and no signal is lost due to exocytosis. Mutants where exocytosis is severely obstructed may take longer to reach a plateau 29 .…”