DictyosteliumLvsB Mutants Model the Lysosomal Defects Associated with Chediak-Higashi Syndrome

Abstract

Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans ("lysosomal trafficking regulator") or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideum contains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal alpha-mannosidase were normal, although lvsB mutants inefficiently retained alpha-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis.

A LYST/beige homolog is involved in biogenesis ofDictyosteliumsecretory lysosomes

“…2A The size and number of lysosomes was also determined in random confocal pictures of cells stained for both H + -ATPase and p80. The diameter of lysosomes in lvsB mutant cells was 27% larger than in WT cells (see Table 1), corroborating previous observations (Harris et al, 2002;Marchetti et al, 2004), but their number was reduced by 28%. Overall the total surface of lysosomal compartments was 23% higher in lvsB than in WT cells (Table 1).…”

“…Two lines of evidence further suggest a defect in the biogenesis of post lysosomes in lvsB mutant cells: first, transfer of internalized particles from lysosomes to post lysosomes is markedly slower in lvsB mutant cells than in WT cells; second, in lvsB mutant cells, p80 is depleted from the cell surface and concentrated in lysosomal compartments, suggesting that the transfer of p80 to post lysosomes is diminished. Further experiments will be necessary to determine whether abnormal maturation of lysosomes is caused by their abnormal fusion properties (Harris et al, 2002).…”

“…of the LYST gene recapitulate several features of the ChediakHigashi syndrome in Dictyostelium cells , notably an enlargement of endocytic compartments, and anomalies in the secretion of lysosomal enzymes (Cornillon et al, 2002;Harris et al, 2002;Marchetti et al, 2004). Here we show that the Dictyostelium LvsB protein is specifically involved in the biogenesis of fusion-competent post-lysosomal compartments.…”

Dictyostelium Finds New Roles to Model

“…Several of these have already been the subject of functional studies (Lee et al 1998;Harris et al 2002;Wessels et al 2006;Langenick et al 2008), and this seems likely to be a major future use of the organism. Also, Dictyostelium has been used to help identify targets for drugs used to treat human diseases: cis-platin, an anticancer agent (Li et al 2000); lithium used to treat bipolar disorder (Williams et al 1999); and bisphosphonates used to treat osteoporosis (Grove et al 2000).…”

RabD, aDictyosteliumRab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion

“…To quantify the rate of phagosome fusion, we pulsed cells in shaking suspension for 10 minutes with FITC-bacteria and allowed the cells to chase for 60 minutes while they settled on coverslips. Under these conditions bacteria enter as single particles (Harris et al, 2002), and 60 minutes of chase corresponds to the linear portion of the phagosome fusion curve. A representative fluorescence and phase contrast micrograph is shown in Fig.…”