Abstract
btn1, the Schizosaccharomyces pombe orthologue of the human Batten disease gene CLN3, exerts multiple cellular effects. As well as a role in vacuole pH homoeostasis, we now show that Btn1p is essential for growth at high temperatures. Its absence results in progressive defects at 37°C that culminate in total depolarized growth and cell lysis. These defects are preceded by a progressive failure to correctly polarize sterol-rich domains after cytokinesis and are accompanied by loss of Myo1p localization. Furthermore, we found that in Sz. pombe, sterol spreading is linked to defective formation/polarization of F-actin patches and disruption of endocytosis and that these processes are aberrant in btn1Δ cells. Consistent with a role for Btn1p in polarized growth, Btn1p has an altered location at 37°C and is retained in actin-dependent endomembrane structures near the cell poles or septum.
Highlights
Juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten disease, a severe neurodegenerative lysosomal storage disease of childhood characterized by accumulation of lipofuscin-like material, is caused by mutations in CLN3
We describe several phenotypes associated with loss of btn1, the Sz. pombe orthologue of CLN3, and investigation of the underlying molecular pathways provided evidence of a link between these and with Btn1p function (Table 1)
Progressive depolarization and spreading of sterol-rich domains commenced after the first cell cycle (7 h) at 378C, and this was preceded by a progressive failure in the formation and polarization of F-actin patches and by endocytic defects that we show are linked
Summary
Juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten disease, a severe neurodegenerative lysosomal storage disease of childhood characterized by accumulation of lipofuscin-like material, is caused by mutations in CLN3. Its function has been linked to many cellular processes, including trafficking [8], cytoskeletal organization [9], lysosomal homoeostasis [10,11], autophagy [12], apoptosis [13] and lipid modification or changes [14,15]. We have previously shown that deletion of btn, the CLN3 orthologue, causes dysregulation of vacuole homoeostasis, with btn1D cells displaying larger and less acidic vacuoles (equivalent to mammalian lysosomes) [16], as in patient cells [10]. Ectopic expression of N-terminally fused green fluorescent protein (GFP)–Btn1p or GFP–CLN3 constructs in btn1D cells complements the vacuole size and pH defects, confirming that Btn1p and CLN3 are functional homologues. Subsequent analysis revealed that at 378C, Btn1p is involved in an F-actin-dependent process that links endocytosis and the polarized localization of sterol-rich membrane domains with polarized growth
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