Souffle/Spastizin regulates secretory granule maturation by sorting lysosomal cargo from immature secretory granule during zebrafish oogenesis

Abstract

Successful reproduction of all sexually reproducing organisms mainly depends on the viability of the oocyte. The making of the oocyte is regulated at multiple level to ensure its quality where it carries the information from mother to the next generation. During oogenesis, apart from meiotic maturation cytoplasm also changes dynamically. The cellular transport pathways play an essential role in importing molecules from the mother and positioning them, which creates the polarity, axis and germplasm for future embryo. Vesicle trafficking is a vital transport process in all cells to import and deliver molecules to interact with the surrounding environment. This cellular mechanism is necessary for many biological processes like signal transduction, cytokinesis, polarity and lysosomal degradation. The discovery of many regulators (Rabs and SNAREs) in unicellular organisms showed the requirement of tight regulation in vesicle fission and fusion process. However, a forward genetic screen for yolk endocytosis in C.elegans discovered many novel regulators and a novel compartment, which are specific to multi-cellular organisms suggesting the presence of more complex regulation in vertebrates. Hence, finding new regulators are necessary to understand the process in vertebrates. We aimed at identifying novel, vertebrate regulators of oogenesis in zebrafish oocytes using a maternal-effect mutagenesis screen. We isolated a group of mutants, which lays opaque rather than transparent embryos. One of the mutants named soufflé (suf) has been mapped to a FYVE- Zinc finger protein by positional cloning. This FYVE domain binds to PI3P lipids, which are predominantly found on endosomal membranes. Biochemical analysis revealed that these mutants have a defect in yolk cleavage during oocyte maturation, which leads to the observed opaqueness (Dosch et al., 2004). Furthermore, electron microscopy analysis revealed that suf accumulates smaller endosomes, whereas in wild types they mature into functional yolk globules in the oocyte. These preliminary results raised the question that how does soufflé regulate trafficking during oogenesis and what is the molecular mechanism behind the defect in suf mutant. Endosomal analysis showed accumulation of Rab11 and fragmented lysosome. In-vivo trafficking assay showed no defect in endocytosis and endosomal recycling. Rab11 localizes to secretory granule and suf mutant accumulates immature secretory granule (ISG). In-vivo assay showed that suf fail to elevate the chorion after fertilization indicating a defect in exocytosis of dense core vesicle (DCV). Further analysis showed that suf mutant accumulate secretory granule without dense core resulted from defective sorting and vesicle fission from ISG. Surprisingly, we discovered the vesicle in cortical region with Abstract 13 many clathrin buds without fission accumulating in suf mutant using electron microscopy, consistent with accumulation of ISG marker VAMP4. Remarkably, chemical inhibition of dynamin function in the wild-type egg mimics suf mutant phenotype in cellular level and failed in chorion elevation. In addition, mis-organised F-actin showed the failure in exocytosis of vesicle. Rescue analysis with mutant construct showed that suf allele is a hypo morph. These results show that suf is necessary for sorting and fission during DCV formation. Sorting from ISG during DCV formation is also necessary for lysosomal biogenesis. We found that suf mutant lysosome did not receive cathepsin B, F and L, yolk-degrading proteases, explaining the opaque phenotype. Furthermore, vATPase subunit d1 is also missing in the vesicle of suf mutant oocytes. Analysis of lysosomal sorting receptors and adaptor revealed that suf mutant does not have a defect in M6PR and sortilin mediated pathway, but they show reduced LAMP carrier intermediate mediated by VPS41 and AP3. In addition, AP3 positive vesicles are reduced while AP3 accumulates more on secretory granule consistent with reduced VPS41 recruitment on secretory granule. VPS41-AP3 mediated lysosomal transport carries lysosomal enzymes, SNAREs and other membrane proteins. The VPS41 and AP3 mutant phenotype explains the reason for fragmented lysosome and failure in DCV formation phenotype in suf mutant. Bioinformatics prediction shows that suf has a clathrin heavy chain domain, which can form clathrin like coat like in VPS41. Suf interaction with AP5 further suggesting that suf may work as a coat protein like VPS41 for lysosomal transport. We discovered a novel function for suf in sorting of lysosomal cargo sorting from ISG results in successful DCV formation and lysosomal function, in the absence of suf both lysosome and DCV formation is affected. Interestingly, souffle mutation has been identified to cause progressive motor neuro-degeneration in human. This defect in DCV formation could be one reason for why the long motor neuron loses its connection and causes spasticity, since DCV is necessary for long-term potentiation. Hence, souffle mutant egg is an excellent model to study the molecular mechanism and disease pathology.