The noncanonical role of the protease cathepsin D as a cofilin phosphatase.

Cathepsin D (cathD) is traditionally regarded as a lysosomal protease that degrades substrates in acidic compartments. Here we report cathD plays an unconventional role as a cofilin phosphatase orchestrating actin remodeling. In neutral pH environments, the cathD precursor directly dephosphorylates and activates the actin-severing protein cofilin independent of its proteolytic activity, whereas mature cathD degrades cofilin in acidic pH conditions. During development, cathD complements the canonical cofilin phosphatase slingshot and regulates the morphogenesis of actin-based structures. Moreover, suppression of cathD phosphatase activity leads to defective actin organization and cytokinesis failure. Our findings identify cathD as a dual-function molecule, whose functional switch is regulated by environmental pH and its maturation state, and reveal a novel regulatory role of cathD in actin-based cellular processes.

Late endosomes promote microglia migration via cytosolic translocation of immature protease cathD.

Organelle transport requires dynamic cytoskeleton remodeling, but whether cytoskeletal dynamics are, in turn, regulated by organelles remains elusive. Here, we demonstrate that late endosomes, a type of prelysosomal organelles, facilitate actin-cytoskeleton remodeling via cytosolic translocation of immature protease cathepsin D (cathD) during microglia migration. After cytosolic translocation, late endosome-derived cathD juxtaposes actin filaments at the leading edge of lamellipodia. Suppressing cathD expression or blocking its cytosolic translocation impairs the maintenance but not the initiation of lamellipodial extension. Moreover, immature cathD balances the activity of the actin-severing protein cofilin to maintain globular-actin (G-actin) monomer pool for local actin recycling. Our study identifies cathD as a key lysosomal molecule that unconventionally contributes to actin cytoskeleton remodeling via cytosolic translocation during adenosine triphosphate-evoked microglia migration.

Lysosomal Hydrolase Cathepsin D Non-proteolytically Modulates Dendritic Morphology in Drosophila.

The main lysosomal protease cathepsin D (cathD) is essential for maintaining tissue homeostasis via its degradative function, and its loss leads to ceroid accumulation in the mammalian nervous system, which results in progressive neurodegeneration. Increasing evidence implies non-proteolytic roles of cathD in regulating various biological processes such as apoptosis, cell proliferation, and migration. Along these lines, we here showed that cathD is required for modulating dendritic architecture in the nervous system independent of its traditional degradative function. Upon cathD depletion, class I and class III arborization (da) neurons in Drosophila larvae exhibited aberrant dendritic morphology, including over-branching, aberrant turning, and elongation defects. Re-introduction of wild-type cathD or its proteolytically-inactive mutant dramatically abolished these morphological defects. Moreover, cathD knockdown also led to dendritic defects in the adult mushroom bodies, suggesting that cathD-mediated processes are required in both the peripheral and central nervous systems. Taken together, our results demonstrate a critical role of cathD in shaping dendritic architecture independent of its proteolytic function.

A Novel Method to Image Macropinocytosis in Vivo.

Here we described an experimental protocol for in vivo imaging of macropinocytosis and subsequent intracellular events. By microinjection, we delivered fluorescence dextrans together with or without ATPγS into transparent Drosophila melanogaster embryos. Using a confocal microscope for live imaging, we monitored the generation of dextran-positive macropinosomes and subsequent intracellular events. Our protocol provides a continent and reliable way for investigating macropinocytosis and its underlying mechanisms, especially when combined with genetic strategies.