Functional complementation of V-ATPase a subunit isoforms in osteoclasts

In osteoclasts, the a3 isoform of the proton-pumping V-ATPase plays essential roles in anterograde trafficking of secretory lysosomes and extracellular acidification required for bone resorption. This study examined functional complementation of the a isoforms by exogenously expressing the a1, a2 and a3 isoforms in a3-knockout (KO) osteoclasts. The expression levels of a1 and a2 in a3KO osteoclasts were similar, but lower than that of a3. a1 significantly localized to lysosomes, whereas a2 slightly did. On the other hand, a2 interacted with Rab7, a regulator of secretory lysosome trafficking in osteoclasts, more efficiently than a1. a1 partly complemented the functions of a3 in secretory lysosome trafficking and calcium phosphate resorption, while a2 partly complemented the former but not the latter function.

Aesculetin Inhibits Bone Resorption Through Down-Regulating Differentiation and Lysosomal Formation in Osteoclasts

Objectives For the optimal resorption of mineralized bone extracellular matrix, osteoclasts require the generation of a resorption lacuna characterized by the presence of specific proteases and a low pH. Thus, bone resorption by osteoclasts highly rely on lysosomes, the organelles specialized in intra- and extracellular material degradation. Aesculetin, a derivative of coumarin, possesses anti-inflammatory and anti-bacterial effects. The purpose of this study was to identify that aesculetin inhibited osteoclast differentiation and bone resorption through down-regulating lysosomal formation. Methods Raw 264.7 cells were cultured for 5 days on α-MEM with 10% FBS in the absence or presence of 50 ng/ml RANKL and 1–10 μM aesculetin. Tartrate-resistance acid phosphatase (TRAP) staining and bone resorption assay were performed by using assay kits. Western blotting was conducted with antibodies of target proteins involved in activation and lysosome biogenesis of osteoclasts. Immunocytochemical analysis employed LysoTracker for lysosome staining and α-tubulin antibody conjugated with FITC. Results Aesculetin inhibited RANKL-treated formation of multinucleated osteoclasts with a reduction of TRAP activity. When 1–10 μM aesculetin was treated to RANKL-exposed osteoclasts, the bone resorption was highly suppressed in osteoclasts. In addition, aesculetin reduced cellular expression of carbonic anhydrase II, vacuolar-type H (+)-ATPase D2 and cathepsin K elevated by RANKL, all involved in the bone resorption. Furthermore, aesculetin curtailed cellular induction of autophagy-related (Atg)5, Atg7 and small GTPase Rab7 elevated by RANKL for lysosome transportation/secretion and bone resorption in osteoclasts. Conclusions Aesculetin was effective in retarding osteoclast differentiation and secretory lysosome formation for osteoclast resorption, indicating that this compound may be a potential agent for the treatment of osteoporosis. Funding Sources No funding sources to report.

Biomechanical control of lysosomal secretion via the VAMP7 hub: a tug-of-war mechanism between VARP and LRRK1

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we found that cells deficient in the secretory lysosome v-SNARE VAMP7 were impaired in adapting to substrate rigidity. Conversely VAMP7-mediated secretion was stimulated by more rigid substrate and this regulation depended on the Longin domain of VAMP7. We further found that the Longin domain bound the kinase and retrograde trafficking adaptor LRRK1 and LRRK1 negatively regulated VAMP7-mediated exocytosis. Conversely, VARP, a VAMP7- and kinesin 1-interacting protein, further controlled the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. We propose a mechanism whereby biomechanical constraints regulate VAMP7- dependent lysosomal secretion via LRRK1 and VARP tug-of-war control of the peripheral readily- releasable pool of secretory lysosomes.

The munc13-4–rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane

Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4–rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4–rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.