Search for: All records

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag) protein and mediates the intercellular RNA transfer through virus-like capsids. However, the regulators and secretion pathway through which Arc capsids maneuver cargos are unclear. Here, we identified that phosphatidylinositol-3-phosphate (PI3P) mediates Arc capsid assembly and secretion through the endosomal–multivesicular body (MVB) pathway. Indeed, reconstituted Arc protein preferably binds to PI3P. In HEK293T cells, Arc forms puncta that colocalize with FYVE, an endosomal PI3P marker, as well as Rab5 and CD63, early endosomal and MVB markers, respectively. Superresolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces the Arc-mediated RNA transfer efficiency. RalA/B double knockdown in cultured rat cortical neurons increases the percentage of mature dendritic spines. Intake of extracellular vesicles purified from Arc-expressing wild-type, but not RalA/B double knockdown, cells in mouse cortical neurons reduces their surface GlutA1 levels. These results suggest that unlike the HIV Gag, whose membrane targeting requires interaction with plasma-membrane-specific phosphatidyl inositol (4,5) bisphosphate (PI(4,5)P2), the assembly of Arc capsids is mediated by PI3P at endocytic membranes. Understanding Arc’s secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins. 

Articular cartilage is a collagen-rich tissue that provides a smooth, lubricated surface for joints and is also responsible for load bearing during movements. The major components of cartilage are water, collagen, and proteoglycans. Osteoarthritis is a degenerative disease of articular cartilage, in which an early-stage indicator is the loss of proteoglycans from the collagen matrix. In this study, confocal Raman microspectroscopy was applied to study the degradation of articular cartilage, specifically focused on spatially mapping the loss of glycosaminoglycans (GAGs). Trypsin digestion was used as a model for cartilage degradation. Two different scanning geometries for confocal Raman mapping, cross-sectional and depth scans, were applied. The chondroitin sulfate coefficient maps derived from Raman spectra provide spatial distributions similar to histological staining for glycosaminoglycans. The depth scans, during which subsurface data were collected without sectioning the samples, can also generate spectra and GAG distributions consistent with Raman scans of the surface-to-bone cross sections. In native tissue, both scanning geometries demonstrated higher GAG content at the deeper zone beneath the articular surface and negligible GAG content after trypsin degradation. On partially digested samples, both scanning geometries detected an ∼100 μm layer of GAG depletion. Overall, this research provides a technique with high spatial resolution (25 μm pixel size) to measure cartilage degradation without tissue sections using confocal Raman microspectroscopy, laying a foundation for potential in vivo measurements and osteoarthritis diagnosis.