␣B-crystallin (␣B) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of ␣B has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that ␣B is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of ␣B in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize ␣B in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100 -200 nm, were characterized by the presence of flotillin-1, ␣-enolase, and Hsp70, the same proteins that associate with detergentresistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of ␣B is predominantly apical. Using OptiPrep gradients we demonstrate that ␣B resides in the DRM fraction. The secretion of ␣B is inhibited by the cholesterol-depleting drug, methyl ␤-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.The small heat shock protein, ␣B-crystallin (␣B) 2 is a developmentally regulated gene product whose association with multiple pathologies of varied antecedents such as neurodegeneration, oncogenesis, and cataracts suggests a vital function for this protein (1-3). Elevated levels of ␣B have been reported in Alexander, Alzheimer, and Parkinson diseases. It is expressed in astrocytes (4) and has been implicated in peripheral nerve myelination (5). It is known to be one of the main antigens involved in multiple sclerosis (6). Its expression in a subset of basal-like breast carcinomas has led to its characterization as a novel oncoprotein (7). It is also a potential tissue biomarker for renal cell carcinoma (8). Interestingly, ␣B has also been shown to activate T cells (9) and inhibit platelet aggregation (10).In the eye, apart from its predominant presence in the ocular lens, ␣B was initially reported in primary cultures of human retinal pigment epithelium (RPE) (11) and has since been shown to be expressed in the retina (1) and during early development of the rat eye in the embryonic RPE (12). It is highly expressed in rod outer segments as well as in the rat RPE, following intense light exposures that lead to photoreceptor cell degeneration (13). In age-related macular degeneration, high concentrations of ␣B transcripts are found in microdissected retinal tissue juxtaposed with subretinal lipoprotein deposits, known as ...

␣B-Crystallin is a developmentally regulated small heat shock protein known for its binding to a variety of denatured polypeptides and suppression of protein aggregation in vitro. Elevated levels of ␣B-crystallin are known to be associated with a number of neurodegenerative pathologies such as Alzheimer disease and multiple sclerosis. Mutations in ␣B-crystallin gene have been linked to desminrelated cardiomyopathy and cataractogenesis. The physiological function of this protein, however, is unknown. Using discontinuous sucrose density gradient fractionation of post-nuclear supernatants, prepared from rat tissues and human glioblastoma cell line U373MG, we have identified discrete membrane-bound fractions of ␣B-crystallin, which co-sediment with the Golgi matrix protein, GM130. Confocal microscopy reveals co-localization of ␣B-crystallin with BODIPY TR ceramide and the Golgi matrix protein, GM130, in the perinuclear Golgi in human glioblastoma U373MG cells. Examination of synchronized cultures indicated that ␣B-crystallin follows disassembly of the Golgi at prometaphase and its reassembly at the completion of cytokinesis, suggesting that this small heat shock protein, with its chaperone-like activity, may have an important role in the Golgi reorganization during cell division.

SummaryThe developing eye lens presents an exceptional paradigm for spatial transcriptomics. It is composed of highly organized long, slender transparent fiber cells, which differentiate from the edges of the anterior epithelium of the lens (equator), attended by high expression of crystallins, which generates transparency. Every fiber cell, therefore, is an optical unit whose refractive properties derive from its gene activity. Here, we probe this tangible relationship between the gene activity and the phenotype by studying the expression of all known 17 crystallins and 77 other non-crystallin genes in single fiber cells isolated from three states/regions of differentiation, allowing us to follow molecular progression at the single-cell level. The data demonstrate highly variable gene activity in cortical fibers, interposed between the nascent and the terminally differentiated fiber cell transcription. These data suggest that the so-called stochastic, highly heterogeneous gene activity is a regulated intermediate in the realization of a functional phenotype.

Purpose All crystallins have non-crystallin catalytic functions. Because catalytic functions do not require large concentrations of protein, as are seen in the lens, there is a perception of dichotomy in the catalytic/physiological function of crystallins within and outside the lens. The status of αB-crystallin, a ubiquitously expressed small heat shock protein (and a crystallin) in the ocular lens, was investigated. Methods Discontinuous sucrose density gradients were used for fractionation of Golgi membranes and vesicles. Light microscopy and confocal microscopy were used for immunolo-calization in cultured cells and the native lens. Results αB-crystallin is highly organized, as indicated by its polar presence in the apical Golgi in lens epithelium and in the perinuclear Golgi streaks in differentiating lens fiber cells. Assessment of the distribution of αB-crystallin in Golgi-enriched and vesicular fractions (characterized by the presence of Golgi membrane protein GM130 and vesicle coat protein γCOP) in the developing lens reveal a gradual transition from Golgi to vesicular fraction, concomitant with the appearance of αB-crystallin as a “soluble” protein. Conclusions These data demonstrate that αB-crystallin, known to be a soluble protein, starts life as a Golgi-associated membrane protein in the fetal and early postnatal lens and that the developmentally controlled physical state of the Golgi determines the status of this protein in the lens. These findings also show the similarity in the localization/ physiological function of αB-crystallin within and outside the ocular lens and suggest that non-crystallin/catalytic function is an innate component of the expression of a crystallin in the lens.

Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein ␣B-crystallin (␣B) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of ␣B via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where ␣B expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and ␣B-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of ␣B results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for ␣B in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.Exosomes are 50 -200-nm nanovesicles produced via the endosomal pathway of protein homeostasis (1-3). The process of endocytosis and the trafficking of endocytosed vesicles to lysosomes are major pathways of protein metabolism that regulate transmembrane protein (receptor) turnover at the plasma membrane. The exosome biogenesis starts with the early endosome that matures into a late endosome concomitant with its intraluminal vesicles, making it a multivesicular body (MVB). 2The MVB is at a branch point of this pathway from where it may progress toward two different cellular compartments with two entirely different physiological consequences. The first one is that the MVB may fuse with the lysosomes (generating the endolysosomal compartment) leading to the degradation of its vesicular contents; the second is that the MVB traffics to and fuses with the plasma membrane, leading to the release of its intraluminal vesicles as exosomes carrying the active macromolecular cargo. What dictates either of the two fates is not known (4).RPE is an important single layer of cells at the interface of the blood-retina barrier. It provides critical physiological support for the maintenance of the photoreceptor neurons (5, 6). It is a polarized epithelium, which on its apical side nourishes the ...