PURPOSE. Retinal degeneration initiated by loss of photoreceptors is the prevalent cause of visual impairment and blindness in industrialized countries. Transplantation of photoreceptor cells represents a possible replacement strategy. This study determined that identification of cell surface antigens can assist in enriching photoreceptor precursors for transplantation. METHODS. The expression profile of rod photoreceptors at postnatal day 4 was investigated by microarray analysis to identify photoreceptor-specific cell surface antigens. For enrichment of transplantable photoreceptors, neonatal retinas from rod photoreceptor-specific reporter mice were dissociated, and the rods were purified by magnetic associated cell sorting (MACS) with CD73 antibodies. MAC-sorted cell fractions were transplanted into the subretinal space of adult wild-type mice. The number of rod photoreceptors contained in unsorted, CD73-negative, and CD73-positive cell fractions were quantified in vitro and after grafting in vivo. RESULTS. Microarray analysis revealed that CD73 is a marker for rod photoreceptors. CD73-based MACS resulted in enrichment of rods to 87%. Furthermore, in comparison with unsorted cell fractions, CD73-positive MAC-sorted cells showed an approximately three-fold increase in the number of integrated, outer segment-forming photoreceptors after transplantation. CONCLUSIONS. CD73-based MACS is a reliable method for the enrichment of integrating photoreceptors. Purification via cell surface markers represents a new tool for the separation of transplantable photoreceptor precursors from a heterogeneous cell population, avoiding the need of reporter gene expression in target cells.
Insulin maintains homeostasis of glucose by promoting its uptake into cells from the blood. Hyperglycemia triggers secretion of insulin from pancreatic -cells. This process is mediated by secretory granule exocytosis. However, how -cells keep granule stores relatively constant is still unknown. ICA512 is an intrinsic granule membrane protein, whose cytosolic domain binds 2-syntrophin, an F-actin-associated protein, and is cleaved upon granule exocytosis. The resulting cleaved cytosolic fragment, ICA512-CCF, reaches the nucleus and up-regulates the transcription of granule genes, including insulin and ICA512. Here, we show that ICA512-CCF also dimerizes with intact ICA512 on granules, thereby displacing it from 2-syntrophin. This leads to increased granule mobility and insulin release. Based on these findings, we propose a model whereby the generation of ICA512-CCF first amplifies insulin secretion. The ensuing reduction of granule stores would then increase the probability of newly generated ICA512-CCF to reach the nucleus and enhance granule biogenesis, thus allowing -cells to constantly adjust production of granules to their storage size and consumption. Pharmacological modulation of these feedback loops may alleviate deficient insulin release in diabetes.
The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.
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