Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging.

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.

RFX6 Maintains Gene Expression and Function of Adult Human Islet α-Cells.

Mutations in the gene encoding the transcription factor regulatory factor X-box binding 6 (RFX6) are associated with human diabetes. Within pancreatic islets, RFX6 expression is most abundant in islet α-cells, and α-cell RFX6 expression is altered in diabetes. However, the roles of RFX6 in regulating gene expression, glucagon output, and other crucial human adult α-cell functions are not yet understood. We developed a method for selective genetic targeting of human α-cells and assessed RFX6-dependent α-cell function. RFX6 suppression with RNA interference led to impaired α-cell exocytosis and dysregulated glucagon secretion in vitro and in vivo. By contrast, these phenotypes were not observed with RFX6 suppression across all islet cells. Transcriptomics in α-cells revealed RFX6-dependent expression of genes governing nutrient sensing, hormone processing, and secretion, with some of these exclusively expressed in human α-cells. Mapping of RFX6 DNA-binding sites in primary human islet cells identified a subset of direct RFX6 target genes. Together, these data unveil RFX6-dependent genetic targets and mechanisms crucial for regulating adult human α-cell function.

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs.

ß cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of ß cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo-labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic ß cells.

Pancreatic ß cells store insulin within secretory granules which undergo exocytosis upon elevation of blood glucose levels. Crinophagy and autophagy are instead responsible to deliver damaged or old granules to acidic lysosomes for intracellular degradation. However, excessive consumption of insulin granules can impair ß cell function and cause diabetes. Atp6ap2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy. Here, we show that Cre recombinase-mediated conditional deletion of Atp6ap2 in mouse ß cells causes a dramatic accumulation of large, multigranular vacuoles in the cytoplasm, with reduction of insulin content and compromised glucose homeostasis. Loss of insulin stores and gigantic vacuoles were also observed in cultured insulinoma INS-1 cells upon CRISPR/Cas9-mediated removal of Atp6ap2. Remarkably, these phenotypic alterations could not be attributed to a deficiency in autophagy or acidification of lysosomes. Together, these data indicate that Atp6ap2 is critical for regulating the stored insulin pool and that a balanced regulation of granule turnover is key to maintaining ß cell function and diabetes prevention.

A 4D view on insulin secretory granule turnover in the ß-cell.

Insulin secretory granule (SG) turnover consists of several highly regulated processes allowing for proper ß-cell function and insulin secretion. Besides the spatial distribution of insulin SGs, their age has great impact on the likelihood of their secretion and their behaviour within the ß-cell. While quantitative measurements performed decades ago demonstrated the preferential secretion of young insulin, new experimental approaches aim to investigate insulin ageing at the granular level. Live-cell imaging, automated image analysis and correlative light and electron microscopy have fostered knowledge of age-defined insulin SG dynamics, their interaction with the cytoskeleton and ultrastructural features. Here, we review our recent work in regards to the connection between insulin SG age, SG dynamics, intracellular location and interaction with other proteins.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Phototropin influence on eyespot development and regulation of phototactic behavior in Chlamydomonas reinhardtii.

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity-dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.