The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER.

This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.

Loss of pancreatic islet b-cell mass and b-cell dysfunction are central in the development of type 2 diabetes (T2DM). We recently showed that mature human insulincontaining b-cells can convert into glucagon-containing a-cells ex vivo. This loss of b-cell identity was characterized by the presence of b-cell transcription factors (Nkx6.1, Pdx1) in glucagon + cells. Here, we investigated whether the loss of b-cell identity also occurs in vivo, and whether it is related to the presence of (pre)diabetes in humans and nonhuman primates. We observed an eight times increased frequency of insulin + cells coexpressing glucagon in donors with diabetes. Up to 5% of the cells that were Nkx6.1 + but insulin 2 coexpressed glucagon, which represents a five times increased frequency compared with the control group. This increase in bihormonal and Nkx6.1 + glucagon + insulin 2 cells was also found in islets of diabetic macaques. The higher proportion of bihormonal cells and Nkx6.1 + glucagon + insulin 2 cells in macaques and humans with diabetes was correlated with the presence and extent of islet amyloidosis. These data indicate that the loss of b-cell identity occurs in T2DM and could contribute to the decrease of functional b-cell mass. Maintenance of b-cell identity is a potential novel strategy to preserve b-cell function in diabetes.Loss of pancreatic b-cell mass and b-cell dysfunction are central in the development of type 2 diabetes (T2DM) and, in combination with peripheral insulin resistance, lead to hyperglycemia (1). Whereas b-cells, on the one hand, fail to properly secrete insulin at a given glucose level, there is also a progressive decline in the number of b-cells (2,3). Loss of b-cell mass has been ascribed to increased apoptosis in T2DM (4). In patients with T2DM, b-cell mass can be up to 40-60% lower than in healthy control subjects (4-6). In addition, abnormal function of glucagon-producing a-cells leading to hyperglucagonemia is associated with T2DM (7). b-cell dedifferentiation and subsequent transition to other islet cell types were suggested as an alternative explanation for the loss of functional b-cell mass in mice (8,9). In this concept, b-cells lose insulin content and insulin secretory capacity followed by the production of other endocrine hormones such as glucagon (8). We recently showed (10) that loss of b-cell identity with the conversion of b-cells into glucagon-containing a-cells can occur in human pancreatic islets ex vivo.A number of transcription factors have been identified to be essential for the development and maintenance of functional b-cells (11,12). Recent reports (13,14) indicate that a selective loss of transcription factors MafA, Nkx6.1, and Pdx1 is associated with b-cell dysfunction and T2DM. Chronic hyperglycemia in rats is accompanied by the loss of b-cell transcription factors (15). Moreover, mouse b-cells that genetically lack FOXO1 can dedifferentiate in vivo under conditions of metabolic stress and subsequently can convert (or transdifferentiate) into glucagonproducing a-cells...

Cargo sorting that promotes the transport of cargo proteins from a membrane compartment has been predicted to be unlikely in the endocytic recycling pathways. We now show that ACAP1 binds specifically and directly to recycling cargo proteins. Reducing this interaction for TfR inhibits its recycling. Moreover, ACAP1 binds to two distinct phenylalanine-based sequences in the cytoplasmic domain of TfR that function as recycling sorting signals to promote its transport from the recycling endosome. Taken together, these findings indicate that ACAP1 promotes cargo sorting by recognizing recycling sorting signals.

Whether coat proteins play a widespread role in endocytic recycling remains unclear. We find that ACAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF) 6, is part of a novel clathrin coat complex that is regulated by ARF6 for endocytic recycling in two key physiological settings, stimulation-dependent recycling of integrin that is critical for cell migration and insulin-stimulated recycling of glucose transporter type 4 (Glut4), which is required for glucose homeostasis. These findings not only advance a basic understanding of an early mechanistic step in endocytic recycling but also shed key mechanistic insights into major physiological events for which this transport plays a critical role.