Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles

The direct identification of the intracellular site where proinsulin is proteolytically processed into insulin has been achieved by immunocytochemistry using an insulin-specific monoclonal antibody. Insulin immunoreactivity is absent from the Golgi stack of pancreatic B-cells and first becomes detectable in clathrin-coated secretory vesicles released from the trans Golgi pole. Clathrin-coated secretory vesicles transform into mature noncoated secretory granules which contain the highest concentration of insulin immunoreactive sites. Maturation of clathrin-coated secretory vesicles is accompanied by a progressive acidification of the vesicular milieu, as evidenced by a cytochemical probe that accumulates in acidic compartments whereupon it can be revealed by immunocytochemistry. Thus packaging of the prohormone in secretory vesicles, and acidification of this compartment, are critical steps in the proper proteolytic maturation of insulin.     

Fatty acyl-coenzyme A is required for budding of transport vesicles from Golgi cisternae

We describe a new role for fatty acylation. Conditions were established under which vesicular transport from the cis to the medial Golgi compartment in vitro depends strongly upon the addition of a fatty acyl-coenzyme A, e.g., palmitoyl-CoA. Using an inhibitor of long-chain acyl-CoA synthetase, we demonstrate that the fatty acid has to be activated by CoA to stimulate transport. A nonhydrolyzable analog of palmitoyl-CoA competitively inhibits transport. Electron microscopy and biochemical studies show that fatty acyl-CoA is required for budding of (non-clathrin-) coated transport vesicles from Golgi cisternae and that budding is inhibited by the nonhydrolyzable analog.     

Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors

We have designed an in vitro system in which Madin-Darby canine kidney (MDCK) epithelial cells are cocultured in collagen gels with fibroblasts under conditions precluding heterocellular contact. Using this experimental approach, we have obtained evidence that fibroblast-derived soluble factors play a crucial role in the control of epithelial morphogenesis. First, MDCK cells suspended alone in collagen gels form spherical cysts, whereas in the presence of fibroblasts they form branching tubules. Second, MDCK cells grown as a monolayer on fibroblast-containing collagen gels invade the underlying matrix, within which they form a network of tubules. Third, fibroblast-conditioned medium mimics the effects of coculture by eliciting tubulogenesis by MDCK cells. These results demonstrate the involvement of diffusible paracrine factors in morphogenetic epithelial-mesenchymal interactions and provide a strategy for their molecular characterization.     

Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor.

We have previously shown that Madin-Darby canine kidney (MDCK) epithelial cells grown in collagen gels in the presence of fibroblasts or fibroblast-conditioned medium (CM) form branching tubules, instead of the spherical cysts that develop under control conditions. We now report that the fibroblast-derived molecule responsible for epithelial tubulogenesis is hepatocyte growth factor (HGF). First, addition of exogenous HGF to cultures of MDCK cells induces formation of epithelial tubules. Second, the tubulogenic activity of fibroblast CM is completely abrogated by antibodies to HGF. These results demonstrate that HGF, a polypeptide that was identified as a mitogen for cultured hepatocytes, has the properties of a paracrine mediator of epithelial morphogenesis, and suggest that it may play important roles in the formation of parenchymal organs during embryonic development.     

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFA's effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.     

Horseradish peroxidase uptake and crinophagy in insulin-secreting cells

Upon exposure of pancreatic B cells to exogenous horseradish peroxidase (HRP), a population of secretory granules becomes HRP-labelled. In isolated islets of Langerhans, we studied the fate of HRP-labelled secretory granules during a pulse-chase experiment with HRP in order to assess their relationship with lysosomes containing secretory granule cores. These structures (crinophagic or multigranular bodies) were previously shown to be a site of insulin degradation (Orci et al., J cell biol 98 (1984) 222) [4]. After a 15-min pulse of peroxidase, the number and volume density of HRP-labelled secretory granules decreased over an 85-min chase period, during which the number and volume density of multigranular bodies labelled with HRP was significantly increased. At both time points, the surface density of HRP-labelled Golgi elements was very small compared with that of unlabelled ones. By autoradiography after a 5-min pulse of [3H]leucine and a 55-min chase, followed by a 15-min pulse of HRP and a 85-min chase, we could show that the majority of HRP-containing secretory granules were not radioactively labelled granules. These results suggest that: The low degree of HRP labelling of the Golgi makes it unlikely that secretory granules derive their HRP by budding from HRP-labelled cisternae. HRP-labelled SGs are preferentially transferred to MGBs (which become HRP-labelled) for prospective degradation. HRP labelling does not involve newly-formed mature secretory granules.     

Calcitonin and vasopressin affect epithelial properties in a renal cell line

We have used a stable clonal variant (D + Sc), isolated from the LLC-PK1 pig kidney-derived cell line and selected for its extensive capacity to form domes, in order to study the hormonal modulation of epithelial permeability in culture. Calcitonin, vasopressin, and other agents that raise intracellular adenosine 3',5'-cyclic monophosphate levels caused a rapid and dramatic decrease in the size and number of domes. This effect was independent of RNA and protein synthesis, and thus appeared unrelated to the production of urokinase, a proteinase synthesized by the cells in response to these agents. Calcitonin caused a decrease in transepithelial electrical resistance, suggesting that the effect of the hormone on domes was due to an increase in the permeability of a paracellular pathway. Thus, in addition to the wellknown effects of vasopressin on collecting duct permeability, part of the in vivo effect(s) of calcitonin and vasopressin on the renal tubule might also involve alterations of epithelial permeability related to those described here.     

The endosomal compartment of rat hepatocytes. Its characterization in the course of [125I]insulin internalization

When freshly isolated hepatocytes are incubated with [125I]insulin in the presence of the microtubule-disrupting agent colchicine, internalization of the labelled hormone is not significantly altered. However, the drug limits the endocytosis of the labelled material to a peripheral band of cytoplasm extending 1 micron beyond the plasma membrane. Both in the presence and absence of colchicine, internalized [125I]insulin preferentially associates with clear vesicles (endosomes) and lysosome-like structures, but the relative amount of labelled material associated with clear vesicles is higher in the presence of the drug than in its absence. An inverse pattern is observed for the lysosome-like structures. As demonstrated by cytochemical methods, clear vesicles do not contain the lysosomal enzyme aryl sulfatase. Moreover, colchicine induces an increase of the clear vesicle diameter without affecting their frequency, while it perturbs multivesicular bodies and dense bodies in an opposite way by increasing their frequency without affecting their size. By reducing and/or delaying the fusion between internalized endocytotic vesicles and lysosomes, colchicine allows better characterization of the endosomal compartment of isolated rat hepatocytes and allows it to be distinguished from other compartments, such as multivesicular bodies and the Golgi apparatus.     

Short and reversible uncoupling evokes little change in the gap junctions of pancreatic acinar cells

Three different preparations of mouse pancreatic fragments where all the cells tested electrophysiologically showed (a) complete electrical coupling (control), (b) complete uncoupling (after 1-to 2-min exposure to 100% CO2), or (c) complete recoupling (1-2 min after removal of 100% CO2) were fixed, with the electrodes in situ, with 0.2% glutaraldehyde and freeze-fractured for quantitative analysis of acinar cell gap junctions. No obvious difference was observed between gap junctions of coupled and uncoupled acinar cells. However, quantitation revealed a small (2.3-5.6%) increase in particle diameter and spacing within junctions of uncoupled cells. Such increase was rapidly reversed upon cell recoupling. In all preparations, most of the gap junctions were made up of disordered arrays of particles but a few of them showed a more tight packing of their particles of which most had lost the usual globular appearance. These "amorphous" gap junctions had larger particle diameter but smaller particle spacing than the other gap junctions and these parameters were not modified during cell uncoupling. However, "amorphous" gap junctions were more frequent in the latter condition.     

Collagen matrix promotes reorganization of pancreatic endocrine cell monolayers into islet-like organoids

To evaluate the capacity of pancreatic endocrine cells to reassociate in vitro according to the characteristic topographical pattern observed in the islets of Langerhans in situ, we cultured cells dissociated from neonatal rat pancreas within a three-dimensional collagen matrix. Cell monolayers grown on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endocrine cells to reorganize into smooth-contoured, three-dimensional aggregates, in which non-B cells (identified by electron microscopy and immunofluorescence) had a preferential distribution at the periphery, whereas B cells were concentrated in a central position. These results show that cultured pancreatic endocrine cells have the capacity to reassociate into islet-like organoids in vitro, and that collagen matrices may have a permissive effect on the expression of this potential.