CRHSP-28 regulates Ca(2+)-stimulated secretion in permeabilized acinar cells

CRHSP-28 is a Ca(2+)-regulated heat-stable phosphoprotein, abundant in the apical cytoplasm of epithelial cells that are specialized in exocrine protein secretion. To define a functional role for the protein in pancreatic secretion, recombinant CRHSP-28 (rCRHSP-28) was introduced into streptolysin-O-permeabilized acinar cells, and amylase secretion in response to elevated Ca(2+) was determined. Secretion was enhanced markedly by rCRHSP-28 over a time course that closely corresponded with the loss of the native protein from the intracellular compartment. No effects of rCRHSP-28 were detected until approximately 50% of the native protein was lost from the cytosol. Secretion was enhanced by rCRHSP-28 over a physiological range of Ca(2+) concentrations with 2-3-fold increases in amylase release occurring in response to low micromolar levels of free Ca(2+). Further, rCRHSP-28 augmented secretion in a concentration-dependent manner with minimal and maximal effects occurring at 1 and 25 microg/ml, respectively. Covalent cross-linking experiments demonstrated that native CRHSP-28 was present in a 60-kDa complex in cytosolic fractions and in a high molecular mass complex in particulate fractions, consistent with the slow leak rate of the protein from streptolysin-O-permeabilized cells. Probing acinar lysates with rCRHSP-28 in a gel-overlay assay identified two CRHSP-28-binding proteins of 35 (pp35) and 70 kDa (pp70). Interestingly, preparation of lysates in the presence of 1 mm Ca(2+) resulted in a marked redistribution of both proteins from a cytosolic to a Triton X-100-insoluble fraction, suggesting a Ca(2+)-sensitive interaction of these proteins with the acinar cell cytoskeleton. In agreement with our previous study immunohistochemically localizing CRHSP-28 around secretory granules in acinar cells, gel-overlay analysis revealed pp70 copurified with acinar cell secretory granule membranes. These findings demonstrate an important cell physiological function for CRHSP-28 in the Ca(2+)-regulated secretory pathway of acinar cells.     

Identification of annexin VI as a Ca2+-sensitive CRHSP-28-binding protein in pancreatic acinar cells

CRHSP-28 is a member of the tumor protein D52 protein family that was recently shown to regulate Ca(2+)-stimulated secretory activity in streptolysin-O-permeabilized acinar cells (Thomas, D. H., Taft, W. B., Kaspar, K. M., and Groblewski, G. E. (2001) J. Biol. Chem. 276, 28866-28872). In the present study, the Ca(2+)-sensitive phospholipid-binding protein annexin VI was purified from rat pancreas as a CRHSP-28-binding protein. The interaction between CRHSP-28 and annexin VI was demonstrated by coimmunoprecipitation and gel-overlay assays and was shown to require low micromolar levels of free Ca(2+), indicating these molecules likely interact under physiological conditions. Immunofluorescence microscopy confirmed a dual localization of CRHSP-28 and annexin VI, which appeared in a punctate pattern in the supranuclear and apical cytoplasm of acini. Stimulation of cells for 5 min with the secretagogue cholecystokinin enhanced the colocalization of CRHSP-28 and annexin VI within regions of acini immediately below the apical plasma membrane. Tissue fractionation revealed that CRHSP-28 is a peripheral membrane protein that is highly enriched in smooth microsomal fractions of pancreas. Further, the content of CRHSP-28 in microsomes was significantly reduced in pancreatic tissue obtained from rats that had been infused with a secretory dose of cholecystokinin for 40 min, demonstrating that secretagogue stimulation transiently alters the association of CRHSP-28 with membranes in cells. Collectively, the Ca(2+)-dependent binding of CRHSP-28 and annexin VI, together with their colocalization in the apical cytoplasm, is consistent with a role for these molecules in acinar cell membrane trafficking events that are essential for digestive enzyme secretion.     

Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces

The enterocyte-like cell line Caco-2 forms a polarized epithelium when grown on filters. We have investigated the interaction of endocytic pathways from the apical and basolateral surfaces. The transferrin receptor was an appropriate marker for the basolateral route; uptake of radiolabeled transferrin was highly polarized, and recycling of this ligand back to the basolateral surface occurred with an efficiency of 95%, even after prolonged incubations with transferrin. Using a transferrin-peroxidase conjugate to delineate the morphological pathway, we have identified an early endocytic compartment in the basolateral cytoplasm of the cells. Longer incubations revealed a deeper endocytic compartment in the apical cytoplasm. Concanavalin A complexed to gold was used to simultaneously label the apical endocytic route. After 60 min, extensive mixing of the two labels was seen in endocytic elements throughout the apical cytoplasm, including in the Golgi area, but never in the basal cytoplasm. Using a second double labeling procedure in which antitransferrin receptor antibody complexed to gold was applied to the basolateral surface for up to 2 h and free peroxidase applied to the apical surface for shorter periods, we demonstrated that this apical marker rapidly (within 5 min) reached endosomes containing antibody-gold. Our results indicate that, in Caco-2 cells, the endocytic pathways from the apical and basolateral surfaces meet in an endosomal compartment from which transferrin can still be recycled.     

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.     

Transport of phagosomal components to an endosomal compartment.

The participation of phagosomes in interorganellar protein and membrane exchange is important to the maturation of phagosomes into phagolysosomes. To investigate this process, we have developed an assay to measure protein transport from phagosomes to other vesicle populations. J774-E clone macrophages phagocytosed 125I-anti-dinitrophenol IgG-coated Staphylococcus aureus for 3 min followed by chase for intervals of 0-30 min. Following cell fractionation, the intracellular distribution of radioiodinated protein was assayed. We observed a time-dependent increase radioiodinated protein in a non-phagosome vesicle fraction which displayed endosome characteristics. Concomitantly, radioiodinated protein within phagosomes decreased over the chase period. As assessed via Percoll density gradient fractionation, the phagocytosed radioiodinated protein migrated to both heavy (lysosome density) and light (endosome density) vesicle populations. Characterization of the fusogenic properties of the transport vesicles demonstrated that they are capable of in vitro fusion with early endosomes. Furthermore, this fusion event shares many of the biochemical requirements identified for phagosome-endosome and endosome-endosome fusion. Morphological analysis of phagosome maturation provides additional evidence for phagosome to endosome transport. These results suggest phagocytosed material is transferred from phagosomes to endosomes and then recycled out of the cell.     

CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells

Ca(2+)-regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) is highly expressed in exocrine glands and was shown to regulate digestive enzyme secretion from pancreatic acinar cells. We found CRHSP-28 highly expressed in cultured mucosal secretory T84 cells, consistent with an important regulatory role in apical membrane trafficking. Stimulation of cells with carbachol (CCh) induced rapid, concentration-dependent phosphorylation of CRHSP-28 on at least two serine residues. Isoelectric focusing and immunoblotting were used to characterize cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation depends on elevated cellular Ca2+, being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTAAM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, phosphopeptide mapping studies demonstrated that CaMKII induced an identical phosphopeptide profile to endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated phosphorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated that CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaMKII was also localized to the subapical cytoplasm and was clearly displaced following actin filament disruption. These data suggest that CRHSP-28 phosphorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells.     

Characterization of an apical ceramide-enriched compartment regulating ciliogenesis

We show that in Madin-Darby canine kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) at the base of primary cilia is colocalized with Rab11a. Ceramide and Rab11a vesicles isolated by magnetic sorting contain a highly similar profile of proteins (atypical protein kinase C [aPKC], Cdc42, Sec8, Rab11a, and Rab8) and ceramide species, suggesting the presence of a ciliogenic protein complex associated with ceramide at the ACEC. It is intriguing that C16 and C18 ceramide, although less abundant ceramide species in MDCK cells, are highly enriched in ceramide and Rab11a vesicles. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppresses ciliogenesis, indicating that the association of ceramide with aPKC is critical for the formation of this complex. Our results indicate that ciliogenic ceramide is derived from apical sphingomyelin (SM) that is endocytosed and then converted to the ACEC. Consistently, inhibition of acid sphingomyelinase with imipramine disrupts ACEC formation, association of ciliogenic proteins with Rab11a vesicles, and cilium formation. Ciliogenesis is rescued by the histone deacetylase (HDAC) inhibitor trichostatin A, indicating that ceramide promotes tubulin acetylation in cilia. Taken together, our results suggest that the ACEC is a novel compartment in which SM-derived ceramide induces formation of a ciliogenic lipid-protein complex that sustains primary cilia by preventing deacetylation of microtubules.     

Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment.

We have examined the pH of the various endosomal compartments in the amoebae of the cellular slime mould Dictyostelium discoideum. This was accomplished both by fluorescence and by in vivo 31P-NMR methods. The fluid-phase marker, fluorescein-labeled dextran, was fed to the amoebae to report the average pH of their endocytic vesicles. During the progressive loading of successive endosomal compartments, we observed an early acidification down to a minimum value of pH < or = 5.3 after 30 min at 20 degrees C followed by an increase to an average pH of 5.8 when all the endosomal compartments were loaded by the fluid-phase marker. The weak fluorescence intensity of FITC-dextran at acidic pH precluded a more detailed investigation and we checked various phosphonate compounds as potential 31P-NMR pH probes for the endosomal compartments. Two molecules, aminomethylphosphonate and 2-aminoethylphosphonate, were selected for this study because of the large amplitudes of their chemical shift variation with pH (2 and 2.5 ppm, respectively) and their acidic pKs of 5.5 and 6.3, respectively. They were only moderately toxic (IC50% approximately 10 mM) towards both the axenic growth and the differentiation program of Dictyostelium amoebae. Internalization of the two aminophosphonates occurred only through the fluid-phase pinocytosis pathway as revealed by the full inhibition of their entry with 1 mM vanadate or 7.5 mM caffeine, two previously characterized inhibitors of endocytosis in Dictyostelium. We found that in vivo 31P-NMR of amoebae suspensions incubated with the aminophosphonates allowed the detection of three distinct intracellular compartments at pH 4.3, 5.8-6.0 and 7.3. Kinetics of aminophosphonate entry were analyzed and the results allowed us to reconstruct the time course for the acidification sequence during endocytosis. The data are consistent with the hypothesis that in Dictyostelium amoebae phosphonates occupy a highly acidic early endosomal compartment (t1/2 = 18 min; pH 4.3) before reaching a less acidic late endosomal/prelysosomal compartment (pH 5.8-6.0) from where they are immediately transported to, and trapped in, the cytoplasm (pH 7.3).     

Targeting of an apical endosomal protein to endosomes in Madin-Darby canine kidney cells requires two sorting motifs

The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin–Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site-directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin-positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate-binding protein rab11, and do not tubulate in response to low concentrations of brefeldin-A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule.     

IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E2 (PGE2) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.     

Concentration of toxins in endosomal compartment of the cells pretreated with ricin and viscumin

Data on volume of the intracellular compartment where the internalized toxin is located and toxin concentration in this compartment have been obtained. Suggestions have been made about a role of toxin molecule aggregates in the translocation of A-chain into cytosol, which leads to cell death.     

Coated endosomal vesicles: sorting and recycling compartment for transferrin in BHK cells.

We have investigated receptor-mediated endocytosis of transferrin (Tf) in baby hamster kidney (BHK) cells, using fluorescence and electron microscopy, and by carrying out colocalization experiments with clathrin antibodies and a fluorescently tagged glycolipid. Early during internalization, Tf was found in small vesicles (100-150 nm in diameter) located at the cell periphery. The ligand remained associated with such vesicles when the latter concentrated towards the cell center, before ending up in the juxtanuclear area. Throughout this vesicular trafficking pathway, clathrin colocalized with Tf. We conclude that Tf is processed intracellularly via small coated endosomal vesicles (CEV) and is not delivered into large tubular endosomes (CURL; compartment for uncoupling receptors and ligands), typical for ligand trafficking to lysosomes. By determining the kinetics of Tf internalization and by comparing the flow of Tf to that of a fluorescent glycolipid, it can also be concluded that CEVs display sorting and recycling properties, implying that small vesicles can be shed from or fuse with CEVs. Acidic pH does not prevent the formation of CEVs, but their intracellular movement, towards the cell center, is impeded.     

Tubular early endosomal networks in AtT20 and other cells

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.     

Nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of HIV-1 infectivity

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.     

Reconstitution of endosomal proteolysis in a cell-free system. Transfer of immune complexes internalized via Fc receptors to an endosomal proteolytic compartment

The presence of acid proteases in the endosomal compartment of macrophages has been recently demonstrated (Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907). This proteolytic activity allows the early degradation of ligands internalized by receptor-mediated endocytosis. To study the early steps that initiate the proteolytic processing of ligands, immune complexes formed with anti-dinitrophenol monoclonal IgG and radiolabeled dinitrophenol-derivatized bovine serum albumin were bound at 4 degrees C to Fc receptors of J774 macrophages. Cells were allowed to internalize immune complexes bound to the plasma membrane for different periods of time at 37 degrees C. Vesicle preparations generated from these cells were incubated in vitro at acidic pH to allow the hydrolysis of ligands located in protease-positive compartments. Ligand hydrolysis was observed after about 5 min of internalization, suggesting that at earlier times immune complexes were located in protease-free vesicles. Upon incubation of cell lysates under conditions that support in vitro endosome-endosome fusion, early protease-free endosomes containing ligand acquire proteolytic activity. Reconstitution of fusion-dependent proteolysis required energy, ions, membrane-associated factors, and cytosol. Cytosol was inactivated by incubation with N-ethylmaleimide. The proteolytic compartment formed upon in vitro incubation colocalized with endosomes in the light region of a Percoll gradient. Reconstitution was also achieved using an endosomal preparation separated from lysosomes in a Percoll gradient. Our results indicate that a fusion step between newly formed endocytic vesicles and a light density, protease-positive compartment triggers the proteolytic processing of ligands internalized by receptor-mediated endocytosis.     

APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.     

Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells

Autophagy is mostly a nonselective bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and killer of the cell depending on the stage of the disease or the surrounding cellular environment (for review see Cuervo, A.M. 2004. Trends Cell Biol. 14:70-77). We found that cytoplasmic vacuoles induced in pancreatic acinar cells by experimental pancreatitis were autophagic in origin, as demonstrated by microtubule-associated protein 1 light chain 3 expression and electron microscopy experiments. To analyze the role of macroautophagy in acute pancreatitis, we produced conditional knockout mice lacking the autophagy-related 5 gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice. Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.     

Transformational process of the endosomal compartment in nephrocytes of Drosophila melanogaster

Summary This study investigates by electron microscopy the transformational process of the endosomal compartment of the Drosophila nephrocyte, the garland cell, which occurs during endocytotic processing of internalized material. The endosomal compartment of the garland cell consists of a prominent tubular/vacuolar complex in the cortical cytoplasm. When internalization of coated pits is blocked at 29°C using the endocytosis mutant, shibire ^ts, the tubules gradually disappear after 7 min at 29°C. By 12 min at 29°C, the vauoles also disappear. Thus, the endosomal compartment appears to constantly undergo a transformational process that necessitates continuous replenishment by coated vesicles. The data suggest that the tubular component of the endosomal compartment gradually transforms into vacuoles by the expansion of the tubular membrane. The vacuoles then transform by invaginating into themselves, creating flattened cisternae. The electron-lucent substance in the lumina of the vacuoles appears to be extruded into the cytoplasm through the invaginating membrane. No shuttle vehicles such as vesicles or tubules could be identified that might have been involved in the transporting of endocytosed materials and membrane from the endosomal compartment to lysosomes or back to the plasma membrane.     

Endocytosis at the apical plasma membrane of pancreatic acinar cells is regulated by tyrosine kinases

We have shown that endocytosis at the apical plasma membrane ofpancreatic acinar cells is regulated by the pH of the acinar lumen andis associated with cleavage of GP2, a glycosylphosphatidylinositol-anchored protein. The aim of this study was todetermine the transduction pathway by which endocytosis is activated.Apical endocytosis was studied in rat pancreatic acini byprestimulation with cholecystokinin followed by measurement ofhorseradish peroxidase (HRP) uptake. Lanthanum, staurosporine, andforskolin had no effect on HRP uptake. Cytochalasin D significantlyinhibited endocytosis, indicating a dependence on actin filamentintegrity. Genistein and the specific tyrphostin inhibitor B42 alsoinhibited HRP uptake, implicating tyrosine kinases in the regulation ofHRP uptake. With the use of an Src kinase-specific substrate, Srckinase activity was temporally related to activation of endocytosis.The tyrosine-dependent phosphorylation of an 85-kDa substrate in bothrat and mouse pancreatic acini correlated with Src kinase activationand pH-dependent regulation of HRP uptake. These results indicate thatapical endocytosis in acinar cells is associated with tyrosine kinaseactivation and is dependent on the actin cytoskeleton.