Ines Anderie

In vivo visualization of actin dynamics and actin interactions by BiFC

The method of bimolecular fluorescence complementation (BiFC) enables selective visualization of protein interactions. While BiFC complex formation under in vitro conditions is considered to be essentially irreversible, there are hints that under in vivo conditions BiFC complex formation can be reversible. In the present study we used the BiFC method to visualize in vivo actin cytoskeleton dynamics. We demonstrate that in living cells formation of actin/actin BiFC complexes is reversible. Furthermore, we show heterologous binding between actin and protein kinase C delta (PKCδ). Treatment with phorbol esters caused translocation of actin/PKCδ complexes from the cytosol to the plasma membrane independent of an intact actin cytoskeleton. Our experiments demonstrate that the BiFC method might be a useful tool to investigate participation of the actin cytoskeleton in regulation of cell function.

Evidence for a 65 kDa sulfonylurea receptor in rat pancreatic zymogen granule membranes

In rat pancreatic zymogen granules (ZG), a K+ selective conductance which can be blocked by ATP has been characterized. Here we show that this pathway can be specifically blocked by glibenclamide. Using a rapid filtration assay, we also found specific binding of [3H]glibenclamide to a low‐affinity site (K d 5.6±1.1 μM) in rat pancreatic zymogen granule membranes (ZGM). In photoaffinity labeling experiments with [3H]glibenclamide, a 65±1.5 kDa polypeptide was specifically labeled. Previously, a ∼65 kDa mdr1 gene product has been demonstrated to be involved in the regulation of the K+ selective conductance of ZG. We conclude that this protein may be a subunit of, or associated with, a ZG KATP channel.

Functional Relationship between a 65-kD mdr1 P-Glycoprotein and ATP-Activated Chloride Conductance in Pancreatic Zymogen Granule Membranes

Zymogen granule (ZG) membranes contain a regulated Cl– conductance which may contribute to secretagogue-induced enzyme, salt and fluid secretion. ATP and nonhydrolyzable ATP analogs, such as adenosine 5’-[βγ-methylene]triphosphate (AMP-PCP) increase ZG membrane (ZGM) Cl– conductance. Here we demonstrate that monoclonal antibodies (mAbs) against multidrug resistance (MDR1) P-glycoprotein inhibit AMP-PCP-activated rat ZG Ch conductance. Moreover, in ZG from mdrla knockout mice, AMP-PCP-activated Cl– conductance is reduced by about 50%. Immunocytochemistry of rat pancreatic tissue using mAbs against MDR1 reveals labeling of the surface of acinar ZG and of apical membranes of pancreatic ducts. A ∼ 180-kD protein is detected in pancreatic duct cell lysates, as expected for full-length P-glycoprotein, and a ∼ 65-kD protein is labeled in ZGM. A high-affinity probe for dihydropyridine-binding domains on MDR1, a benzoyldihydrocinnamic acid derivative of dihydropyridine ((-)-[3H]BZDC-DHP), binds to ZGM with high affinity (Kd ∼ 8 nM), which resembles the binding affinity of full-length MDR1 P-glycoprotein. However, the profile of pharmacological modulation of (-)-[3H]BZDC-DHP binding differs from that of full-length MDR1. The data suggest that ZGM express a marl gene product distinct from the known mdr1 proteins, that may be functionally associated with AMP-PCP-activated ZG Cl- conductance.

Change in the Characteristics of Ca 2+ Signaling in Pancreatic Acinar Cells in Culture

In this study we studied Ca2+ responses to different stimuli in pancreatic acinar cells subjected to culture conditions. Cells were isolated from adult mice pancreas and were subjected to culture conditions along one week. Changes in intracellular free Ca2+ concentration were monitored by single cell fluorescence analysis of fura-2-loaded cells. Mitochondrial distribution was analyzed by confocal microscopy study of MitoTracker Green FM-loaded cells. Expression of amylase-containing cytoplasmic vesicles was analyzed by confocal microscopy study of cells transfected with a plasmid encoding amylase linked to a green fluorescent protein. Cell viability was analyzed employing the AlamarBlue test. Our results show that pancreatic cells in culture retain a mitochondrial network and amylase-positive vesicles. However, cells dropped their ability to mobilize Ca2+ in response to activation of cell membrane receptors. Ca2+ mobilization in response to the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was not altered. Cell viability was not affected by treatment with cholecystokinin, but was reduced in the presence of thapsigargin or hydrogen peroxide. We conclude that primary culture of pancreatic cells may be a suitable model to be used in studies where the involvement of mechanisms linked to the activation of specific cell membrane receptors is not required.