The Distribution of Phosphatidylinositol 4,5-Bisphosphate in Acinar Cells of Rat Pancreas Revealed with the Freeze-Fracture Replica Labeling Method

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a phospholipid that has been implicated in multiple cellular activities. The distribution of PI(4,5)P2 has been analyzed extensively using live imaging of the GFP-coupled phospholipase C-δ1 pleckstrin homology domain in cultured cell lines. However, technical difficulties have prevented the study of PI(4,5)P2 in cells of in vivo tissues. We recently developed a method to analyze the nanoscale distribution of PI(4,5)P2 in cultured cells by using the quick-freezing and freeze-fracture replica labeling method. In principle, this method can be applied to any cell because it does not require the expression of artificial probes. In the present study, we modified the method to study cells of in vivo tissues and applied it to pancreatic exocrine acinar cells of the rat. We found that PI(4,5)P2 in the plasma membrane is distributed in an equivalent density in the apical and basolateral domains, but exists in a significantly higher concentration in the gap junction. The intracellular organelles did not show labeling for PI(4,5)P2. The results are novel or different from the reported distribution patterns in cell lines and highlight the importance of studying cells differentiated in vivo

Retrograde traffic in the biosynthetic-secretory route

In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments’ balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations