cytochalasin-d and Laryngeal-Neoplasms

We have addressed the following question: what is the mechanism behind the delivery of internalized molecules from mature endosomes to lysosomes in HEp-2 cells, and which role does the cytoskeleton play in this process? Quantitative electron microscopy and immunogold labeling revealed that whereas the cytoskeleton was not of importance for endosome maturation, actin filaments facilitated fusion of mature endosomes with preexisting lysosomes. Delivery to lysosomal degradation was not dependent on protein synthesis as determined biochemically, but was reduced by cytochalasin D. Observations made by electron microscopy as well as by video microscopy of living cells showed that the concerted action of actin filaments and microtubules was responsible for the random distribution and movement of endocytic organelles throughout the cell. Actin microfilaments, however, seem to facilitate perinuclear clustering and frequent fusion of mature endosomes and lysosomes, while microtubules play a role in preventing formation of large lysosome aggregates by separating endosomes and lysosomes and moving them toward the cell periphery. Taken together, our data suggest that delivery of internalized molecules to lysosomal proteolysis takes place by fusion of mature endosomes with preexisting lysosomes and that actin microfilaments somehow facilitate this step.

Topics: Actins; Biological Transport; Biomarkers; Colchicine; Cytochalasin D; Dextrans; Endosomes; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Laryngeal Neoplasms; Lysosomes; Microscopy, Video; Microtubules; Nocodazole; Protein Synthesis Inhibitors; Proteins; Ricin; Tumor Cells, Cultured

Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection. A plasmid-mediated EPEC adherence factor (EAF) is responsible for initial and localized adherence. A chromosomally encoded E. coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic "pedestal" formation. By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined. While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines. Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine. Afimbrial adhesion or type I pili from uropathogenic strains of E. coli substituted for EAF in EAF-Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation.

Topics: Adhesins, Escherichia coli; Bacterial Adhesion; Bacterial Outer Membrane Proteins; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Colchicine; Cytochalasin D; Cytoskeleton; Escherichia coli; Humans; Laryngeal Neoplasms; Microscopy, Electron; Microvilli; Plasmids; Tumor Cells, Cultured