We have employed an interferometric technique for the local measurement of bending modulus, membrane tension, and adhesion energy of motile cells adhering to a substrate. Wild-type and mutant cells of Dictyostelium discoideum were incubated in a flow chamber. The flow-induced deformation of a cell near its adhesion area was determined by quantitative reflection interference contrast microscopy (RICM) and analyzed in terms of the elastic boundary conditions: equilibrium of tensions and bending moments at the contact line. This technique was employed to quantify changes caused by the lack of talin, a protein that couples the actin network to the plasma membrane, or by the lack of cortexillin I or II, two isoforms of the actin-bundling protein cortexillin. Cells lacking either cortexillin I or II exhibited reduced bending moduli of 95 and 160 k(B)T, respectively, as compared to 390 k(B)T, obtained for wild-type cells. No significant difference was found for the adhesion energies of wild-type and cortexillin mutant cells. In cells lacking talin, not only a strongly reduced bending modulus of 70 k(B)T, but also a low adhesion energy one-fourth of that in wild-type cells was measured.

Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones. Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton. The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed. Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent. The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface. With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells.

Cells of the unicellular eukaryote Dictyostelium discoideum take up all their nutrients by endocytosis. Both particle- and fluid-containing vacuoles are transiently surrounded by a cytoskeletal coat [1] [2]. When this coat has dissociated, acidification and digestion of the vesicle contents occur, followed by exocytosis of the indigestible remnants after 60-90 minutes. At least nine compartments are needed for mathematical modelling of endocytic transit [3], suggesting that markers associate for only a few minutes with a specific endocytic compartment. Among the proteins that have been identified as components of endocytic vesicles are actin, subunits of the V-H+ ATPase and small GTP-binding proteins of the Rab family [4] [5] [6] [7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corresponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves vacuolin, we generated a fusion protein of the green fluorescent protein (GFP) and vacuolin. GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-lysosomal compartment was identified by the binding of a tagged cytoskeletal protein, coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway, and the integrity of the actin coat was required for exocytosis.

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins of interest to elucidate their biological function. CALI has potential applications to a wide array of biological questions, and its efficiency allows for high-throughput application. A solid understanding of its underlying photochemical mechanism is still missing. In this study, we address the CALI mechanism using a simplified model system consisting of the enzyme beta-galactosidase as target protein and the common dye fluorescein. We demonstrate that protein photoinactivation is independent from dye photobleaching and provide evidence that the first singlet state of the chromophore is the relevant transient state for the initiation of CALI. Furthermore, the inactivation process was shown to be dependent on oxygen and likely to be based on photooxidation of the target protein via singlet oxygen. The simple model system used in this study may be further applied to identify and optimize other CALI chromophores.

Proteins are the molecules that fulfil most cellular functions and represent over 90% of drug targets in the market. Chromophore-assisted laser inactivation (CALI) provides a timely and locally restricted protein inactivation and has proven to specifically destroy protein function using dye-coupled ligands and laser irradiation. CALI involves the generation of short-lived radicals thus limiting the radius of covalent modifications to spatially restricted sites on the target molecule. A transient functional inactivation occurs if the radicals modify amino acids of the target protein that are responsible for function. Here we show specific inactivation of several protein targets, that are members of relevant signal transduction pathways. For each of these targets, simple and high throughput screening-scaleable assays have been developed, making it possible to quantify the observed inactivation. Activities of target proteins have been addressed in cell-free as well as cell-based assays employing human primary and tumor-derived cell lines. In all cases, at least 50% inactivation was achieved. The data presented here demonstrate that CALI is a highly versatile tool for validating disease relevant targets at the protein level. This approach also takes into account post-translational modifications like phosphorylation, glycosylation or acylation, thereby enlarging its applicability for many different types of targets.