Clathrin seems to be dispensable for some endocytic processes and, in several instances, no cytosolic coat protein complexes could be detected at sites of membrane invagination. Hence, new principles must in these cases be invoked to account for the mechanical force driving membrane shape changes. Here we show that the Gb3 (glycolipid)-binding B-subunit of bacterial Shiga toxin induces narrow tubular membrane invaginations in human and mouse cells and model membranes. In cells, tubule occurrence increases on energy depletion and inhibition of dynamin or actin functions. Our data thus demonstrate that active cellular processes are needed for tubule scission rather than tubule formation. We conclude that the B-subunit induces lipid reorganization that favours negative membrane curvature, which drives the formation of inward membrane tubules. Our findings support a model in which the lateral growth of B-subunit-Gb3 microdomains is limited by the invagination process, which itself is regulated by membrane tension. The physical principles underlying this basic cargo-induced membrane uptake may also be relevant to other internalization processes, creating a rationale for conceptualizing the perplexing diversity of endocytic routes.

No abstract

Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membrane-associated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70 were at least 260-fold more effective than free recombinant protein in inducing TNF-α production as an indicator of macrophage activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within membranous structures from intact cells, which could act as a danger signal to activate the immune system.

We present in this paper that porous silicon can be used as a large surface area matrix as well as the transducer of biomolecular interactions. We report the fabrication of heavily doped p-type porous silicon with pore diameters that can be tuned, depending on the etching condition, from approximately 5 to 1200 nm. The structure and porosity of the matrixes were characterized by scanning force microscopy (SFM) and scanning electron microscopy (SEM), Brunnauer-Emmett-Teller nitrogen adsorption isotherms, and reflectance interference spectroscopy. The thin porous silicon layers are transparent to the visible region of the reflectance spectra due to their high porosity (80-90%) and are smooth enough to produce Fabry-Perot fringe patterns upon white light illumination. Porous silicon matrixes were modified by ozone oxidation, functionalized in the presence of (2-pyridyldithiopropionamidobutyl)dimethylmethoxysilane, reduced to unmask the sulfhydryl functionalities, and coupled to biotin through a disulfide-bond-forming reaction. Such functionalized matrixes display considerable stability against oxidation and corrosion in aqueous media and were used to evaluate the utility of porous silicon in biosensing. The streptavidin-biotin interactions on the surface of porous silicon could be monitored by the changes in the effective optical thickness calculated from the observed shifts in the Fabry-Perot fringe pattern caused by the change in the refractive index of the medium upon protein-ligand binding. Porous silicon thus combines the properties of a mechanically and chemically stable high surface area matrix with the function of an optical transducer and as such may find utility in the fabrication of biosensor devices.