Diverse membrane fusion reactions in biology involve close contact between two lipid bilayers, followed by the local distortion of the individual bilayers and reformation into a single, merged membrane. We consider the structures and energies of the fusion intermediates identified in experimental and theoretical work on protein-free lipid bilayers. On the basis of this analysis, we then discuss the conserved fusion-through-hemifusion pathway of merger between biological membranes and propose that the entire progression, from the close juxtaposition of membrane bilayers to the expansion of a fusion pore, is controlled by protein-generated membrane stresses.

Disparate biological processes involve fusion of two membranes into one and fission of one membrane into two. To formulate the possible job description for the proteins that mediate remodeling of biological membranes, we analyze the energy price of disruption and bending of membrane lipid bilayers at the different stages of bilayer fusion. The phenomenology and the pathways of the well-characterized reactions of biological remodeling, such as fusion mediated by influenza hemagglutinin, are compared with those studied for protein-free bilayers. We briefly consider some proteins involved in fusion and fission, and the dependence of remodeling on the lipid composition of the membranes. The specific hypothetical mechanisms by which the proteins can lower the energy price of the bilayer rearrangement are discussed in light of the experimental data and the requirements imposed by the elastic properties of the bilayer.

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48 -60 and (Arg) 9 . We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.During the last decade, several proteins, such as HIV-1 Tat, Drosophila Antennapedia homeoprotein, and HSV-1 VP22 have been shown to traverse the cell membrane by a process called protein transduction and to reach the nucleus while retaining their biological activity (1-5). Short "protein-transduction domains" are responsible for the cellular uptake of these proteins (6, 7). Although the biological relevance of protein transduction remains to be understood, it has attracted much interest. Indeed, it was discovered that short peptides derived from protein-transduction domains (cell-penetrating peptides or CPPs) 1 can be internalized in most cell types and, more importantly, allow the cellular delivery of conjugated (or fused) biomolecules (8, 9). A wide range of biomolecules such as antigenic peptides (10) The mechanism of internalization of CPPs and their cargo is not well understood and has recently been the subject of controversies. It has been described in the literature that internalization of these peptides is not significantly inhibited by incubation at low temperature, by depletion of the cellular ATP pool, or by inhibitors of endocytosis (7, 20 -22). Moreover, structure-activity studies indicate that the internalization of CPPs do not depend on its specific primary sequence, which implies independence of receptor recognition (20,23,24). Based on these results, it has been commonly accepted that the internalization of CPPs do not involve endocytosis or specific protein transporters. Instead, a direct transport through the lipid bilayer of membranes has been proposed as a possible mechanism of translocation (7,20). If correct, this mechanism would require a radical revision of current ideas on the properties of lipid bilayers, taking into account the hydrophilic nature of CPPs such as Tat or (Arg) 9 and the fact that there is no indication of increased membrane permeability in the p...

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell–cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATP-and temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolindependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.Recent advances in the identification of new molecular therapy targets and disease-relevant proteins, accelerated by the completion of the human genome project, emphasized an importance of high molecular weight information-rich biomolecules, such as peptides, proteins, antisense DNA, and small interfering RNA, for molecular therapy. However, the delivery of proteins and nucleic acids into cells is greatly hampered by the low permeability of the cell plasma membrane to polar molecules. Not surprisingly, the discovery that a number of cationic peptides known as protein transduction domains (PTDs) 1 can facilitate cytoplasmic and nuclear delivery of a conjugated cargo has attracted a lot of interest (1-3). Up to date, a wide range of cargo molecules, including low molecular weight drugs (4), oligonucleotides (5), peptides (6) and even full-length proteins (7-10), have been successfully delivered into cells using PTDs and, most importantly, the functional activity of the delivered cargo has been observed (7-10).Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using PTDs, the underlying mechanisms remain under active debate. Until recently, it was widely assumed that the internalization of cationic PTDs is an energy-and receptor-independent process based on direct transport through the lipid bilayer (11-15). On the other hand, there have been indications that uptake of full-length TAT protein, from which one of the most commonly used PTDs referred to as TAT peptide is derived, occurs via endocytosis and depends on cell surface heparan sulfate receptors (16). Moreover, the validity of some of the important data, supporting a direct transport model for synthetic TAT peptide, has been questioned in several recent ...

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During membrane fusion, the outer leaflets of the two membranes merge first, whereas the distal membrane leaflets remain separate until the opening of a fusion pore. This intermediate stage, called hemifusion, is a critical event shared by exocytosis, protein trafficking, and viral entry.