Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs

Membrane bilayer fusion has been shown to be mediated by v- and t-SNAREs initially present in separate populations of liposomes and to occur with high efficiency at a physiologically meaningful rate. Lipid mixing was demonstrated to involve both the inner and the outer leaflets of the membrane bilayer. Here, we use a fusion assay that relies on duplex formation of oligonucleotides introduced in separate liposome populations and report that SNARE proteins suffice to mediate complete membrane fusion accompanied by mixing of luminal content. We also find that SNARE-mediated membrane fusion does not compromise the integrity of liposomes.

Spectral bifurcations in dispersive wave turbulence

Dispersive wave turbulence is studied numerically for a class of one-dimensional nonlinear wave equations. Both deterministic and random (white noise in time) forcings are studied. Four distinct stable spectra are observed—the direct and inverse cascades of weak turbulence (WT) theory, thermal equilibrium, and a fourth spectrum (MMT; Majda, McLaughlin, Tabak). Each spectrum can describe long-time behavior, and each can be only metastable (with quite diverse lifetimes)—depending on details of nonlinearity, forcing, and dissipation. Cases of a long-live MMT transient state dcaying to a state with WT spectra, and vice-versa, are displayed. In the case of freely decaying turbulence, without forcing, both cascades of weak turbulence are observed. These WT states constitute the clearest and most striking numerical observations of WT spectra to date—over four decades of energy, and three decades of spatial, scales. Numerical experiments that study details of the composition, coexistence, and transition between spectra are then discussed, including: (i) for deterministic forcing, sharp distinctions between focusing and defocusing nonlinearities, including the role of long wavelength instabilities, localized coherent structures, and chaotic behavior; (ii) the role of energy growth in time to monitor the selection of MMT or WT spectra; (iii) a second manifestation of the MMT spectrum as it describes a self-similar evolution of the wave, without temporal averaging; (iv) coherent structures and the evolution of the direct and inverse cascades; and (v) nonlocality (in k-space) in the transferral process.

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 ± 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

Mixing of connexins in gap junction membrane channels.

Gap junctions are plaque-like clusters of intercellular channels that mediate intercellular communication. Each of two adjoining cells contains a connexon unit which makes up half of the whole channel. Gap junction channels are formed from a multigene family of proteins called connexins, and different connexins may be coexpressed by a single cell type and found within the same plaque. Rodent gap junctions contain two proteins, connexins 32 and 26. Use of a scanning transmission electron microscope for mass analysis of rodent gap junction plaques and split gap junctions prvided evidence consistent with a model in which the channels may be made from (i) solely connexin 26, (ii) solely connexin 32, or (iii) mixtures of connexin 26 and connexin 32 in which the two connexons are made entirely of connexin 26 and connexin 32. The different types of channels segregate into distinct domains, implying tha connexon channels self-associate to give a non-random distribution within tissues. Since each connexin confers distinct physiological properties on its membrane channels, these results imply that the physiological properties of channels can be tailored by mixing the constituent proteins within these macromolecular structures.