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Natural gas hydrates are a potential source of energy and may play a role in climate change and geological hazards. Most natural gas hydrate appears to be in the form of 'structure I', with methane as the trapped guest molecule, although 'structure II' hydrate has also been identified, with guest molecules such as isobutane and propane, as well as lighter hydrocarbons. A third hydrate structure, 'structure H', which is capable of trapping larger guest molecules, has been produced in the laboratory, but it has not been confirmed that it occurs in the natural environment. Here we characterize the structure, gas content and composition, and distribution of guest molecules in a complex natural hydrate sample recovered from Barkley canyon, on the northern Cascadia margin. We show that the sample contains structure H hydrate, and thus provides direct evidence for the natural occurrence of this hydrate structure. The structure H hydrate is intimately associated with structure II hydrate, and the two structures contain more than 13 different hydrocarbon guest molecules. We also demonstrate that the stability field of the complex gas hydrate lies between those of structure II and structure H hydrates, indicating that this form of hydrate is more stable than structure I and may thus potentially be found in a wider pressure-temperature regime than can methane hydrate deposits.

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Organelle transport to the periphery of the cell involves coordinated transport between the processive motors kinesin and myosin V. Long-range transport takes place on microtubule tracks, whereas final delivery involves shorter actin-based movements. The concept that motors only function on their appropriate track required further investigation with the recent observation that myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of myosin V's diffusion on microtubules is a significant enhancement of the processive run length of kinesin when both motors are present on the same cargo. The degree of run length enhancement correlated with the net positive charge in loop 2 of myosin V. On actin, myosin V also undergoes longer processive runs when kinesin is present on the same cargo. The process that causes run length enhancement on both cytoskeletal tracks is electrostatic. We propose that one motor acts as a tether for the other and prevents its diffusion away from the track, thus allowing more steps to be taken before dissociation. The resulting run length enhancement likely contributes to the successful delivery of cargo in the cell.actin ͉ microtubule ͉ molecular motor ͉ Qdot ͉ electrostatic M ovement of membrane-bound organelles from the center of the cell to its periphery and back involves the interplay between processive molecular motors on both microtubule (MT) and actin tracks. In amphibian melanophores, where pigment granules disperse and aggregate to cause skin color changes, long-range transport toward the plus-end of microtubules is carried out by kinesin-2, whereas myosin V (myoV) takes over in the actin-rich cell cortex to deliver melanosomes to the cell periphery during dispersion (1). This process requires switching between cytoskeletal tracks. We have shown, at the singlemolecule level, that myoV could effectively navigate actin-actin intersections that normally exist within cells, either by executing a turn, or by stepping over the crossing actin filament (2). The frequency of these two events appeared to be related to myoV's inherent flexibility and the availability of actin-binding sites on the intersecting filaments that were within reach of the leading head. Surprisingly, myoV could also undergo a diffusional search on MTs, resulting from an electrostatic interaction between the myoV head and the negatively charged tubulin E-hook. We hypothesized that this diffusive process helps myoV find cargo that is undergoing MT-based movement, and/or facilitates the binding of myoV to kinesin if the two motors directly interact (3).Can myoV's interaction with the MT affect kinesin's processivity when both motors are present on the same cargo? Here we show that this interaction enhances the processivity of kinesinbased cargo transport. We also provide evidence that the structural element on myoV that acts as an electrostatic tether is loop 2, a charged surface loop that also influences myoV's processive run length on actin (...

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.actin | myosin II | smooth muscle | thoracic aortic aneurysms | vascular disease T horacic aortic aneurysms and dissections (TAAD) are the 18th most common cause of death in individuals in the United States (1). The high degree of mortality is partly due to the fact that aneurysms tend to be asymptomatic until a lifethreatening acute aortic dissection occurs. Familial TAAD is an autosomal dominant disorder with variable penetrance, which is characterized by enlargement or dissection of the thoracic aorta (reviewed in 2). The most prevalent genetic cause of familial TAAD, responsible for ∼15% of all cases, are mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2. More than 40 mutations in ACTA2 have been identified to date (3-5). Intriguingly, ACTA2 mutations also differentially predispose individuals to occlusive vascular diseases, such as premature coronary artery disease and strokes (6). ACTA2 mutations thus can lead to either dilation of large elastic arteries like the aorta or occlusion of smaller muscular arteries.SM α-actin is the most abundant protein in vascular smooth muscle cells, constituting ∼40% of the total protein and ∼70% of the total actin, with the rest composed of β-and γ-cytoplasmic actin. Actin is critical for contraction and force production by smooth muscle cells, as well as for their proliferation and migration. Dissected aortas show s...