Transition metal ion FRET between a noncanonical fluorescent amino acid incorporated into TRPV1 and metal ions bound to the cell plasma can be used to measure distances and dynamics between cytosolic domains of proteins and the membrane.

Background: Whether phosphoinositide 4,5-bisphosphate (PI(4,5)P 2 ) activates or inhibits TRPV1 is controversial. Results: PI(4,5)P 2 in the intracellular leaflet activates TRPV1, whereas PI(4,5)P 2 in the extracellular leaflet inhibits TRPV1. Conclusion: Inhibition by PI(4,5)P 2 in the extracellular leaflet may explain previous findings that TRPV1 reconstituted into PI(4,5)P 2 -containing liposomes is inhibited. Significance: PI(4,5)P 2 in the physiologically relevant leaflet (the intracellular leaflet) of the membrane activates TRPV1.

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus–centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Gα proteins GOA-1/GPA-16. In centration- defective let-99(−) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Gα signaling and actomyosin. During wild-type centration, NMY-2–GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16–depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(−) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Gα signaling and LET-99 control centration by regulating polarized actomyosin contraction.

A novel method is presented to measure short distances in cell plasma membranes using transition metal ion FRET with metal ions bound to introduced sites in the membrane.

The dynamic microenvironment of cells depends on macromolecular architecture, equilibrium fluctuations, and nonequilibrium forces generated by cytoskeletal proteins. We studied the influence of these factors on the motions of mitochondria in mating S. cerevisiae using Fourier imaging correlation spectroscopy (FICS). Our measurements provide detailed length-scale dependent information about the dynamic behavior of mitochondria. We investigate the influence of the actin cytoskeleton on mitochondrial motion and make comparisons between conditions in which actin network assembly and disassembly is varied either by using disruptive pharmacological agents or mutations that alter the rates of actin polymerization. Under physiological conditions, nonequilibrium dynamics of the actin cytoskeleton leads to 1.5-fold enhancement of the long-time mitochondrial diffusion coefficient and a transient subdiffusive temporal scaling of the mean-square displacement (MSD ∝ τ α , with α ¼ 2∕3). We find that nonequilibrium forces associated with actin polymerization are a predominant factor in driving mitochondrial transport. Moreover, our results lend support to an existing model in which these forces are directly coupled to mitochondrial membrane surfaces.anomalous diffusion | Arp2/3-mediated actin polymerization | Fourier imaging correlation spectroscopy | intracellular transport | mitochondrial dynamics T he intracellular environment is a dynamic multicomponent fluid with relaxations spanning a broad range of length and time scales. As in ordinary fluids, in cells thermally generated inertial forces give rise to stochastic particle motions, which may be characterized by the particle mean-square displacement (MSD). Under many circumstances, thermal diffusion is a primary mechanism of intracellular transport. However, nonequilibrium forces that are generated by protein polymerization, motor proteins, and gradients in thermodynamic potentials can additionally influence particle motion. A central question in modeling cell transport is whether the cytoplasm may be viewed as a simple extension of a complex fluid at equilibrium or if nonequilibrium effects dominate the motions of intracellular species.For a spatially homogeneous fluid at equilibrium, the MSD scales linearly in time (i.e. MSD ∝ τ α with α ¼ 1). However, microscopic heterogeneity and viscoelasticity of multicomponent fluids results in "anomalous" particle motion (1-5). In the context of cell dynamics the motion of a macromolecule or larger particle may become hindered by obstacles in its immediate environment, leading to subdiffusive scaling of the MSD (i.e. α < 1), over an appreciable time range (4). For example, the motions of small proteins in bacteria appear to be diffusive (6-8), whereas those of larger particles, such as mRNA-protein clusters in E. coli and yolk granules in yeast, appear to be subdiffusive (2, 3). These differences in the dynamics of intracellular species and host organisms may be reconciled by accounting for the relative size of the mobile particles...