Structural evidence for a two-regime photobleaching mechanism in a reversibly switchable fluorescent protein.

Photobleaching, the irreversible photodestruction of a chromophore, severely limits the use of fluorescent proteins (FPs) in optical microscopy. Yet, the mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of FPs that can be repeatedly photoswitched between nonfluorescent and fluorescent states, photobleaching limits the achievable number of switching cycles, a process known as photofatigue.

Local motion analysis reveals impact of the dynamic cytoskeleton on intracellular subdiffusion.

Intracellular transport is a complex interplay of ballistic transport along filaments and of diffusive motion, reliably delivering material and allowing for cell differentiation, migration, and proliferation. The diffusive regime, including subdiffusive, Brownian, and superdiffusive motion, is of particular interest for inferring information about the dynamics of the cytoskeleton morphology during intracellular transport. The influence of dynamic cytoskeletal states on intracellular transport are investigated in Dictyostelium discoideum cells by single particle tracking of fluorescent nanoparticles, to relate quantitative motion parameters and intracellular processes before and after cytoskeletal disruption.

Low-temperature chromophore isomerization reveals the photoswitching mechanism of the fluorescent protein Padron.

Photoactivatable fluorescent proteins are essential players in nanoscopy approaches based on the super-localization of single molecules. The subclass of reversibly photoswitchable fluorescent proteins typically activate through isomerization of the chromophore coupled with a change in its protonation state. However, the interplay between these two events, the details of photoswitching pathways, and the role of protein dynamics remain incompletely understood.

Chemotactic cell trapping in controlled alternating gradient fields.

Directed cell migration toward spatio-temporally varying chemotactic stimuli requires rapid cytoskeletal reorganization. Numerous studies provide evidence that actin reorganization is controlled by intracellular redistribution of signaling molecules, such as the PI4,5P2/PI3,4,5P3 gradient. However, exploring underlying mechanisms is difficult and requires careful spatio-temporal control of external chemotactic stimuli.

Colchicine-loaded lipid bilayer-coated 50 nm mesoporous nanoparticles efficiently induce microtubule depolymerization upon cell uptake.

We report on a one-step assembly route where supported lipid bilayers (SLB) are deposited on functionalized colloidal mesoporous silica (CMS) nanoparticles, resulting in a core-shell hybrid system (SLB@CMS). The supported membrane acts as an intact barrier against the escape of encapsulated dye molecules. These stable SLB@CMS particles loaded with the anticancer drug colchicine are readily taken up by cells and lead to the depolymerization of microtubules with remarkably enhanced efficiency as compared to the same dose of drug in solution.

Axonal guidance by surface microstructuring for intracellular transport investigations.

Intracellular transport, a complex interplay of diverse processes, is fundamental for the development, function and survival of cells. Passive diffusion and active transport phases alternate in living cells, with active phases arising from molecular motors, such as kinesin or dynein, pulling cargoes along microtubules. A better understanding of stochasic mechanisms involved in motor-microtubule interactions and in diffusion processes, which enable efficient active transport over long distances in motor neurons, requires a better link between theoretical models and live-cell experiments.

Impact of external stimuli and cell micro-architecture on intracellular transport states.

A living cell is a complex out-of-equilibrium system, in which a great variety of biochemical and physical processes have to be coordinated to ensure viability. We investigate properties of intracellular transport in single cells of the amoeba Dictyostelium discoideum, a relevant model organism due to its cytoskeleton simplicity. In the cells, vesicles undergo two types of motion: directed transport, driven by molecular motors on filaments, or thermal diffusion in a crowded active medium.

Temporal analysis of active and passive transport in living cells.

The cellular cytoskeleton is a fascinating active network, in which Brownian motion is intercepted by distinct phases of active transport. We present a time-resolved statistical analysis dissecting phases of directed motion out of otherwise diffusive motion of tracer particles in living cells. The distribution of active lifetimes is found to decay exponentially with a characteristic time tauA = 0.