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 "A ¼ 0:65 s. The velocity distribution of active events exhibits several peaks, in agreement with a discrete number of motor proteins acting collectively.

The interplay of membrane proteins is vital for many biological processes, such as cellular transport, cell division, and signal transduction between nerve cells. Theoretical considerations have led to the idea that the membrane itself mediates protein self-organization in these processes through minimization of membrane curvature energy. Here, we present a combined experimental and numerical study in which we quantify these interactions directly for the first time. In our experimental model system we control the deformation of a lipid membrane by adhering colloidal particles. Using confocal microscopy, we establish that these membrane deformations cause an attractive interaction force leading to reversible binding. The attraction extends over 2.5 times the particle diameter and has a strength of three times the thermal energy (−3.3 kBT). Coarse-grained Monte-Carlo simulations of the system are in excellent agreement with the experimental results and prove that the measured interaction is independent of length scale. Our combined experimental and numerical results reveal membrane curvature as a common physical origin for interactions between any membrane-deforming objects, from nanometre-sized proteins to micrometre-sized particles.

The behaviour of an organism often reflects a strategy for coping with its environment. Such behaviour in higher organisms can often be reduced to a few stereotyped modes of movement due to physiological limitations, but finding such modes in amoeboid cells is more difficult as they lack these constraints. Here, we examine cell shape and movement in starved Dictyostelium amoebae during migration toward a chemoattractant in a microfluidic chamber. We show that the incredible variety in amoeboid shape across a population can be reduced to a few modes of variation. Interestingly, cells use distinct modes depending on the applied chemical gradient, with specific cell shapes associated with shallow, difficult-to-sense gradients. Modelling and drug treatment reveals that these behaviours are intrinsically linked with accurate sensing at the physical limit. Since similar behaviours are observed in a diverse range of cell types, we propose that cell shape and behaviour are conserved traits.

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. We designed a microfluidic setup to generate alternating chemotactic gradient fields for simultaneous multicell exposure, greatly facilitating statistical analysis. For a quantitative description of intracellular response dynamics, we apply alternating time sequences of spatially homogeneous concentration gradients across 300 μm, reorienting on timescales down to a few seconds. Dictyostelium discoideum amoebae respond to gradient switching rates below 0.02 Hz by readapting their migration direction. For faster switching, cellular repolarization ceases and is completely stalled at 0.1 Hz. In this "chemotactically trapped" cell state, external stimuli alternate faster than intracellular feedback is capable to respond by onset of directed migration. To investigate intracellular actin cortex rearrangement during gradient switching, we correlate migratory cell response with actin repolymerization dynamics, quantified by a fluorescence distribution moment of the GFP fusion protein LimEΔcc. We find two fundamentally different cell polarization types and we could reveal the role of PI3-Kinase for cellular repolarization. In the early aggregation phase, PI3-Kinase enhances the capability of D. discoideum cells to readjust their polarity in response to spatially alternating gradient fields, whereas in aggregation competent cells the effect of PI3-Kinase perturbation becomes less relevant.eukaryotic chemotaxis | pseudopod-based motility | gradient sensing | flow chamber

The cell wall is a shape-defining structure that envelopes almost all bacteria. One of its main functions is to serve as a protection barrier to environmental stresses. Bacteria can be forced in a cell wall-deficient state under highly specialized conditions, which are invariably aimed at interrupting cell wall synthesis. Therefore, the relevance of such cells has remained obscure. Here we show that many filamentous actinomycetes have a natural ability to generate a new, cell wall-deficient cell type in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the canonical mycelial mode-of-growth. Remarkably, prolonged exposure of S-cells to hyperosmotic stress yielded variants that are able to proliferate indefinitely without their cell wall. This is the first report that demonstrates the formation of wall-deficient cells as a natural adaptation strategy and their potential transition into stable wall-less forms solely caused by prolonged exposure to osmotic stress. Given that actinomycetes are potent antibiotic producers, our work also provides important insights into how biosynthetic gene clusters and resistance determinants may disseminate into the environment.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

A living cell's interior is one of the most complex and intrinsically dynamic systems, providing an elaborate interplay between cytosolic crowding and ATP-driven motion that controls cellular functionality. Here, we investigated two distinct fundamental features of the merely passive, non-biomotor-shuttled material transport within the cytoplasm of Dictyostelium discoideum cells: the anomalous non-linear scaling of the mean-squared displacement of a 150-nm-diameter particle and non-Gaussian distribution of increments. Relying on single-particle tracking data of 320,000 data points, we performed a systematic analysis of four possible origins for non-Gaussian transport: 1) sample-based variability, 2) rarely occurring strong motion events, 3) ergodicity breaking/aging, and 4) spatiotemporal heterogeneities of the intracellular medium. After excluding the first three reasons, we investigated the remaining hypothesis of a heterogeneous cytoplasm as cause for non-Gaussian transport. A, to our knowledge, novel fit model with randomly distributed diffusivities implementing medium heterogeneities suits the experimental data. Strikingly, the non-Gaussian feature is independent of the cytoskeleton condition and lag time. This reveals that efficiency and consistency of passive intracellular transport and the related anomalous scaling of the mean-squared displacement are regulated by cytoskeleton components, whereas cytoplasmic heterogeneities are responsible for the generic, non-Gaussian distribution of increments.

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. A local mean-square displacement (MSD) analysis separates ballistic motion phases, which we exclude here, from diffusive nanoparticle motion. In this study, we focus on intracellular subdiffusion and elucidate lag-time dependence, with particular focus on the impact of cytoskeleton compartments like microtubules and actin filaments. This method proves useful for binary motion state distributions. Experimental results are compared to simulations of a data-driven Langevin model with finite velocity correlations that captures essential statistical features of the local MSD algorithm. Specifically, the values of the mean MSD exponent and effective diffusion coefficients can be traced back to negative correlations of the motion's increments. We clearly identify both microtubules and actin filaments as the cause for intracellular subdiffusion and show that actin-microtubule cross talk exerts viscosifying effects at timescales larger than 0.2 s. Our findings might give insights into material transport and information exchange in living cells, which might facilitate gaining control over cell functions.