Filopodia are thin, spike-like cell surface protrusions containing bundles of parallel actin filaments. So far, filopodial dynamics has mainly been studied in the context of cell motility on coverslipadherent filopodia by using fluorescence and differential interference contrast (DIC) microscopy. In this study, we used an optical trap and interferometric particle tracking with nanometer precision to measure the three-dimensional dynamics of macrophage filopodia, which were not attached to flat surfaces. We found that filopodia act as cellular tentacles: a few seconds after binding to a particle, filopodia retract and pull the bound particle toward the cell. We observed F-actin-dependent stepwise retraction of filopodia with a mean step size of 36 nm, suggesting molecular motor activity during filopodial pulling. Remarkably, this intracellular stepping motion, which was measured at counteracting forces of up to 19 pN, was transmitted to the extracellular tracked particle via the filopodial F-actin bundle and the cell membrane. The pulling velocity depended strongly on the counteracting force and ranged between 600 nm/s at forces <1 pN and Ϸ40 nm/s at forces >15 pN. This result provides an explanation of the significant differences in filopodial retraction velocities previously reported in the literature. The measured filopodial retraction force-velocity relationship is in agreement with a model for force-dependent multiple motor kinetics.actin filaments ͉ interferometric three-dimensional particle tracking ͉ molecular motors ͉ nanomechanics ͉ optical trapping

The mechanics of the actin cytoskeleton have a central role in the regulation of cells and tissues, but the details of how molecular sensors recognize deformations and forces are elusive. By performing cytoskeleton laser nanosurgery in cultured epithelial cells and fibroblasts, we show that the retraction of stress fibers (SFs) is restricted to the proximity of the cut and that new adhesions form at the retracting end. This suggests that SFs are attached to the substrate. A new computational model for SFs confirms this hypothesis and predicts the distribution and propagation of contractile forces along the SF. We then analyzed the dynamics of zyxin, a focal adhesion protein present in SFs. Fluorescent redistribution after laser nanosurgery and drug treatment shows a high correlation between the experimentally measured localization of zyxin and the computed localization of forces along SFs. Correlative electron microscopy reveals that zyxin is recruited very fast to intermediate substrate anchor points that are highly tensed upon SF release. A similar acute localization response is found if SFs are mechanically perturbed with the cantilever of an atomic force microscope. If actin bundles are cut by nanosurgery in living Drosophila egg chambers, we also find that zyxin redistribution dynamics correlate to force propagation and that zyxin relocates at tensed SF anchor points, demonstrating that these processes also occur in living organisms. In summary, our quantitative analysis shows that force and protein localization are closely correlated in stress fibers, suggesting a very direct force-sensing mechanism along actin bundles.

SummaryAfter internalization into macrophages non-pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phagolysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill 'non-pathogenic' Mycobacterium smegmatis . This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V-ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.

Microplastic particles ubiquitously found in the environment are ingested by a huge variety of organisms. Subsequently, microplastic particles can translocate from the gastrointestinal tract into the tissues likely by cellular internalization. The reason for cellular internalization is unknown, since this has only been shown for specifically surface-functionalized particles. We show that environmentally exposed microplastic particles were internalized significantly more often than pristine microplastic particles into macrophages. We identified biomolecules forming an eco-corona on the surface of microplastic particles, suggesting that environmental exposure promotes the cellular internalization of microplastics. Our findings further indicate that cellular internalization is a key route by which microplastic particles translocate into tissues, where they may cause toxicological effects that have implications for the environment and human health.