Any cell within a tissue is constantly confronted with a variety of mechanical stimuli. Sensing of these diverse stimuli plays an important role in cellular regulation. Besides shear stress, cells of the vascular endothelium are particularly exposed to a permanent cyclic straining originating from the interplay of outwards pushing blood pressure and inwards acting contraction by smooth musculature. Perpendicular alignment of cells as structural adaptation to this condition is a basic prerequisite in order to withstand deformation forces. Here, we combine live cell approaches with immunocytochemical analyses on single cell level to closely elucidate the mechanisms of cytoskeletal realignment to cyclic strain and consolidate orientation analyses of actin fibres, microtubules (MTs) and vimentin. We could show that strain-induced reorientation takes place for all cytoskeletal systems. However, all systems are characterized by their own, specific reorientation time course with actin filaments reorienting first followed by MTs and finally vimentin. Interestingly, in all cases, this reorientation was faster than cell body realignment which argues for an active adaptation mechanism for all cytoskeletal systems. Upon actin destabilization, already smallest alterations in actin kinetics massively hamper cell morphology under strain and therefore overall reorientation. Depolymerization of MTs just slightly influences actin reorientation velocity but strongly affects cell body reorientation.
In mammalian cells, actin, microtubules, and various types of cytoplasmic intermediate filaments respond to external stretching. Here, we investigated the underlying processes in endothelial cells plated on soft substrates from silicone elastomer. After cyclic stretch (0.13 Hz, 14% strain amplitude) for periods ranging from 5 min to 8 h, cells were fixed and double-stained for microtubules and either actin or vimentin. Cell images were analyzed by a two-step routine. In the first step, micrographs were segmented for potential fibrous structures. In the second step, the resulting binary masks were auto- or cross-correlated. Autocorrelation of segmented images provided a sensitive and objective measure of orientational and translational order of the different cytoskeletal systems. Aligning of correlograms from individual cells removed the influence of only partial alignment between cells and enabled determination of intrinsic cytoskeletal order. We found that cyclic stretching affected the actin cytoskeleton most, microtubules less, and vimentin mostly only via reorientation of the whole cell. Pharmacological disruption of microtubules had barely any influence on actin ordering. The similarity, i.e., cross-correlation, between vimentin and microtubules was much higher than the one between actin and microtubules. Moreover, prolonged cyclic stretching slightly decoupled the cytoskeletal systems as it reduced the cross-correlations in both cases. Finally, actin and microtubules were more correlated at peripheral regions of cells whereas vimentin and microtubules correlated more in central regions.
Dendritic filopodia are actin-filled highly dynamic subcellular structures that sprout densely on neuronal dendrites during early brain development. A fraction of filopodia undergo a transition to dendritic spines that later mature into synapses -a process crucial for memory formation and learning, and deficient in neurodevelopmental and neurodegenerative diseases. The dynamics of dendritic filopodia is also different from that of conventional filopodia: the former exhibit sustained length fluctuations and cease their dynamic behavior after transition into spines. While actin retrograde flow in dendritic filopodia was measured previously, there has been no detailed theoretical description of how the flow is maintained and how length oscillations are sustained in dendritic filopodia. We apply mathematical modeling and experimental techniques to dissect and analyze the components of actin-based motility in dendritic filopodia, and suggest its role in the subsequent transition into the spine shape. The simulations demonstrate that the movement of actin network influenced by myosin contractility and viscous shear stresses lead to the myosin build up at the base of the filopodium and its consequent retraction. When myosin is inactive, the filopodium grows with the rate of actin polymerization. However, when myosin is active, the processes of polymerization at the tip, and cytoskeletal contraction due to myosin, and resistance to the flow out of the filopod at the base, conspire to produce an actin flow gradient along the filopodial axis. We have measured average rates of growth and retraction of filopodia in cultured hippocampal neurons using a custom tip-tracking algorithm. The simulated length fluctuations and actin retrograde flow compare well with the experimental data. We estimate the resistive force at the base of filopodia necessary to maintain the pattern of growth and shrinking and suggest new experiments to test the proposed mechanism. Using a standard actin filament gliding assay, rhodamine-phalloidin labeled actin filaments were visualized moving over a myosin-II coated surface by fluorescence microscopy and filament tracks were analyzed by computer. When micromolar amounts of non-fluorescent phalloidin-stabilised actin were added, filament motion became aligned into a common direction (Butt et al., 2010, J Biol Chem285:4964-4974). Experiments conducted at low filament densities show that, on average, there is a small angular deflection that tends to align the paths of individual colliding filaments. Path alignment is independent of relative track directionality and increases with collision incident angle. Filament elastic deformation during collision events indicates the collision bending energy is~20k B T. As increasing amounts of bulk actin are added, individual filament paths become straighter and start to move in a common direction. Spatial correlation analysis shows that increased alignment is due to incremental recruitment of filament tracks into one orientation rather than fusion of seed domains t...
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