Decomposition cross-correlation for analysis of collagen matrix deformation by single smooth muscle cells

Microvascular remodeling is known to depend on cellular interactions with matrix tissue. However, it is difficult to study the role of specific cells or matrix elements in an in vivo setting. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local collagen matrix remodeling by single smooth muscle cells. We combined a motorized microscopic setup and time-lapse video microscopy with a new cross-correlation based image analysis algorithm to enable automated recording of cell-induced matrix reorganization. This method rendered 60–90 single cell studies per experiment, for which collagen deformation over time could be automatically derived. Thus, the current setup offers a tool to systematically study different components active in matrix remodeling

Complexity of the Tensegrity Structure for Dynamic Energy and Force Distribution of Cytoskeleton during Cell Spreading

Cytoskeleton plays important roles in intracellular force equilibrium and extracellular force transmission from/to attaching substrate through focal adhesions (FAs). Numerical simulations of intracellular force distribution to describe dynamic cell behaviors are still limited. The tensegrity structure comprises tension-supporting cables and compression-supporting struts that represent the actin filament and microtubule respectively, and has many features consistent with living cells. To simulate the dynamics of intracellular force distribution and total stored energy during cell spreading, the present study employed different complexities of the tensegrity structures by using octahedron tensegrity (OT) and cuboctahedron tensegrity (COT). The spreading was simulated by assigning specific connection nodes for radial displacement and attachment to substrate to form FAs. The traction force on each FA was estimated by summarizing the force carried in sounding cytoskeletal elements. The OT structure consisted of 24 cables and 6 struts and had limitations soon after the beginning of spreading by declining energy stored in struts indicating the abolishment of compression in microtubules. The COT structure, double the amount of cables and struts than the OT structure, provided sufficient spreading area and expressed similar features with documented cell behaviors. The traction force pointed inward on peripheral FAs in the spread out COT structure. The complex structure in COT provided further investigation of various FA number during different spreading stages. Before the middle phase of spreading (half of maximum spreading area), cell attachment with 8 FAs obtained minimized cytoskeletal energy. The maximum number of 12 FAs in the COT structure was required to achieve further spreading. The stored energy in actin filaments increased as cells spread out, while the energy stored in microtubules increased at initial spreading, peaked in middle phase, and then declined as cells reached maximum spreading. The dynamic flows of energy in struts imply that microtubules contribute to structure stabilization

Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background: Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood. Principal Findings: In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize. Conclusions: Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin

Membrane Invaginations Reveal Cortical Sites that Pull on Mitotic Spindles in One-Cell C. elegans Embryos

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell

Axon Extension Occurs Independently of Centrosomal Microtubule Nucleation

Microtubules are polymeric protein structures and components of the cytoskeleton. Their dynamic polymerization is important for diverse cellular functions. The centrosome is the classical site of microtubule nucleation and is thought to be essential for axon growth and neuronal differentiation-processes that require microtubule assembly. We found that the centrosome loses its function as a microtubule organizing center during development of rodent hippocampal neurons. Axons still extended and regenerated through acentrosomal microtubule nucleation, and axons continued to grow after laser ablation of the centrosome in early neuronal development. Thus, decentralized microtubule assembly enables axon extension and regeneration, and, after axon initiation, acentrosomal microtubule nucleation arranges the cytoskeleton, which is the source of the sophisticated morphology of neurons

Dynein-mediated pulling forces drive rapid mitotic spindle elongation in Ustilago maydis

Spindle elongation segregates chromosomes and occurs in anaphase, an essential step in mitosis. Dynein-mediated pulling forces position the spindle, but their role in anaphase is a matter of debate. Here, we demonstrate that dynein is responsible for rapid spindle elongation in the model fungus Ustilago maydis. We show that initial slow elongation is supported by kinesin-5, which is located in the spindle mid-zone. When the spindle reaches ∼2 μm in length, the elongation rate increases four-fold. This coincides with the appearance of long and less-dynamic microtubules (MTs) at each pole that accumulate dynein at their tips. Laser-mediated nanosurgery revealed that these MTs exert pulling forces in control cells, but not in dynein mutants. In addition, dynein mutants undergo initial slow anaphase, but fail to establish less-dynamic MTs and do not perform rapid spindle elongation, suggesting that dynein drives anaphase B. This is most likely mediated by cortical sliding of astral MTs along stationary dynein, which is off-loaded from the MT plus-end to the cortex

Myosin Vs organize actin cables in fission yeast.

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces