Resolving the Role of Actoymyosin Contractility in Cell Microrheology

Einstein's original description of Brownian motion established a direct relationship between thermally-excited random forces and the transport properties of a submicron particle in a viscous liquid. Recent work based on reconstituted actin filament networks suggests that nonthermal forces driven by the motor protein myosin II can induce large non-equilibrium fluctuations that dominate the motion of particles in cytoskeletal networks. Here, using high-resolution particle tracking, we find that thermal forces, not myosin-induced fluctuating forces, drive the motion of submicron particles embedded in the cytoskeleton of living cells. These results resolve the roles of myosin II and contractile actomyosin structures in the motion of nanoparticles lodged in the cytoplasm, reveal the biphasic mechanical architecture of adherent cells—stiff contractile stress fibers interdigitating in a network at the cell cortex and a soft actin meshwork in the body of the cell, validate the method of particle tracking-microrheology, and reconcile seemingly disparate atomic force microscopy (AFM) and particle-tracking microrheology measurements of living cells

Integrating transposable elements in the 3D genome

Chromosome organisation is increasingly recognised as an essential component of genome regulation, cell fate and cell health. Within the realm of transposable elements (TEs) however, the spatial information of how genomes are folded is still only rarely integrated in experimental studies or accounted for in modelling. Whilst polymer physics is recognised as an important tool to understand the mechanisms of genome folding, in this commentary we discuss its potential applicability to aspects of TE biology. Based on recent works on the relationship between genome organisation and TE integration, we argue that existing polymer models may be extended to create a predictive framework for the study of TE integration patterns. We suggest that these models may offer orthogonal and generic insights into the integration profiles (or "topography") of TEs across organisms. In addition, we provide simple polymer physics arguments and preliminary molecular dynamics simulations of TEs inserting into heterogeneously flexible polymers. By considering this simple model, we show how polymer folding and local flexibility may generically affect TE integration patterns. The preliminary discussion reported in this commentary is aimed to lay the foundations for a large-scale analysis of TE integration dynamics and topography as a function of the three-dimensional host genome

Localisation of RNAs into the germ plasm of vitellogenic xenopus oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others

Equilibria of Idealized Confined Astral Microtubules and Coupled Spindle Poles

Positioning of the mitotic spindle through the interaction of astral microtubules with the cell boundary often determines whether the cell division will be symmetric or asymmetric. This process plays a crucial role in development. In this paper, a numerical model is presented that deals with the force exerted on the spindle by astral microtubules that are bent by virtue of their confinement within the cell boundary. It is found that depending on parameters, the symmetric position of the spindle can be stable or unstable. Asymmetric stable equilibria also exist, and two or more stable positions can exist simultaneously. The theory poses new types of questions for experimental research. Regarding the cases of symmetric spindle positioning, it is necessary to ask whether the microtubule parameters are controlled by the cell so that the bending mechanics favors symmetry. If they are not, then it is necessary to ask what forces external to the microtubule cytoskeleton counteract the bending effects sufficiently to actively establish symmetry. Conversely, regarding the cases with asymmetry, it is now necessary to investigate whether the cell controls the microtubule parameters so that the bending favors asymmetry apart from any forces that are external to the microtubule cytoskeleton

Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM)

Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 µm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The ‘patchy’ structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments

Quantitative imaging of concentrated suspensions under flow

We review recent advances in imaging the flow of concentrated suspensions, focussing on the use of confocal microscopy to obtain time-resolved information on the single-particle level in these systems. After motivating the need for quantitative (confocal) imaging in suspension rheology, we briefly describe the particles, sample environments, microscopy tools and analysis algorithms needed to perform this kind of experiments. The second part of the review focusses on microscopic aspects of the flow of concentrated model hard-sphere-like suspensions, and the relation to non-linear rheological phenomena such as yielding, shear localization, wall slip and shear-induced ordering. Both Brownian and non-Brownian systems will be described. We show how quantitative imaging can improve our understanding of the connection between microscopic dynamics and bulk flow.Comment: Review on imaging hard-sphere suspensions, incl summary of methodology. Submitted for special volume 'High Solid Dispersions' ed. M. Cloitre, Vol. xx of 'Advances and Polymer Science' (Springer, Berlin, 2009); 22 pages, 16 fig

The Intermediate Filament Network in Cultured Human Keratinocytes Is Remarkably Extensible and Resilient

The prevailing model of the mechanical function of intermediate filaments in cells assumes that these 10 nm diameter filaments make up networks that behave as entropic gels, with individual intermediate filaments never experiencing direct loading in tension. However, recent work has shown that single intermediate filaments and bundles are remarkably extensible and elastic in vitro, and therefore well-suited to bearing tensional loads. Here we tested the hypothesis that the intermediate filament network in keratinocytes is extensible and elastic as predicted by the available in vitro data. To do this, we monitored the morphology of fluorescently-tagged intermediate filament networks in cultured human keratinocytes as they were subjected to uniaxial cell strains as high as 133%. We found that keratinocytes not only survived these high strains, but their intermediate filament networks sustained only minor damage at cell strains as high as 100%. Electron microscopy of stretched cells suggests that intermediate filaments are straightened at high cell strains, and therefore likely to be loaded in tension. Furthermore, the buckling behavior of intermediate filament bundles in cells after stretching is consistent with the emerging view that intermediate filaments are far less stiff than the two other major cytoskeletal components F-actin and microtubules. These insights into the mechanical behavior of keratinocytes and the cytokeratin network provide important baseline information for current attempts to understand the biophysical basis of genetic diseases caused by mutations in intermediate filament genes

Dynamics of Mechanical Signal Transmission through Prestressed Stress Fibers

Transmission of mechanical stimuli through the actin cytoskeleton has been proposed as a mechanism for rapid long-distance mechanotransduction in cells; however, a quantitative understanding of the dynamics of this transmission and the physical factors governing it remains lacking. Two key features of the actin cytoskeleton are its viscoelastic nature and the presence of prestress due to actomyosin motor activity. We develop a model of mechanical signal transmission through prestressed viscoelastic actin stress fibers that directly connect the cell surface to the nucleus. The analysis considers both temporally stationary and oscillatory mechanical signals and accounts for cytosolic drag on the stress fibers. To elucidate the physical parameters that govern mechanical signal transmission, we initially focus on the highly simplified case of a single stress fiber. The results demonstrate that the dynamics of mechanical signal transmission depend on whether the applied force leads to transverse or axial motion of the stress fiber. For transverse motion, mechanical signal transmission is dominated by prestress while fiber elasticity has a negligible effect. Conversely, signal transmission for axial motion is mediated uniquely by elasticity due to the absence of a prestress restoring force. Mechanical signal transmission is significantly delayed by stress fiber material viscosity, while cytosolic damping becomes important only for longer stress fibers. Only transverse motion yields the rapid and long-distance mechanical signal transmission dynamics observed experimentally. For simple networks of stress fibers, mechanical signals are transmitted rapidly to the nucleus when the fibers are oriented largely orthogonal to the applied force, whereas the presence of fibers parallel to the applied force slows down mechanical signal transmission significantly. The present results suggest that cytoskeletal prestress mediates rapid mechanical signal transmission and allows temporally oscillatory signals in the physiological frequency range to travel a long distance without significant decay due to material viscosity and/or cytosolic drag

In Vivo Determination of Fluctuating Forces during Endosome Trafficking Using a Combination of Active and Passive Microrheology

BACKGROUND: Regulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes) can be assessed in living cells by using a combination of active and passive microrheology. METHODOLOGY/PRINCIPAL FINDINGS: This dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking. CONCLUSIONS/SIGNIFICANCE: In this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures. Finally a computational model gives estimates of the force itself and hence of the number of the motors pulling on endosomes

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