Structure and dynamics of actin network at the leading edge of migrating cells

Crawling motion is characteristic of most animal cells and is principally based on actin-myosin II cytoskeletal system. The major components and reactions contributing to motility have been identified, but the overall picture of how these molecular events are orchestrated to result in the motion of the cell is still missing. We used a combination of experimental imaging, image analysis, and numerical modeling to analyze at a quantitative level how the elements of the actin-myosin machinery are coordinated in several important aspects of the crawling motion: the assembly of the characteristic pattern of actin network in the leading lamellipodium, the interaction between retrograde flow of the actin network and substrate adhesions, and the coordination of motion and assembly of actin and myosin II polymers over the entire cell during steady-state migration. First, we used enhanced phase contrast microscopy correlated with fluorescence and electron microscopy to investigate the supra-filament organization of actin in the lamellipodium of motile fish epidermal keratocytes. We showed a close correspondence between individual filament orientations and optical criss-cross network pattern by quantifying the orientational distribution of features in electron microscopy and optical microscopy images. We tested the hypothesis that the criss-cross pattern observed by optical microscopy results from variation of actin density, which is due to stochastic events of branching and capping of filaments. Based on this hypothesis, we produced simulated images of lamellipodium similar to experimental images. These simulations provided quantitative tools to estimate structural and dynamical parameters of the actin network in lamellipodia, notably the filament length, the actin concentration, the curvature of filaments and capping rate. Protrusion at the edge of the cell is coupled to the substrate adhesion, but at the same time is associated with the retrograde flow of the actin network away from the edge. We observed that focal adhesions were mostly localized at the boundary between the fast retrograde flow zone (the lamellipodium) and the slower flow zone (the lamellum). We analyzed simultaneously the actin flow and the formation of focal adhesion. Based on these results, we proposed a multi-stage model for the advance of the leading edge of migrating cell: advance of the lamellipodium is followed by the formation of the nascent adhesion complexes, which results in the reduction of the flow rate, advance of the lamellum, and then protrusion of the lamellipodium from a new base. We finally focused on the dynamics of the acto-myosin system in entire migrating cell. For this purpose, we developed a tracking program which allowed quantifying motion, contraction and assembly of cytoskeletal components. We measured the dynamics of actin, myosin II and their relative motion in migrating fish keratocytes. These results supported the dynamic network contraction model suggesting that the actin network deformed by myosin II drives the translocation of cell body. Focusing on the different aspects of the crawling motion, and balancing experimental and theoretical approaches, we obtained original results, which contributed to a better understanding of the dynamics of the lamellipodium and crawling motion in general.

Mechanical properties of peripheral stress fibers in adherent cells spreading on patterned substrates

Céline Jeanne Labouesse

Many cellular processes require or depend on mechanical feedback from the cell's micro-environment. For example, the local organization of the actin cytoskeleton is known to adapt to the physical properties of the extracellular matrix. To gain better understanding of the mechanisms controlling this process, we have studied the particular case of peripheral stress fibers, thick concave contractile bundles of actin filaments. We have employed micropatterned adhesive substrates to constrain cell shape and adhesion layout. We have then used fluorescence microscopy, traction force microscopy and cantilever-based force measurements to experimentally characterize the assembly and mechanical tension of these peripheral stress fibers. We combined these experimental data with numerical simulations of cell shape and cell tension to propose a general mechanical model of stress fiber behavior. We first identified the formation of peripheral stress fibers as significant of a behavioral switch triggered by non-adhesive gaps in the substrate geometry. These bundles indeed form above a threshold distance of 3-4µm, necessary to stimulate adhesion maturation, and lead to a redirection of cellular tension parallel to the cell edge. Additional analysis of cells on rigid and soft substrates showed that the radius and tension of peripheral bundles weakly increase with spanning distance. Thus both the cytoskeleton local organization and its mechanical properties can be controlled to some extent by non-adhesive features in substrate geometry. The peripheral fibers' concave curvature results from the balance of the line and surface tensions. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, are not clear. To get insight into this force balance, we inhibited myosin activity and monitored the shape and tension responses. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that line tension dropped to a lesser extent than surface tension. These results suggest a myosin-independent component in the tension of peripheral arcs. We propose a simple physical model in which the line tension is the sum of a myosin-dependent active component and of a passive elastic component. Numerical simulations of this model reproduce well the measured shape dynamics and allow estimating the relative contributions of elasticity and contractility to the arc line tension. We then developed an alternative setup to directly probe the elasticity of semi-isolated peripheral fibers, using micromanipulation techniques. A soft cantilever was used to measure the force with which the fiber resisted a transverse deformation. The overall mechanical response of the fiber can be represented by a 3-parameter-solid model, i.e. a spring in series with a Kelvin-Voigt unit. The response was separated into two regimes: first a fast traction during which elastic response dominated, followed by a viscoelastic relaxation. The elastic and viscoelastic coefficients were extracted from the force-elongation curves and from the elongation velocity during relaxation. Inhibiting myosin activity decreased the active pre-tension, but had no clear effect on the elasticity. Our results show that myosin is an important actor, but not the only one, in the response of peripheral fibers to external mechanical constraints (active deformation) or geometrical constraints (limited adhesive area).

EPFL2015

Dynamics of actin and myosin II during cell polarization and directional locomotion

Sophie Bohnet

Crawling cell motility is characteristic of most animal cells and is involved in many important biological processes such as embryogenesis, immune response, and wound healing. It involves several steps: protrusion (dynamic surface extension) at the front of the cell, attachment to the substratum, and forward translocation of the cell body accompanied by the detachment and retraction of the rear portion of the cell. Crawling motion is believed to be based on actin, a major component of the cytoskeleton, and myosin II, a motor protein that is believed to produce contraction force by moving along actin filaments. Actin and myosin II are the two major structural and force-generating components of the cell's motile machinery. Protrusion is thought to be driven by the assembly of a dense actin network that is attached to the substrate through integrin-containing adhesions. Previous studies estimated the forces generated by actin polymerization and the adhesion forces, but the net force developed by the protruding leading edge has not been yet determined. The forward translocation of the cell body is believed to depend on the interaction of myosin with the actin network, but the exact mechanisms behind this interaction are still unclear. Moreover, it is still poorly understood how the molecular reactions involved in each step of the crawling motion are coordinated to result in the integrated cellular response. In this thesis work, we have quantitatively characterized the assembly and motion of actin and myosin II in the stationary and migratory states of the cell, and during the transition between these two steady-states. We have also measured the force developed by the protruding leading edge. The experimental system used is the fish epidermal keratocytes, one of the most spectacular models of cell locomotion. These cells are characterized by fast and persistent migration, and a stable and simple cytoskeletal organization. Enhanced phase contrast, interference reflection, classical fluorescence and fluorescent speckles microscopy were used as imaging techniques along with computer-based tracking approaches. We have first studied actin movement and assembly in the lamellipodium of keratocyte (thin, sheet-like extension at the leading edge of the cell). Actin dynamics in the lamellipodia of many cell types are characterized by retrograde flow of the actin network away from the leading edge. This flow is believed to result from membrane resistance to actin assembly and contractile forces in the network, and was speculated to play a role in controlling the cell shape and motion. Retrograde flow was previously observed in every crawling cell with the exception of keratocytes. We have for the first time detected actin retrograde flow throughout the keratocyte lamellipodium at velocities of 1–3 µm/min and analyzed its organization and relation to the cell motion during both unobstructed persistent migration and events of cell collision. We have found that freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium, whereas in colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of the flow in other lamellipodial regions. Our findings support the universality of the flow phenomenon and indicate that maintenance of the keratocyte shape during locomotion depends on the regulation of retrograde flow and actin polymerization. We have subsequently quantitatively characterized the assembly and movement of actin and myosin II throughout the entire keratocyte. Actin exhibited a slow backward motion in the lamellipodium and a faster forward motion in the trailing cell body. The change of direction occurred along the line parallel to the leading edge of the cell just in front of the front boundary of the cell body. Myosin II also moved forward in the cell body, but no motion with respect to the substratum was detected in the lamellipodium. At the cell sides, actin and myosin II exhibited motion towards the cell center with velocities increasing with the distance from the center. Net assembly of actin occurred locally at the leading edge of the lamellipodium, and disassembly at the back of the lamellipodium. Myosin II assembled in a distributed manner in the lamellipodium and disassembled in the cell body. Eventually, a simultaneous tracking of actin and myosin II revealed that myosin II moved forward relatively to actin throughout the cell. Taken together, these results suggest a longitudinal and transverse contraction along the lamellipodium/cell body junction. To understand how the polarized patterns of the organization of actin/myosin machinery were first established, we have studied in the next part of the work random cell polarization, i.e. the spontaneous transition between the stationary and the migratory state of a cell in the absence of external directional stimuli. Cell polarization and directional locomotion are believed to involve asymmetry in actin assembly, contractility, and substrate adhesion. We have analyzed the distribution and dynamics of actin and myosin II and the dynamics of substrate adhesions during cell polarization. In the stationary state, the cells exhibited lamellipodia with a uniform rate of actin retrograde flow all around the cell perimeter. Upon initiation of directional movement, retrograde flow accelerated at the prospective rear of the cell, but the lamellipodia initially persisted all around the perimeter. The assembly of actin network also continued all around the cell. Local areas of detachment from the substrate were observed at the base of rear lamellipodia. Treatment with myosin inhibitor blebbistatin nearly completely blocked the polarization. Depolymerization of microtubules with nocodazole resulted in attenuated polarization. Taken together, these results suggest that initial polarization results from asymmetry in actin retrograde flow, which is induced by myosin-dependent contraction and stabilized by microtubules. Finally, we have estimated the force necessary to stall the protruding leading edge of the keratocytes. We arrested the leading edge of a moving cell with a hydrodynamic load generated by a fluid flow from a micropipette. The flow arrested the protrusion locally, as the cell approached the pipette, causing an arc-shaped indentation and upward folding of the leading edge. Modeling of the fluid flow gave a surprisingly low value for the arresting force of just a few piconewtons per micrometer. This small force did not abolish actin polymerization nor disrupt the adhesions formed before the arrest, but rather interfered with the weak nascent adhesions at the very front of the cell. We conclude that a weak external force is sufficient to reorient the growing actin network at the leading edge and to stall the cell protrusion. Our study of the dynamics of actin and myosin II in polarizing and migrating cells provides novel and important insights into the cell motility mechanism and establishes a fundamental quantitative data set necessary to test and improve theoretical models of cell motility.

EPFL2006

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

In this thesis new imaging approaches for optical microscopy are proposed and studied. They are based on the use of dynamic structured illumination in combination with a demodulation detection concept employing CMOS image detectors. Two particular implementations are suggested: real-time optical sectioning microscopy and whole field quantitative polarization microscopy. Optical sectioning, i.e depth resolved imaging, allows observation of thin sections in volumetric samples. In its classical configuration the wide-field microscope does not allow optical sectioning of the investigated objects. In this thesis the optical sectioning in a wide-field microscope is achieved by illuminating the sample with a continuously moving single spatial frequency pattern, which temporarily modulates the signal obtained on the photodetector. Only the object parts that are in the focus have a good contrast and consequently generate stronger signal on the detector. The optically sectioned images are obtained employing a CMOS image detector, which demodulates the time-dependent component of the image. Two different systems for real-time acquisition of optically sectioned images are proposed. The first one is based on a specially designed smart-pixel-detector-array (SPDA), which allows performing the signal processing by an electronic circuit integrated to each pixel of the sensor. The second system utilizes a commercial CMOS image sensor. Here, the images are treated by a digital signal processor (DSP) integrated into the camera (iMVS-155). Both approaches provide real-time acquisition of optically sectioned images. The proof of principle for the new method is demonstrated by imaging artificial three-dimensional reflective samples. The optical sectioning imaging of biological samples in bright-field mode and in fluorescence mode is also demonstrated. The optical sectioning performances of the studied systems are similar to that of the confocal microscope. However the new method has some advantages over the classical confocal system: e.g. the difficulties with the alignment and the post-processing inherent to the confocal system are avoided. The theoretical framework presented in the thesis describes the image formation in structured illumination for both reflective and fluorescent samples. The influence of longitudinal chromatic aberration and spherical aberration caused by specimen induced index mismatch on depth discrimination property are studied. In extension to the work related to optically sectioned microscopy, the quantitative polarization microscopy imaging method is proposed as a new application for CMOS detectors. The polarization state of the illuminating light is dynamically changed and the demodulation detection approach is employed to extract the retardance and azimuth of the birefringent sample in real-time; for a whole field of view. Examples of polarization sensitive measurements for different samples are presented. The theoretical analysis is performed using the Jones formalism. Finally, the potentials and limitations of the CMOS detectors for applications in optical microscopy are discussed. Thanks to constant advancements in CMOS technology, the acquisition speed, resolution and sensitivity of new CMOS detectors generations have been improved. This will allow adapting the methods proposed in this thesis for investigations of fast dynamic process where high temporal and spatial resolution is required.

EPFL2004