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1.
J R Soc Interface ; 21(214): 20230439, 2024 May.
Article in English | MEDLINE | ID: mdl-38807527

ABSTRACT

We present a novel approach to traction force microscopy (TFM) for studying the locomotion of 10 cm long walking centipedes on soft substrates. Leveraging the remarkable elasticity and ductility of kudzu starch gels, we use them as a deformable gel substrate, providing resilience against the centipedes' sharp leg tips. By optimizing fiducial marker size and density and fine-tuning imaging conditions, we enhance measurement accuracy. Our TFM investigation reveals traction forces along the centipede's longitudinal axis that effectively counterbalance inertial forces within the 0-10 mN range, providing the first report of non-vanishing inertia forces in TFM studies. Interestingly, we observe waves of forces propagating from the head to the tail of the centipede, corresponding to its locomotion speed. Furthermore, we discover a characteristic cycle of leg clusters engaging with the substrate: forward force (friction) upon leg tip contact, backward force (traction) as the leg pulls the substrate while stationary, and subsequent forward force as the leg tip detaches to reposition itself in the anterior direction. This work opens perspectives for TFM applications in ethology, tribology and robotics.


Subject(s)
Arthropods , Locomotion , Locomotion/physiology , Animals , Arthropods/physiology , Microscopy/methods
2.
Phys Rev Lett ; 125(8): 088102, 2020 Aug 21.
Article in English | MEDLINE | ID: mdl-32909763

ABSTRACT

We perform a bidimensional Stokes experiment in an active cellular material: an autonomously migrating monolayer of Madin-Darby canine kidney epithelial cells flows around a circular obstacle within a long and narrow channel, involving an interplay between cell shape changes and neighbor rearrangements. Based on image analysis of tissue flow and coarse-grained cell anisotropy, we determine the tissue strain rate, cell deformation, and rearrangement rate fields, which are spatially heterogeneous. We find that the cell deformation and rearrangement rate fields correlate strongly, which is compatible with a Maxwell viscoelastic liquid behavior (and not with a Kelvin-Voigt viscoelastic solid behavior). The value of the associated relaxation time is measured as τ=70±15 min, is observed to be independent of obstacle size and division rate, and is increased by inhibiting myosin activity. In this experiment, the monolayer behaves as a flowing material with a Weissenberg number close to one which shows that both elastic and viscous effects can have comparable contributions in the process of collective cell migration.


Subject(s)
Cell Movement/physiology , Epithelial Cells/chemistry , Epithelial Cells/cytology , Models, Biological , Viscoelastic Substances/chemistry , Animals , Dogs , Madin Darby Canine Kidney Cells
3.
Lab Chip ; 20(21): 4016-4030, 2020 11 07.
Article in English | MEDLINE | ID: mdl-32975276

ABSTRACT

Emerging evidence suggests the importance of mechanical stimuli in normal and pathological situations for the control of many critical cellular functions. While the effect of matrix stiffness has been and is still extensively studied, few studies have focused on the role of mechanical stresses. The main limitation of such analyses is the lack of standard in vitro assays enabling extended mechanical stimulation compatible with dynamic biological and biophysical cell characterization. We have developed an agarose-based microsystem, the soft cell confiner, which enables the precise control of confinement for single or mixed cell populations. The rigidity of the confiner matches physiological conditions and its porosity enables passive medium renewal. It is compatible with time-lapse microscopy, in situ immunostaining, and standard molecular analyses, and can be used with both adherent and non-adherent cell lines. Cell proliferation of various cell lines (hematopoietic cells, MCF10A epithelial breast cells and HS27A stromal cells) was followed for several days up to confluence using video-microscopy and further documented by Western blot and immunostaining. Interestingly, even though the nuclear projected area was much larger upon confinement, with many highly deformed nuclei (non-circular shape), cell viability, assessed by live and dead cell staining, was unaffected for up to 8 days in the confiner. However, there was a decrease in cell proliferation upon confinement for all cell lines tested. The soft cell confiner is thus a valuable tool to decipher the effects of long-term confinement and deformation on the biology of cell populations. This tool will be instrumental in deciphering the impact of nuclear and cytoskeletal mechanosensitivity in normal and pathological conditions involving highly confined situations, such as those reported upon aging with fibrosis or during cancer.


Subject(s)
Cell Nucleus , Cytoskeleton , Cell Proliferation , Extracellular Matrix , Sepharose
4.
Phys Rev E ; 99(6-1): 062401, 2019 Jun.
Article in English | MEDLINE | ID: mdl-31330615

ABSTRACT

Mechanical strain and stress play a major role in biological processes such as wound healing or morphogenesis. To assess this role quantitatively, fixed or live images of tissues are acquired at a cellular precision in large fields of views. To exploit these data, large numbers of cells have to be analyzed to extract cell shape anisotropy and cell size. Most frequently, this is performed through detailed individual cell contour determination, using so-called segmentation computer programs, complemented if necessary by manual detection and error corrections. However, a coarse-grained and faster technique can be recommended in at least three situations: first, when detailed information on individual cell contours is not required; for instance, in studies which require only coarse-grained average information on cell anisotropy. Second, as an exploratory step to determine whether full segmentation can be potentially useful. Third, when segmentation is too difficult, for instance due to poor image quality or too large a cell number. We developed a user-friendly, Fourier-transform-based image analysis pipeline. It is fast (typically 10^{4} cells per minute with a current laptop computer) and suitable for time, space, or ensemble averages. We validate it on one set of artificial images and on two sets of fully segmented images, one from a Drosophila pupa and the other from a chicken embryo; the pipeline results are robust. Perspectives include in vitro tissues, nonbiological cellular patterns such as foams and xyz stacks.


Subject(s)
Cell Shape , Epithelium/diagnostic imaging , Fourier Analysis , Molecular Imaging , Animals , Anisotropy , Biomechanical Phenomena , Cell Size , Chick Embryo , Drosophila melanogaster/cytology , Pupa/cytology , Stress, Mechanical , Time Factors
5.
Phys Rev Lett ; 105(24): 248103, 2010 Dec 10.
Article in English | MEDLINE | ID: mdl-21231559

ABSTRACT

We present a 4D (x; y; z; t) force map of Dictyostelium cells crawling on a soft gel substrate. Vertical forces are of the same order as the tangential ones. The cells pull the substratum upward along the cell, medium, or substratum contact line and push it downward under the cell except for the pseudopods. We demonstrate quantitatively that the variations in the asymmetry in cortical forces correlates with the variations of the direction and speed of cell displacement.


Subject(s)
Cell Movement , Dictyostelium/cytology , Dictyostelium/physiology , Microscopy, Atomic Force/methods , Biomechanical Phenomena/physiology , Elastomers/chemistry , Fluorescence , Stress, Mechanical , Time Factors
6.
Cell Motil Cytoskeleton ; 65(4): 314-31, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18205201

ABSTRACT

The distribution of forces exerted by migrating Dictyostelium amebae at different developmental stages was measured using traction force microscopy. By using very soft polyacrylamide substrates with a high fluorescent bead density, we could measure stresses as small as 30 Pa. Remarkable differences exist both in term of the magnitude and distribution of forces in the course of development. In the vegetative state, cells present cyclic changes in term of speed and shape between an elongated form and a more rounded one. The forces are larger in this first state, especially when they are symmetrically distributed at the front and rear edge of the cell. Elongated vegetative cells can also present a front-rear asymmetric force distribution with the largest forces in the crescent-shaped rear of the cell (uropod). Pre-aggregating cells, once polarized, only present this last kind of asymmetric distribution with the largest forces in the uropod. Except for speed, no cycle is observed. Neither the force distribution of pre-aggregating cells nor their overall magnitude are modified during chemotaxis, the later being similar to the one of vegetative cells (F(0) approximately 6 nN). On the contrary, both the force distribution and overall magnitude is modified for the fast moving aggregating cells. In particular, these highly elongated cells exert lower forces (F(0) approximately 3 nN). The location of the largest forces in the various stages of the development is consistent with the myosin II localization described in the literature for Dictyostelium (Yumura et al.,1984. J Cell Biol 99:894-899) and is confirmed by preliminary experiments using a GFP-myosin Dictyostelium strain.


Subject(s)
Dictyostelium/growth & development , Dictyostelium/physiology , Myosin Type II/metabolism , Acrylic Resins , Animals , Cell Movement , Chemotaxis , Cyclic AMP/metabolism
7.
Phys Rev Lett ; 93(10): 108102, 2004 Sep 03.
Article in English | MEDLINE | ID: mdl-15447454

ABSTRACT

We study in detail the properties of fingers, a particular type of cell-cell adhesive structures appearing in adherens junctions. These periodic patterns break the symmetry of cell-cell contacts. We show that finger formation is driven by cadherin interactions and actin growth. A theoretical model is introduced in which the growth of fingers is limited by membrane tension. The steady shape and formation kinetics of fingers are experimentally measured and compared with the theoretical predictions.


Subject(s)
Actin Cytoskeleton/physiology , Adherens Junctions/physiology , Cadherins/physiology , Cell Adhesion/physiology , Membrane Fluidity/physiology , Membrane Fusion/physiology , Models, Biological , Actin Cytoskeleton/ultrastructure , Adherens Junctions/ultrastructure , Animals , CHO Cells , Cadherins/ultrastructure , Computer Simulation , Cricetinae , Cricetulus , Periodicity
8.
Proc Natl Acad Sci U S A ; 101(8): 2229-34, 2004 Feb 24.
Article in English | MEDLINE | ID: mdl-14982992

ABSTRACT

Physicists have studied the aggregation of adhesive proteins, giving a central role to the elastic properties of membranes, whereas cell biologists have put the emphasis on the cytoskeleton. However, there is a dramatic lack of experimental studies probing both contributions on cellular systems. Here, we tested both mechanisms on living cells. We compared, for the same cell line, the growth of cadherin-GFP patterns on recombinant cadherin-coated surfaces, with the growth of vinculin-GFP patterns on extracellular matrix protein-coated surfaces by using evanescent wave microscopy. In our setup, cadherins are not linked to actin, whereas vinculins are. This property allows us to compare formation of clusters with proteins linked or not to the cytoskeleton and thus study the role of membrane versus cytoskeleton in protein aggregation. Strikingly, the motifs we obtained on both surfaces share common features: they are both elongated and located at the cell edges. We showed that a local force application can impose this symmetry breaking in both cases. However, the origin of the force is different as demonstrated by drug treatment (butanedione monoxime) and hypotonic swelling. Cadherins aggregate when membrane tension is increased, whereas vinculins (cytoplasmic proteins of focal contacts) aggregate when acto-myosin stress fibers are pulling. We propose a mechanism by which membrane tension is localized at cell edges, imposing flattening of membrane and enabling aggregation of cadherins by diffusion. In contrast, cytoplasmic proteins of focal contacts aggregate by opening cryptic sites in focal contacts under acto-myosin contractility.


Subject(s)
Actomyosin/physiology , Cell Adhesion/physiology , Membrane Proteins/physiology , Myosins/physiology , Animals , CHO Cells , Cadherins/genetics , Cadherins/physiology , Cell Line , Cell Membrane/physiology , Cricetinae , Humans , Kinetics , Recombinant Proteins/metabolism
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