Eukaryotic CRFK cells motion characterized with atomic force microscopy (original) (raw)
Related papers
Gradient of Rigidity in the Lamellipodia of Migrating Cells Revealed by Atomic Force Microscopy
Biophysical Journal, 2005
Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.
1987
Cell locomotion begins with a protrusion from the leading periphery of the cell. What drives this extension? Here we present a model for the extension of cell protuberances that unifies certain aspects of this phenomenon, and is based on the hypothesis that osmotic pressure drives cell extensions. This pressure arises from membrane-associated reactions, which liberate osmoticallv active particles, and from the swelling of the actin network that underlies the membrane. in t r o d u c t io n : w hat are the forces driving cell motility? Cells possess several mechanisms for exerting forces on their surroundings. In particular, a number of mechanochemical enzymes have been identified, including myosin, dynein and kinesin; other molecules will probably be identified in the future. All of these molecules share a common characteristic: they enable the cell to exert only contractile forces. This is a puzzling situation, since in order to move about cells must also be capable of generating protrusive forces. Placing cells in hypertonic media seems to suppress all protrusive activity, suggesting that protrusive force generation may be produced by simple osmotic pressure (Harris, 1973; Trinkaus, 1984 Trinkaus, , 1985. But osmotic pressure is an isotropic force: it acts equally in all directions. Therefore, in order to use pressure for protrusion, the cell must devise means to focus the force in particular directions. In the next section we propose a mechanism by which osmotic forces drive cell protrusion, and which is coordinated with the polymerization of the actin network that fills such protrusions. This model is an extension of two previous models we have proposed for lamellipod and acrosomal extension.
Slow cellular dynamics in MDCK and R5 cells monitored by time-lapse atomic force microscopy
Biophysical Journal, 1994
We have examined dynamic events that occur on a time scale of minutes in an epithelial monolayer of Madine-Darby Canine KKiney (MDCK) cells and in ras-transformed MDCK cells by atomic force microscopy (AFM). Cells were imaged under physiological condKions, and timelapse movies represenbng -60 s real time perframe were assembled. In normal MDCK cells, two types of prorusion in the apical plasma membrane exhibit dynamic behavior. First, smooth bulges fofmed tansiently over the time scale of minutes to tens of minutes. Second, spike-like proitrusions appear initially as bulges, extend well above the apical surface and, finally, seem to detach. R5, an oncogenictrarsformant derived from MDCK cells, grows very flat on glass.
Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2008
The motility of living eukaryotic cells is a complex process driven mainly by polymerization and depolymerization of actin filaments underneath the plasmatic membrane (actin cytoskeleton). However, the exact mechanisms through which cells are able to control and employ 'actin-generated' mechanical forces, in order to change shape and move in a well-organized and coordinated way, are not quite established. Here, we summarize the experimental results obtained by our research group during recent years in studying the motion of living cells, such as macrophages and erythrocytes. By using our recently developed defocusing microscopy technique, which allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope, we were able to analyse morphological and dynamical parameters of membrane ruffles and small membrane fluctuations, study the process of phagocytosis and also measure values for cell refractive index, membrane bending modulus and cell viscosity. Although many questions still remain unanswered, our data seem to corroborate some aspects of recent physical models of cell membranes and motility.
Cytoskeleton of living, unstained cells imaged by scanning force microscopy
Biophysical Journal, 1993
Subsurface cytoskeletal structure can be visualized in either fixed or living mammalian cells in aqueous medium with -50 nm resolution using the Scanning Force Microscope (SFM). In living cells, changes in cell topography, or subsurface cytoskeleton caused by the introduction of drugs (colchicine) or cross-linking of surface receptors (by antibodies against IgE bound to the IgE receptor) can be followed in time. Contrast in SFM images of cell surfaces result from both topographic features of the cell and from variations in cell surface "stiffness". The SFM is therefore capable of measuring local compliance and stress in living cells, and so should make it possible to map the cytoskeletal forces used to generate cell motions and changes in cell shape. ment the cells were stimulated by injecting 1.0 gg/ml DNP-BSA in 0006 3495/93/04
Nature Protocols, 2012
Forces in cells Physical forces are crucial to a number of cellular processes such as cell division 1,2 , stem cell differentiation 3 , cell migration 4-6 , tissue formation 7,8 and development 9,10 , wound healing 11 , tumor growth 12,13 , progression of disease states 14-21 , biomechanics 22 and mechanotransduction 23. In all of these processes, forces must be generated and transmitted through the components of the cells themselves, and therefore it is crucial to understand the mechanical properties of the cell. Mechanical properties can be assessed by studying the relationship between stress (force/area) and strain (deformation) as a function of time 24. These concepts, which were originally developed from materials science and engineering of inanimate materials, have been adapted to the cellular context 25. Historical beginnings of cell mechanics Some of the first single-cell micromechanical studies were carried out on the relatively large (diameter ~100 µm) egg cells of marine and amphibian organisms. Methods involved the sessile drop approach 26 , centrifuge microscopy 27,28 , compression with microbeams 29 and microplates 30,31 , micropipette aspiration 32,33 , microneedle poking 34 and tensile stretching with beads 35. These pioneering efforts inspired studies on the much smaller and more delicate red blood cells; these studies exploited technological improvements in microscopy and mechanical techniques such as micropipette aspiration 36,37 and fluid shear stress assays 38. From the results of these studies, researchers made apparent the connection between the mechanical properties of individual blood cells and their contribution to pathophysiology 39,40 , and these effects are currently well documented 41. A diversity of methods in cell mechanics After these promising beginnings, the challenge was to expand the field of animal cell mechanics to cover a broader range of cell types and biological contexts 42. At present, progress has been enabled by the development and optimization of techniques to measure and apply forces and displacements with picoNewton and nanometer sensitivity in combination with enhanced live cell microscopy 43. Methods currently in use can be roughly split into two categories. The first category involves active imposition of force or deformation on the cell and includes micropipette aspiration 44 , parallel plate devices 45,46 , magnetic twisting cytometry 47,48 , active microrheology 49 , optical tweezers 50-52 , optical cell stretching 53 , cell-populated substrate or gel deformation devices 54,55 , fluid shear stress 56,57 , microfluidics 58,59 , micro-electro-mechanical systems 60,61 and various modes of AFM 62-67. The second category of methods instead teases out mechanical information from optical or acoustic data and includes passive microrheology by tracking probe particles 49 or subcellular components 68,69 , analysis of contractile release dynamics 70,71 , acoustic microscopy 72,73 , laser ablation of subcellular structures 74 , optical mapping of intracellular force distribution 75 and traction force microscopy in two dimensions using deformable substrates 76,77 and flexible micropillar arrays 78,79 or in three dimensions with elastic gel matrices containing embedded fiduciary markers 80,81. In general, the first category of techniques is more straightforward and involves fewer assumptions, whereas the second category involves substantial computational and data analysis but has a better capability to probe beyond the surface. AFM in cell mechanics Of all the above-mentioned techniques, one of the most versatile is AFM, which provides a multifaceted platform to the study forces and mechanics in cell biology. Commercial AFM instruments designed for biological research are becoming increasingly adaptable and include cell-friendly options such as temperature control, liquid perfusion and compatibility with high-end light microscopes. Moreover, the experimental procedure can be tailored to probe local or global mechanical properties of cells over a wide range of forces from picoNewtons to microNewtons at nanometer precision, and it can provide high temporal resolution
Spatio-temporal analysis of eukaryotic cell motility by improved force cytometry
Proceedings of the National Academy of Sciences, 2007
Cell motility plays an essential role in many biological systems, but precise quantitative knowledge of the biophysical processes involved in cell migration is limited. Better measurements are needed to ultimately build models with predictive capabilities. We present an improved force cytometry method and apply it to the analysis of the dynamics of the chemotactic migration of the amoeboid form of Dictyostelium discoideum. Our explicit calculation of the force field takes into account the finite thickness of the elastic substrate and improves the accuracy and resolution compared with previous methods. This approach enables us to quantitatively study the differences in the mechanics of the migration of wildtype (WT) and mutant cell lines. The time evolution of the strain energy exerted by the migrating cells on their substrate is quasiperiodic and can be used as a simple indicator of the stages of the cell motility cycle. We have found that the mean velocity of migration v and the period of the strain energy T cycle are related through a hyperbolic law v ؍ L/T, where L is a constant step length that remains unchanged in mutants with adhesion or contraction defects. Furthermore, when cells adhere to the substrate, they exert opposing pole forces that are orders of magnitude higher than required to overcome the resistance from their environment.
Atomic force microscopy combined with confocal laser scanning microscopy: A new look at cells
Bioimaging, 1993
A stand-a lone atomic fo rce microscope (AFM) has been developed, which features a la rge sca n area a nd which allows opera tion under liquid. This system was combined with a co nfoca l laser sca nning microscope (C LSM). Information about cell structures, obtained by CLSM, can be complemented with images of the cell surface obtained with the AFM. This is illustra ted by stud ying the pseudopodia of cells from a human cell line (K562-cells, predecessor of eryth ro bl as ts) and the cytoskeleton of monk ey kidney cells (in air and under liquid), both stained with F-actin-specific Auo rescent pro bes. Im ages of the cytoskeleton during the cytotox ic interaction betwee n a na tu ral killer and a K562 target cell are presented. O ur results show that co mbination of these techniques ca n provid e new information about cells and cellular structu res. Key words: a to mic fo rce microscopy, co nfoca l lase r sca nnin g microscopy, cy toskeleto n, cy totox ic. interacti o n.