Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Lim, Soon-Mi; Kreipe, Bryan A; Trzeciakowski, Jerome P.; Dangott, Larry; Trache, Andreea

doi:10.1016/j.yexcr.2010.06.010

Cited by 27 publications

(33 citation statements)

References 99 publications

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“…In recent years, optical microscopy techniques including bright field, confocal microscopy, and total internal reflection fluorescence microscopy (TIRF) have been combined with the AFM for real-time imaging of cells to mechanical stimuli. 41,42 These setups also allow simultaneous measurements of cell mechanics and imaging of the cytoskeletal components. Correlating the local stiffness data to the distribution of local cytoskeletal components will provide insightful information on how the cytoskeletal components contribute to cell mechanics.…”

Section: Discussionmentioning

confidence: 99%

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Thomas

Burnham

Camesano³

et al. 2013

JoVE

134

127

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Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Thomas

Burnham

Camesano³

et al. 2013

JoVE

134

127

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“…Consequently, activated and clustered integrins lead to the recruitment and organization of several different cytoskeletal proteins and reinforces focal adhesion formation. Moreover, RhoA activity was also suggested to involve the increased activation of stress fibers and FA formation [57]. Nevertheless, cells adhered to PLL-coated or non-coated glass slides, via non-integrin-mediated binding, exhibit different cytoskeleton structures.…”

Section: Discussionmentioning

confidence: 99%

The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment

et al. 2013

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Atomic force microscopy provides a novel technique for differentiating the mechanical properties of various cell types. Cell elasticity is abundantly used to represent the structural strength of cells in different conditions. In this study, we are interested in whether physical or physiological cues affect cell elasticity in Atomic force microscopy (AFM)-based assessments. The physical cues include the geometry of the AFM tips, the indenting force and the operating temperature of the AFM. All of these cues show a significant influence on the cell elasticity assessment. Sharp AFM tips create a two-fold increase in the value of the effective Young’s modulus (Eeff) relative to that of the blunt tips. Higher indenting force at the same loading rate generates higher estimated cell elasticity. Increasing the operation temperature of the AFM leads to decreases in the cell stiffness because the structure of actin filaments becomes disorganized. The physiological cues include the presence of fetal bovine serum or extracellular matrix-coated surfaces, the culture passage number, and the culture density. Both fetal bovine serum and the extracellular matrix are critical for cells to maintain the integrity of actin filaments and consequently exhibit higher elasticity. Unlike primary cells, mouse kidney progenitor cells can be passaged and maintain their morphology and elasticity for a very long period without a senescence phenotype. Finally, cell elasticity increases with increasing culture density only in MDCK epithelial cells. In summary, for researchers who use AFM to assess cell elasticity, our results provide basic and significant information about the suitable selection of physical and physiological cues.

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“…4B); (i) a single, polarized protrusion was evident in the majority of control and rescued cells whereas most of the Nck-depleted cells presented numerous protrusions oriented in multiple directions, and (ii) prominent, well organized actin fibers were present in control and rescued cells but not in Nck-depleted cells. Quantitative analysis (Lim et al, 2010) showed that the F-actin area relative to cell area was reduced in cells with disruption of Nck signaling (Fig. 4C).…”

Section: Nck Regulates Directional Motility Through Modulation Of Promentioning

confidence: 98%

“…Cell and actin area were determined using the masking tool and image statistics tools available in the SlideBook software (Intelligent Imaging Innovations, Denver, CO). For comparisons, the protein area was normalized to the total cell area (Lim et al, 2010).…”

Section: Analysis Of Actin Areamentioning

confidence: 99%

Nck enables directional cell migration through the coordination of polarized membrane protrusion with adhesion dynamics

Chaki¹,

Barhoumi²,

Berginski³

et al. 2013

Journal of Cell Science

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SummaryDirectional migration requires the coordination of cytoskeletal changes essential for cell polarization and adhesion turnover. Extracellular signals that alter tyrosine phosphorylation drive directional migration by inducing reorganization of the actin cytoskeleton. It is recognized that Nck is an important link between tyrosine phosphorylation and actin dynamics; however, the role of Nck in cytoskeletal remodeling during directional migration and the underlying molecular mechanisms remain largely undetermined. In this study, a combination of molecular genetics and quantitative live cell microscopy was used to show that Nck is essential in the establishment of front-back polarity and directional migration of endothelial cells. Time-lapse differential interference contrast and total internal reflection fluorescence microscopy showed that Nck couples the formation of polarized membrane protrusions with their stabilization through the assembly and maturation of cell-substratum adhesions. Measurements by atomic force microscopy showed that Nck also modulates integrin a5b1-fibronectin adhesion force and cell stiffness. Fluorescence resonance energy transfer imaging revealed that Nck depletion results in delocalized and increased activity of Cdc42 and Rac. By contrast, the activity of RhoA and myosin II phosphorylation were reduced by Nck knockdown. Thus, this study identifies Nck as a key coordinator of cytoskeletal changes that enable cell polarization and directional migration, which are crucial processes in development and disease.

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Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Cited by 27 publications

References 99 publications

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment

Nck enables directional cell migration through the coordination of polarized membrane protrusion with adhesion dynamics

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