Quantification of the actin cytoskeleton is of prime importance to unveil the cellular force sensing and transduction mechanism. Although fluorescence imaging provides a convenient tool for observing the morphology of the actin cytoskeleton, due to the lack of approaches to accurate actin cytoskeleton quantification, the dynamics of mechanotransduction is still poorly understood. Currently, the existing image-based actin cytoskeleton analysis tools are either incapable of quantifying both the orientation and the quantity of the actin cytoskeleton simultaneously or the quantified results are subject to analysis artifacts. In this study, we propose an image recognition-based actin cytoskeleton quantification (IRAQ) approach, which quantifies both the actin cytoskeleton orientation and quantity by using edge, line, and brightness detection algorithms. The actin cytoskeleton is quantified through three parameters: the partial actin-cytoskeletal deviation (PAD), the total actin-cytoskeletal deviation (TAD), and the average actin-cytoskeletal intensity (AAI). First, Canny and Sobel edge detectors are applied to skeletonize the actin cytoskeleton images, then PAD and TAD are quantified using the line directions detected by Hough transform, and AAI is calculated through the summational brightness over the detected cell area. To verify the quantification accuracy, the proposed IRAQ was applied to six artificially-generated actin cytoskeleton mesh work models. The average error for both the quantified PAD and TAD was less than 1.22 ∘ . Then, IRAQ was implemented to quantify the actin cytoskeleton of NIH/3T3 cells treated with an F-actin inhibitor (latrunculin B). The quantification results suggest that the local and total actin-cytoskeletal organization became more disordered with the increase of latrunculin B dosage, and the quantity of the actin cytoskeleton showed a monotonically decreasing relation with latrunculin B dosage.
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Effect of F-actin and Microtubules on Cellular Mechanical Behavior Studied Using Atomic Force Microscope and an Image Recognition-Based Cytoskeleton Quantification Approach
Cytoskeleton morphology plays a key role in regulating cell mechanics. Particularly, cellular mechanical properties are directly regulated by the highly cross-linked and dynamic cytoskeletal structure of F-actin and microtubules presented in the cytoplasm. Although great efforts have been devoted to investigating the qualitative relation between the cellular cytoskeleton state and cell mechanical properties, comprehensive quantification results of how the states of F-actin and microtubules affect mechanical behavior are still lacking. In this study, the effect of both F-actin and microtubules morphology on cellular mechanical properties was quantified using atomic force microscope indentation experiments together with the proposed image recognition-based cytoskeleton quantification approach. Young’s modulus and diffusion coefficient of NIH/3T3 cells with different cytoskeleton states were quantified at different length scales. It was found that the living NIH/3T3 cells sense and adapt to the F-actin and microtubules states: both the cellular elasticity and poroelasticity are closely correlated to the depolymerization degree of F-actin and microtubules at all measured indentation depths. Moreover, the significance of the quantitative effects of F-actin and microtubules in affecting cellular mechanical behavior is depth-dependent.
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- NSF-PAR ID:
- 10177919
- Date Published:
- Journal Name:
- International Journal of Molecular Sciences
- Volume:
- 21
- Issue:
- 2
- ISSN:
- 1422-0067
- Page Range / eLocation ID:
- 392
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The measurement of local mechanical properties of living cells by nano/micro indentation relies on the foundational assumption of locally isotropic cellular deformation. As a consequence of assumed isotropy, the cell membrane and underlying cytoskeleton are expected to locally deform axisymmetrically when indented by a spherical tip. Here, we directly observe the local geometry of deformation of membrane and cytoskeleton of different living adherent cells during nanoindentation with the integrated Atomic Force (AFM) and spinning disk confocal (SDC) microscope. We show that the presence of the perinuclear actin cap (apical stress fibers), such as those encountered in cells subject to physiological forces, causes a strongly non-axisymmetric membrane deformation during indentation reflecting local mechanical anisotropy. In contrast, axisymmetric membrane deformation reflecting mechanical isotropy was found in cells without actin cap: cancerous cells MDA-MB-231, which naturally lack the actin cap, and NIH 3T3 cells in which the actin cap is disrupted by latrunculin A. Careful studies were undertaken to quantify the effect of the live cell fluorescent stains on the measured mechanical properties. Using finite element computations and the numerical analysis, we explored the capability of one of the simplest anisotropic models – transverse isotropy model with three local mechanical parameters (longitudinal and transverse modulus and planar shear modulus) – to capture the observed non-axisymmetric deformation. These results help identifying which cell types are likely to exhibit non-isotropic properties, how to measure and quantify cellular deformation during AFM indentation using live cell stains and SDC, and suggest modelling guidelines to recover quantitative estimates of the mechanical properties of living cells.
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Cell–substrate interaction plays an important role in intracellular behavior and function. Adherent cell mechanics is directly regulated by the substrate mechanics. However, previous studies on the effect of substrate mechanics only focused on the stiffness relation between the substrate and the cells, and how the substrate stiffness affects the time-scale and length-scale of the cell mechanics has not yet been studied. The absence of this information directly limits the in-depth understanding of the cellular mechanotransduction process. In this study, the effect of substrate mechanics on the nonlinear biomechanical behavior of living cells was investigated using indentation-based atomic force microscopy. The mechanical properties and their nonlinearities of the cells cultured on four substrates with distinct mechanical properties were thoroughly investigated. Furthermore, the actin filament (F-actin) cytoskeleton of the cells was fluorescently stained to investigate the adaptation of F-actin cytoskeleton structure to the substrate mechanics. It was found that living cells sense and adapt to substrate mechanics: the cellular Young’s modulus, shear modulus, apparent viscosity, and their nonlinearities (mechanical property vs. measurement depth relation) were adapted to the substrates’ nonlinear mechanics. Moreover, the positive correlation between the cellular poroelasticity and the indentation remained the same regardless of the substrate stiffness nonlinearity, but was indeed more pronounced for the cells seeded on the softer substrates. Comparison of the F-actin cytoskeleton morphology confirmed that the substrate affects the cell mechanics by regulating the intracellular structure.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/ijms21020392
