A spatiotemporal characterization method for the dynamic cytoskeleton

Abstract

The significant gap between quantitative and qualitative understanding of cytoskeletal function is a pressing problem; microscopy and labeling techniques have improved qualitative investigations of localized cytoskeleton behavior, whereas quantitative analyses of whole cell cytoskeleton networks remain challenging. Here we present a method that accurately quantifies cytoskeleton dynamics. Our approach digitally subdivides cytoskeleton images using interrogation windows, within which box-counting is used to infer a fractal dimension (D f ) to characterize spatial arrangement, and gray value intensity (GVI) to determine actin density. A partitioning algorithm further obtains cytoskeleton characteristics from the perinuclear, cytosolic, and periphery cellular regions. We validated our measurement approach on Cytochalasin-treated cells using transgenically modified dermal fibroblast cells expressing fluorescent actin cytoskeletons. This method differentiates between normal and chemically disrupted actin networks, and quantifies rates of cytoskeletal degradation. Furthermore, GVI distributions were found to be inversely proportional to D f , having several biophysical implications for cytoskeleton formation/degradation. We additionally demonstrated detection sensitivity of differences in D f and GVI for cells seeded on substrates with varying degrees of stiffness, and coated with different attachment proteins. This general approach can be further implemented to gain insights on dynamic growth, disruption, and structure of the cytoskeleton (and other complex biological morphology) due to biological, chemical, or physical stimuli. V C 2016The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.

Probing Single-Cell Mechanical Allostasis Using Ultrasound Tweezers

“…Moreover, we quantified the actin structures in both cell types using actin fractal dimensions (D f ), which describes complex patterns of F-actin filaments in cells. 1 As shown in Figs. 5e and 5f, there is a distinct difference in distribution (D f ) of F-actin filaments in VSMCs and T2DM-VSMCs with a significantly larger D f value in T2DM-VSMCs, indicating a more chaotic F-actin structure and a distinct mechanophenotype in T2DM-VSMCs.…”

Nanotopographic Regulation of Human Mesenchymal Stem Cell Osteogenesis

“…Automated image processing was used to perform fast and standardized quantification of the spatial distribution of the cell actin CSK. 63 From the raw images, the program identifies two sets of coordinate points that defines perimeters of the entire cell and the nucleus. The located boundaries are then re-defined through an offset by a predetermined distance of 5 μm.…”

“…Distinct cellular mechanical properties arise from different actomyosin CSK structures and could be indicative of measures of cellular response to the immediate microenvironment. 63,64 Specifically, we quantified fractal dimensions (D f ) describing complex patterns of cytoskeletal filaments in hMSCs (see Materials and Methods for details). As shown, there is a distinct distribution (D f ) of actin fibers in hMSCs on a smooth (R q = 1 nm) and a nanorough (R q = 200 nm) surface (Figure 4 A,B).…”

“…Here, integrating live cell imaging with an in house MATLAB processing method, we are able to noninvasively decipher intracellular properties for cellular mechanotransduction on our nanotopographic surface (see Materials and Methods for details). 63,64 With this method, we examined the cellular biomechanics of hMSCs on surfaces of different nanoroughness (see Materials and Methods for details). 64 Time-lapse images of live hMSCs on smooth (R q = 1 nm) and nanorough (R q = 200 nm) were acquired for 12 h under standard cell culture environment for the subsequent single-cell biomechanical properties analysis.…”