Abstract
Numerous biological functions such as cytokinesis, changes in cell shape and cell migration require actomyosin-driven cellular contractility. However, the detailed mechanism of how contractile forces drive cellular processes are difficult to decipher due to the complexity of the intracellular environment. In particular, the mesoscopic description of the myosin II-dependent actin retrograde flow in cell lamellum is missing. Here, we describe a methodology for detergent extraction of cell, which preserves integrity of the actin cytoskeleton. This semi-in vitro cell model allows for the observation, using light microscopy, and quantification of changes in the actin cytoskeleton resulting from the activation of cellular contractility upon addition of ATP. This assay also allows for the evaluation of the effects of actin-associated proteins and other related factors in the modulation of the actin contractile activities. Here, we demonstrate the retrograde flow of a well-known actin-based structures- transverse arcs, which are myosin IIA-containing structures that emerge at the boundary between lamellipodium-lamellum and move centripetally in myosin II-dependent fashion.
Keywords: Actin fibers, Actin flow, Actomyosin contractility, Micropatterning, Triton-insoluble cytoskeleton
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 1. Semi-in vitro detergent-permeabilized cell system. Human foreskin fibroblasts were spread on micro-contact printed circular fibronectin island for 5 h. Actin cytoskeleton (labeled by AlexaFluor-488-phalloidin) organized into a radially symmetrical system with actin radial fibers (RFs) oriented perpendicular to the cell edge and actin transverse arcs (magenta arrowheads) arranged perpendicular to the radial fibers. (A). Actin cytoskeleton remained inactive in contractility buffer without ATP. No movements of the transverse arcs were observed. Yellow arrow points to a radial fiber (RF). (B). Centripetal movement of transverse arcs along the radial fibers was seen in 2 mM ATP containing contractility buffer. Magenta arrowheads indicate the positions of transverse arcs. Magenta dotted lines indicate the initial positions of transverse arcs. Kymograph analysis in C is performed along the white lines indicated. Scale bar, 10 μm. (C). Kymograph analysis of transverse arcs in various experimental conditions: (i) without ATP, (ii) addition of 2 mM ATP, (iii) addition of 2 mM AMP-PNP and (iv) addition of 2 mM ATP together with 100 μM of blebbistatin. Centripetal movement of transverse arcs in permeabilized cells is ATP- and myosin-dependent since movement was only seen following the addition of ATP but not in the conditions with AMP-PNP, a non-hydrolyzable ATP analog, nor in the presence of blebbistatin, a myosin II inhibitor. Vertical scale bar, 2 μm.
Notes
Recipes
Acknowledgments
This protocol was adapted from the previously reported in Tint et al. (1991). This work was supported by the National Research Foundation Singapore, Ministry of Education of Singapore, Grant R-714-006-006-271, and administrated by the National University of Singapore.
References
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