Experimental methods to control pinned and coupled actomyosin contraction events

TL;DR

This work introduces two experimental strategies to study actomyosin contractility under controlled environmental boundaries: a pinned contraction setup with two rigid boundaries and a coupled contraction setup using a flexure hinge as a compliant boundary. By selectively nucleating actin at predefined sites and anchoring the gel transversely, the authors enable precise measurement of contractile forces and mechanical work, converting flexure deflection into force via a calibrated spring constant. The flexure-based method furnishes quantitative force and energy data, while the pinned system provides insight into directional stress generation and tissue-like boundary interactions. Together, these approaches advance mechanistic understanding of actomyosin actuation in contexts that mimic tissue mechanics, with potential extensions to 3D matrices and higher-throughput configurations.

Abstract

Actin and myosin drive many instances of force generation, deformation, and shape change in cells, tissues, and organisms. In particular, cytoskeletal actomyosin is remarkable in its adaptive architecture, responding to a host of actin-binding proteins. Equally important, however, is actomyosin's interaction with its mechanical environment. Actomyosin contractility and environmental properties, such as geometry and stiffness, are inherently coupled. To understand this coupling, novel experimental techniques are needed. Here we describe methods to spatially control the anchoring of reconstituted contractile actomyosin networks to two, opposing surfaces ("transverse anchoring"). The two surfaces can be either rigid ("pinned contraction"), or one of the surfaces may be compliant ("coupled contraction"). We introduce compliance by manufacturing flexure hinges, and describe their calibration. Calibration permits a direct measurement of the contractile force and mechanical work that actomyosin exerts on the environment. The methods described here provide an avenue toward a more complete characterization of actomyosin's role as an actuator, an essential property in its context of driving deformation and shape change in living systems.

Figures (7)

• Figure 1: Pinned contraction chamber A schematic of the sample chamber used in pinned contraction assays. (a) Top view of the chamber wings constructed from 316 L stainless steel. (b) Front view of the chamber wings. Depicted with 2:1 aspect ratio (height:width) for clarity. (c) Top view of the assembled pinned contraction chamber, with steel wings (dark gray) Parafilm (yellow) and cover glass (blue). (d) Front view of the assembled chamber. Depicted with 2:1 aspect ratio (height:width) for clarity.
• Figure 2: Flexure Chamber A schematic of the sample chamber used in flexure experiment assays. (a) Top view of the flexure, made of acrylic. (b) Side view of the flexure. (c) Top view of the assembled flexure chamber, with flexure (light gray), steel wings (dark gray), Parafilm (yellow), and cover glass (blue). (d) Transverse cross section of the flexure chamber (cf. panel c, dashed line). Depicted with 2:1 aspect ratio (height:width) for clarity.
• Figure 3: Selective nucleation of actin Steps to initiate actin nucleation at defined adhesion sites. (a) Surface of the sample chamber. (b) KOH-treated surfaces coated with PLL-PEG (light blue). (c) Selective exposure of UV (purple) to PLL-PEG of actin adhesion site specified by a mask (brown). (d) Degradation of PLL-PEG passivation (dark blue) at the adhesion site. (e) Attachment of GST-VCA (green) at the adhesion site. (f) Interaction of Arp2/3 (pink) in protein assay with GST-VCA on the adhesion site. (g) Actin nucleation (red) at the adhesion site.
• Figure 4: Epifluorescence Microscopy Schematic of the epifluorescence microscopy setup. Excitation light (filtered using an mPlum filter) is directed through the objective onto the sample, where fluorophores absorb the light and re-emit fluorescence. The emitted light is filtered for the desired wavelength before being captured by the camera.
• Figure 5: Flexure Calibration The flexure is calibrated using atomic force microscopy (AFM) with a probe of known stiffness to determine the spring constant of the flexure for force calculations. (a) Schematic of calibration setup. (b) Combined reference measurements of $n=2$ datasets of the photodiode signal as a function of Z-piezo displacement in Angstroms. (c) Single flexure measurement dataset of the photodiode voltage signal as a function of Z-piezo displacement in Angstroms. (d) Combined measurements of multiple flexure positions along the same flexure ($n=29$). (e) Histogram showing the frequency distribution of fitted slope, $S^m$. $\bar{S}^m$= -1.47e-04$\pm$5.06e-08