In Vitro Reconstitution of Cytoskeleton Networks: A Collection of Methods

Collection Overview

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The cytoskeleton is a dynamic composite network of interacting biopolymers, including semiflexible actin filaments, rigid microtubules, and intermediate filaments that provide structural and mechanical support to the cells. Associated molecular motors and binding proteins restructure and adapt the cytoskeleton to allow cells to grow, change shape, stiffen, move, and even self-heal - enabling myriad cellular processes ranging from migration and division to mechanosensing. Beyond its significance in cellular biophysics, the cytoskeleton is also a quintessential example of active matter with potential materials applications ranging from wound healing and drug delivery to filtration and soft robotics. In an effort to dissect and demystify the interactions and contributions from the different cytoskeletal constituents that lead to signature cellular properties, researchers have developed powerful in vitro reconstitution methods to build and study cytoskeleton systems outside the cells. However, due to the complexity and non-equilibrium nature of these systems, as well as the labile nature of their constituents, in vitro reconstitution methods are often difficult to replicate from lab to lab. Assays for characterizing the mechanical and structural properties of reconstituted cytoskeleton systems are likewise inherently complicated and require careful optimization and expertise. This collection highlights the different reconstitution assays and experimental methods that researchers at the forefront of cytoskeleton research are using to recreate and elucidate cytoskeleton systems in an effort to advance biophysics and materials science alike.  

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Featured Method:

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

Sheng Chen1, Zachary Gao Sun2,3, Michael P. Murrell1,2,3

Abstract

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

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Cytoskeleton proteins, actin and tubulin, are two of the most well-characterized proteins and the networks they form have been actively studied over the past decade.  Interestingly, these actin and tubulin networks interact extensively in cells, yet our understanding of how these networks affect the other’s dynamics is just beginning to come to light.  Work by Dr. Ross and Dr. Robertson-Anderson has led the way towards unmasking this mystery.  In their recent study these groups sought to determine how the interactions between actin and microtubule networks impact actomyosin activity.  They utilized cutting-edge technologies like particle image velocimetry and dynamic differential microscopy to measure the network dynamics of actin and microtubules that are co-entangled in the presence of myosin II.  Interestingly they observed that microtubules facilitated organized contraction of actomyosin networks that were otherwise disjointed and displayed disordered dynamics. Importantly, they also observed that these co-entangled networks can undergo ballistic contraction with indistinguishable characteristics. 

Cytoskeleton proteins, actin and tubulin, are two of the most well-characterized proteins and the networks they form have been actively studied over the past decade.  Interestingly, these actin and tubulin networks interact extensively in cells, yet our understanding of how these networks affect the other’s dynamics is just beginning to come to light.  Work by Dr. Ross and Dr. Robertson-Anderson has led the way towards unmasking this mystery.  In their recent study these groups sought to determine how the interactions between actin and microtubule networks impact actomyosin activity.  They utilized cutting-edge technologies like particle image velocimetry and dynamic differential microscopy to measure the network dynamics of actin and microtubules that are co-entangled in the presence of myosin II.  Interestingly they observed that microtubules facilitated organized contraction of actomyosin networks that were otherwise disjointed and displayed disordered dynamics. Importantly, they also observed that these co-entangled networks can undergo ballistic contraction with indistinguishable characteristics. 

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