Bermudez JG et al. (2017), Probing the biology of cell boundary conditions...

XB-ART-52997

Genesis 2017 Jan 01;551-2:. doi: 10.1002/dvg.23013.

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Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

Bermudez JG , Chen H , Einstein LC , Good MC .

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Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery.

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Species referenced: Xenopus laevis
Genes referenced: acta1

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Figure 1. Xenopus cell-free egg cytoplasmic extracts and strategies for 2D confinement or 3D encapsulation. (A) Eggs collected from the frog, Xenopus laevis, are fractionated to generate cell-free cytoplasm. (B) The cytoplasmic extract is capable of carrying out complex cell biological processes in vitro in the absence of cell boundaries. Microliter volumes of cytoplasm are often activated in test tubes and imaged on microscope slides. (C) Configuration for confinement of cellular reactions to a supported lipid bilayer surrounded by cytoplasm. The process of interest is initiated at or signals to a two-dimensional membrane whose composition is controllable. (D) Encapsulation of cytoplasmic extract reactions within cell-like boundaries. The stiffness of these boundaries varies: PDMS wells (rigid), water-in-oil emulsion (intermediate, lipid monolayer), or liposomes (soft, lipid bilayers). Importantly, compartment dimensions can be specified to encapsulate cell-size volumes of material; from picoliters to nanoliters

Figure 2. Encapsulation of Xenopus cytoplasmic extracts in microfabricated chambers and cell-like compartments. (A) Growth of radial microtubule asters is restricted by the droplet boundary in cytoplasm-filled water-in-oil emulsions. Below a threshold emulsion size, the microtubule growth causes the aster structure to buckle. (B) Spatial control of actin nucleation. Boundary-localized ActA initiates cortical actin polymerization in a cytoplasm-filled water-in-oil emulsions. (C) Nucleus growth scales with the cross-sectional area of the microchannel in which it is confined. (D) Metaphase spindle length is regulated by cytoplasmic volume. This scaling effect, in water-in-oil emulsions, is independent of compartment geometry. (E) Cell cycle duration is dependent on spatial position within cytoplasm-filled tubing. Citations: i: Pinot et al. (2009), ii: Jimenez et al. (2011), iii: Shah et al. (2014), iv: Pinot et al. (2012), v: Hara & Merten (2015), vi: Good et al. (2016), vii: Good et al. (2013), viii: Hazel et al. (2013), ix: Chang & Ferrell (2013). Images courtesy of Z. Gueroui, K. Keren, C. Merten, J. Ferrell.

References [+] :

Abu Shah, Reconstitution of cortical actin networks within water-in-oil emulsions. 2015, Pubmed, Xenbase