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

Abstract

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.

Organelle size scaling over embryonic development

“…A variety of new techniques and approaches may lend themselves to studies of organelle size. Bottom-up cellular reconstitution allows for precise control of synthetic cell size and shape to address how these properties influence organelle size scaling (Bermudez, Chen, Einstein, & Good, 2017;Gopfrich, Platzman, & Spatz, 2018;Sonnen & Merten, 2019;Vahey & Fletcher, 2014). Identifying the parts list for a given organelle will facilitate studies of size because the presence and levels of individual components can be precisely modulated.…”

Elucidating nuclear size control in the Xenopus model system

“…Its extensive use has contributed many important discoveries in the fields of molecular biology, cell biology, developmental biology, physiology, and neurobiology ( Dominguez-Sola et al, 2007 ; Fuller et al, 2008 ; Raschle et al, 2008 ; Bajpai et al, 2010 ; Cruciat et al, 2010 ; Hellsten et al, 2010 ; Levy & Heald, 2010 ; Poulsen et al, 2010 ). Advances in existing and emerging technologies, such as microfluidics ( Liu & Singh, 2013 ; Bermudez et al, 2017 ), microscopy ( Zumbusch et al, 2013 ; Puah et al, 2017 ), and high-throughput imaging ( Shachar et al, 2015 ), will offer many new approaches for studying nuclear size regulation in the Xenopus system.…”

Chemical Screening Using Cell-Free Xenopus Egg Extract

“…Other specific XE preparation protocols are available for the study of cytoskeletal dynamics (Lebensohn et al 2006; Maresca and Heald 2006), nucleocytoplasmic transport (Chan and Forbes 2006), DNA replication (Walter et al 1998), nuclear assembly (Newport 1987), nuclear size regulation (Edens and Levy 2016), DNA damage (Willis et al 2012), and apoptosis (Kornbluth and Evans 2001; Deming and Kornbluth 2006). XE can also be used to study signal transduction (see Bermudez et al [2017] and references therein), DNA replication (e.g., Raspelli et al 2017), ubiquitin metabolism (Verma et al 2004), and protein turnover (Shennan 2006). In particular, XE screens have successfully identified small molecule inhibitors of actin polymerization (Peterson et al 2004), proteasomal degradation (Verma et al 2004), cyclin turnover (Salic and King 2005), DNA damage-repair (Landais et al 2009), and Wnt signaling (Thorne et al 2011).…”