In Vitro Reconstitution of theEndoplasmic Reticulum

摘要

Reconstitution of cellular organelles in vitro offers the possibility to performquantitative and qualitative experiments in a controlled environment that cannotbe done with the same accuracy in living cells. Following a previous report,the subsequent list of protocols describes how to reconstitute and quantify atubular ER network in vitro based on purified microsomes from culture cellsand cytosol from Xenopus laevis egg extracts. Biological material preparationand reconstitution assays require mostly basic laboratory instrumentation andchemicals, and can be executed without any specific training, making themappealing to a wide range of laboratories. Moreover, to promote conditions thatare markedly more reflective of in vivo environments, this method describes forthe first time in the literature, the purification of microsomes from HeLa cells insome detail. Basic Protocol 1 in this article describes the reconstitution processon different substrates including the associated fluorescence imaging process.Purification of ER microsomes and cytosol, both of which are needed for thisapproach, are described in detail in Support Protocols 1 and 2, respectively.Coating of surfaces with polyacrylamide gels is described in Support Protocol 3.Basic Protocol 2 outlines how to segment and skeletonize fluorescence imagesof ER networks, and how to quantify segment lengths between the network’sbranching points. The described quantitative evaluation provides a meaningfulapproach to analyze the topology and geometry of organelle structures.