In Vitro Reconstitution of the Endoplasmic Reticulum

Csilla‐Maria Ferencz1, Gernot Guigas2, Andreas Veres3, Brigitte Neumann4, Olaf Stemmann4, Matthias Weiss3

1 Current address: Max Planck Institute of Colloids and Interfaces, Department of Theory and Bio‐Systems, Potsdam, 2 Current address: Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), Karlsruhe, 3 Department of Experimental Physics I, University of Bayreuth, Bayreuth, 4 Department of Genetics, University of Bayreuth, Bayreuth
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.22
DOI:  10.1002/cpcb.30
Online Posting Date:  September, 2017
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Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, 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 images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.

Keywords: endoplasmic reticulum; self‐assembly; ER reconstitution; in‐vitro assay; ER network; microsomes; quantitative microscopy

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Table of Contents

  • Basic Protocol 1: In‐Vitro Reconstitution of ER Networks
  • Basic Protocol 2: Image Analysis to Quantify Geometrical and Topological Features of Reconstituted Networks
  • Support Protocol 1: Isolation of Endoplasmic Reticulum Microsomes from HeLa Culture Cells
  • Support Protocol 2: Preparation of Xenopus laevis Extract
  • Support Protocol 3: Preparation of PA Hydrogel Substrates of Various Stiffness
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: In‐Vitro Reconstitution of ER Networks

  • 1 mg/ml fibronectin stock solution
  • Tris‐salt buffer (see recipe)
  • Isolated ER microsomes (see protocol 3)
  • Isolated cytosol (see protocol 4)
  • Ice
  • Energy regeneration system to enhance membrane fusion (ATP and GTP as described in Comerford & Dawson, ; Paiement, Beaufay, & Godelaine, ; Watkins, Hermanowski, & Balch, )
  • Membrane dye (e.g., DiIC 18)
  • Silicone oil
  • Glass coverslips
  • µ‐Slide 15‐well (Ibidi), bare or coated with polyacrylamide gel (see protocol 5)
  • 35‐mm ESS microdishes precoated with a gel of 1.5 kPa, 15 kPa, 28 kPa stiffness (Ibidi)
  • Pipets
  • 0.5‐ml microcentrifuge tubes
  • Glass microscopy slides (thickness 150 to 200 µm)
  • Confocal fluorescence microscope, e.g., Leica TCS SP5 confocal laser scanning microscope

Basic Protocol 2: Image Analysis to Quantify Geometrical and Topological Features of Reconstituted Networks

  • Two‐dimensional fluorescence images of ER networks in TIF format (obtained, for example, by protocol 1)
  • Standard computer or laptop running Matlab (The Mathworks)

Support Protocol 1: Isolation of Endoplasmic Reticulum Microsomes from HeLa Culture Cells

  Additional Materials (also see Basic Protocol 3)
  • 145 mm × 20 mm culture dishes containing 90% confluent monolayer HeLa cells
  • Phosphate‐buffered saline (PBS; see recipe), 1×
  • Hypotonic lysis buffer (see recipe)
  • Ultrapure water
  • 70% ethanol
  • Ice
  • Sucrose gradients (see recipe)
  • 25%, 23%, 21%, and 19% iodixanol (see recipe)
  • Liquid nitrogen
  • Cell scraper
  • 15‐ml and 50‐ml of conical Falcon tubes with caps
  • Low‐speed refrigerated centrifuge with swinging‐bucket rotor and adaptors for appropriate Falcon tubes (Heraeus Megafuge 16 Centrifuge)
  • Pipettes
  • 250‐ml glass beakers
  • Bandelin Sonopuls with microtip MS 73, diameter 3 mm
  • Kimwipes
  • 5‐ml centrifuge tubes
  • 14‐ml thin‐walled, polypropylene, ultracentrifuge tubes
  • Beckman ultracentrifuge with SW‐41Ti swinging‐bucket rotor
  • 5‐ml syringe equipped with a 19‐G needle
  • 7‐ml, 150 K MWCO, Pierce Protein concentrator
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and immunblotting (Gallagher, )

Support Protocol 2: Preparation of Xenopus laevis Extract

  • Xenopus laevis frogs
  • 1000 i.U./ml human Chorionic Gonadotropin (hCG; Sigma) in H 2O
  • 200 U/ml pregnant mare chorionic gonadotropin (PMSG; Sigma) in H 2O
  • MMR (see recipe)
  • Dejellying solution (see recipe)
  • CSF XB buffer (see recipe)
  • 10 mg/ml cytochalasin B (Sigma) in dimethyl sulfoxide (DMSO)
  • 25× CaCl 2 (see recipe)DAPI‐fix (see recipe)
  • Liquid nitrogen
  • 27‐G needle
  • 200‐ml crystallizing dishes with spout
  • Pasteur pipets
  • Vortex mixer
  • 4‐ml Beckman ultra‐clear centrifuge tubes
  • Beckman centrifuge with JS13.1 swinging bucket rotor
  • 4‐ml thin‐walled, polypropylene tubes + custom made adaptors for JS13.1 rotor
  • 18‐G needles
  • 5‐ml syringes
  • Glass slides and coverslips
  • Fluorescence microscope
  • 6‐ml thin‐walled, polypropylene SW‐60Ti ultracentrifuge tubes
  • Beckman ultracentrifuge with SW‐60Ti swinging‐bucket rotor

Support Protocol 3: Preparation of PA Hydrogel Substrates of Various Stiffness

  • 40% (w/v) acrylamide stock solution
  • 2% (w/v) bis‐acrylamide stock solution
  • 10% (w/v) Ammonium persulfate stock solution (APS)
  • Tetramethylethylenediamine solution (TEMED)
  • Phosphate‐buffered saline (PBS; see recipe), 1×
  • 2‐ml tubes
  • Vortex mixer
  • 15‐well µ‐slide (Ibidi)
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Literature Cited

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