ER membranes exhibit phase behavior at sites of organelle contact

Christopher King, Prabuddha Sengupta, Arnold Seo, Jennifer Lippincott-Schwartz

Posted on: 19 November 2019

Preprint posted on 19 July 2019

Article now published in Proceedings of the National Academy of Sciences at

Blowing cells with water enables an easy detection of contacts and phase behavior of membrane bound organelles.

Selected by Berrak Ugur

Categories: biophysics, cell biology

Written in collaboration with Dragomir Milovanovic


 The plasma membrane is constituted from various lipid species of different concentrations and thousands of membrane spanning and membrane associated proteins. Early models suggested that the differences in lipid composition and concentration lead to the formation of liquid-ordered and liquid-disordered regions within the plasma membrane (Singer and Nicolson, 1972, Simons and Ikonen, 1997). Over the decades, it became clear that the protein-protein, protein-lipid, and lipid-lipid interactions are all responsible for the formation of so-called microdomains in the plasma membrane (Sezgin et al., 2017). These microdomains represent signaling hubs on the plasma membrane for diverse cellular functions.

Similar to plasma membrane, the endoplasmic reticulum (ER) membrane is also composed of various lipids with different concentrations that may form microdomains acting as signaling hubs (Sunshine and Iruela-Arispe, 2017). However, the presence and potential function of such microdomains within the ER membranes remain ill-studied.

Key Findings:

Swelling cells with water leads to organelles forming Large Intra-Cellular Vesicles

To study the physical properties of the ER, the authors treated cells with hypotonic media that induces the membrane-bound organelles to transform into Large Intra-Cellular Vesicles (LICVs). To follow the identity of Large Intracellular Vesicles, the authors labelled the organelle with specific markers. For example, they use two different markers for ER, for the ER membrane and ER lumen, respectively. Under normal conditions, these markers are overlapping due to the ER’s tubular nature. However, under hypotonic conditions, the ER transforms into large vesicles resembling the giant unilamellar vesicles prepared in vitro. Consequently, the ER membrane marker forms a circle that is filled with ER luminal marker. This observation indicates that ER membrane integrity is not affected by swelling, as the luminal marker stays intact. Moreover, when photobleached, fluorescence intensity in these vesicular structures does not recover, indicating that swollen ER does not remain in its reticulous and connected structure under hypotonic conditions. When the hypotonic condition is reversed back, the cell can fully recover. These observations provide a framework to study ER dynamics as well as contacts between ER and other membrane-bound organelles.

 Cooling down the cells leads to distinct properties within the ER membrane

To study if the ER is able to phase separate, the authors use two distinct ER markers with different physical properties; one that is an ER-resident transmembrane protein and the other that is an ER-retained protein concentrated at cholesterol-enriched regions. Under normal temperature (37°C), both proteins are homogenously distributed along the ER membrane. However, at low temperature (10°C), the protein that favors cholesterol-enriched environment displays discrete spots with liquid ordered properties. These spots are separated from the otherwise homogenous distribution of the ER-resident protein, indicating that ER is able to phase-separate in a temperature-dependent manner.

Swollen cells are able to maintain inter-organelle contact

 To study if these liquid-order/disorder properties are also present in different organelles, the authors use various organellar markers and show that these properties are present in most of the organelles. During this experiment, the authors also observe that some markers form stable contacts between different organelles, which may be further explored in follow-up studies.

What I liked about this study:

This study provides a simple but elegant method to study phase behavior at membrane contact sites in cells. We enjoyed their refreshing creativity, and several members of our group have already used this method successfully. Therefore, we think that this method will make it easier to study physical properties of membrane contact sites and the protein tethers within cells.

Open Questions:

  1. Compared to the other organelles, the ER is very abundant within cells and forms a network. Is it possible that the ER-LICV are overcrowding and therefore leading to a constriction within cells that promotes more contacts compared to normal conditions?
  1. Is the probability of two organelles contacting each other altered after hypotonic treatment? How to assure that the contact between two organelles is not a consequence of a sudden stress (i.e., strong hypotonic treatment) rather than a depiction of the equilibrium state under physiological conditions?
  1. What is the effect of the hypotonic treatment on the cytoskeleton? Do you think that polymerization-depolymerization of cytoskeletal elements due to shifts in temperature and osmolarity affect LICV formation?
  2. Similarly to question 2, how to assure that a change in osmolarity does not disrupt the protein complexes present in the intracellular membranes (including the enzymes involved in lipid metabolism) thereby changing the distribution of lipids within the membrane? Such an alteration of lipid dynamics would artificially induce phase separation.


Singer, S. J.; Nicolson, G. L. The Fluid Mosaic Model of the Structure of Cell Membranes. Science 1972, 175 (4023), 720–731.

Simons, K.; Ikonen, E. Functional Rafts in Cell Membranes. Nature 1997, 387 (6633), 569–572.

Sezgin E, Levental I, Mayor S, Eggeling C. The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nat Rev Mol Cell Biol. 2017, 18(6):361-374

Sunshine H, Iruela-Arispe ML. Membrane lipids and cell signaling. Curr Opin Lipidol. 2017, 28(5):408-413.

Tags: er, membrane contact sites, phase separation


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