ER-PM contacts regulate apical domain formation in hepatocytes

Gary Hong Chun Chung, Jemima J. Burden, Maëlle Lorvellec, Paul Gissen, Christopher J. Stefan

Posted on: 4 June 2020

Preprint posted on 24 April 2020

Membrane contacts meet cell polarity: ER-PM contacts are asymmetrically distributed in epithelial cells and contribute to lipid organisation

Selected by Helen Zenner

Categories: cell biology, developmental biology

Background

Cells polarise by segregating their plasma membrane and cortical components into distinct domains. Enrichment of phosphatidylinositol 4,5 phosphate (PI(4,5)P₂) and phosphatidylserine (PS) occurs at the forming apical domain in epithelial cells, and then is maintained once the cell is polarised. Both PI(4,5)P₂ and PS are involved in recruiting apical polarity factors, including Cdc42 and Par complex. Surprisingly, how these lipids first accumulate asymmetrically is unknown.

Recently, extensive efforts have been made to characterise the proteins involved in lipid transfer at membrane contact sites throughout the cell. One of the best studied contacts is that between the plasma membrane (PM) and endoplasmic reticulum (ER). Two proteins found at these contacts, extended synaptotagmim protein-1 (E-Syt1) and oxysterol-binding protein related protein 5 (ORP5) have been shown to regulate PI(4,5)P₂ and PS levels, although data for this is mixed. In this preprint, Chung et al. hypothesise that lipid transfer proteins (LTPs) play a role in setting up the lipid asymmetry in epithelial cells. This, of course, would require types of ER-PM contacts, or densities of contacts to be also be asymmetrically distributed within polarised cells.

Main findings

Analysing beautiful electron microscopy (EM) serial sections from mouse hepatocytes, the authors find striking differences in the density of ER-PM between the different plasma membrane domains. The polarity of hepatocytes in a little more complicated than ‘textbook’ epithelial cells but can be divided into equivalent domains of apical (that forms bile canaliculi, BC), lateral (cell-cell junctional zone) and basal. Quantification of the EM images shows 20% of the lateral membrane is associated with ER contact sites, whilst the basal membrane has a mere 5.4%, and the apical membrane only 0.6%. Furthermore, the extent and morphology of each contact varies substantially, where the average contact length on the lateral surface is four times greater than that observed for the apical contacts. Even within the lateral membrane the localisation of the contacts is not uniform suggesting they are under precise regulation. Similar data was observed in mouse renal inner medullary collecting duct spheroids.

Using hepatoblastoma couplets (liver derived cells that form polarised pairs), the authors then checked for a functional role of the LTPs E-Syt1 and ORP5 in apical domain establishment. Upon overexpression of a mutant of E-Syt1 assumed to be defective in Ca²⁺ dependent membrane tethering, fewer developing and mature stage couplets were observed. Coincident with this, intracellular accumulations of PS-positive membrane compartments were observed suggesting that E-Syt1 may be involved in transfer of PS to the plasma membrane. Overexpression of wild type ORP5 also impaired apical (BC) development. This supports a model where E-Syt1 and ORP5 are involved in regulated lipid composition during apical initiation as it is known that overexpression of wild type ORP5 reduces of the presence PI(4,5)P₂ and PI(4)P at the plasma membrane. of impaired apical (BC) development.

Overall this paper provides exciting data suggesting that control of ER-PM contacts may be an upstream component of both polarity initiation and establishment in epithelial cells.

Questions for the authors

Does overexpression of E-Syt1 or Orp5 have any impact of spheroid establishment or maintenance?
The lateral and basal membranes are described in the literature to have similar lipid composition, but from this work very different densities of contacts, why might this be?
Is the size of the contacts constrained by the morphology of the plasma membrane rather than by the composition of contacts?
Why is the density of ER-PM contacts lowest at the apical membrane, where lipid composition is changing most during polarisation?

Tags: er-pm, hepatocytes, membrane contacts, phospholipids, polarity

doi: https://doi.org/10.1242/prelights.21722

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Author's response

Christopher J. Stefan, Jemima J. Burden, Gary Hong Chun Chung shared

Thank you for your interest in our findings, complimentary remark about the beautiful electron microscopy images, and for your thoughtful questions.

We are excited that our initial observations reveal the organisation, distribution, and features of ER-PM contacts are conserved in polarised epithelial cells. This suggests that ER-PM contacts may influence various aspects of epithelial development, function, and possibly even remodelling. By extension, this may imply that proteins that form and function at ER-PM contacts, including the E-Syt proteins and ORP family members, also have universal roles in epithelial cells. However, involvement of the E-Syts and ORPs during epithelial development remains to be validated and it is premature to make strong conclusions. We are further investigating the localisation and roles of the E-Syts and ORPs in epithelial spheroids and tissues. Of course, we are also keen to uncover additional activities taking place at ER-PM contacts during epithelial cell development and function.

As you point out, it is truly striking that the majority of extensive ER-PM contacts are found in the junctional (lateral) domain within a few microns of the tight junction. This must be of significance, but for now we can only speculate. Previous intriguing findings by other groups including Philippe Bastiaens’ have suggested that ER-PM contacts may be enriched at cell-cell junctions. Perhaps inter-cellular signalling pathways regulate where ER-PM contacts form. In other pioneering studies by several groups including Ole Petersen’s, it is now thought that store-operated calcium entry (SOCE) occurs at ER-PM contacts in baso-lateral domains of polarised cells. Calcium may then ‘tunnel’ through the ER lumen to the sub-apical ER network where its release generates a signal for regulated exocytosis at the apical domain. Possibly, clustering of ER-PM contacts just outside the apical domain facilitates fast repetitive responses to secretory stimuli as calcium ions would have a shorter distance to ‘tunnel’. In the discussion of our manuscript, we even speculate that ER-PM contacts may regulate the stability or dynamics of junctional complexes in the lateral domain. We are continuing to investigate how ER-PM might be involved in epithelial tissue remodelling during normal development and disease. While it is tempting to consider each of these interesting possibilities, they are beyond the scope of our initial study and future work is needed to test these ideas in further detail.

In addition to ER-PM contact proteins, PM composition and architecture may dictate ER-PM size and distribution, as you suggest. Indeed, the apical domain has a thick underlying actin cortex that may impede extensive ER-PM contacts in this region. We have also observed examples of mutually exclusive actin-rich ER-free zones and actin-depleted ER-PM contact zones in the basal domains of hepatocytes that neighbour cholangiocytes. Membrane curvature may also be an important determinant. Curiously, the PM is quite planar in lateral domains where extensive ER-PM contacts form. In contrast, the apical PM is highly curved and scarce in ER-PM contacts.

Finally, it should be noted that while ER-PM contacts are rare at the apical domain in fully mature polarised epithelial cells, this may not be the case during early stages of polarisation. For example, our recent findings suggest that E-Syt2 is enriched at sites of PM apposition that become apical and lateral domains. Thus, the E-Syts may act very early on in the control of membrane lipid dynamics in the regulation of epithelial cell polarity.

Thank you again for your interest in our work. We hope our study will also be appreciated by the broad cell biology community. We also look forward to providing more detailed answers to your questions and our speculative ideas in future studies.

Sincerely,

Gary, Jemima, and Chris

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ER-PM contacts regulate apical domain formation in hepatocytes

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