Self-contact elimination by membrane fusion

Abstract

Mutual, homophilic cell-cell adhesion between epithelial cells is required for proper maintenance of epithelial barrier function. Whereas opposing membranes from neighboring cells rapidly assemble junctional complexes, self-contacting membranes curiously do not, suggesting that cells have the ability to prevent the maturation of self-junctions. Using a self-contact-inducing microfabricated substrate, we show that self-contacts of normal epithelial cells are rapidly eliminated by membrane fusion between two opposing plasma membranes of a single cell. This membrane fusion is most frequently observed in E-cadherin-expressing epithelial cells, but not in fibroblasts. The efficiency of self-contact elimination depends on extracellular calcium concentration and the level of E-cadherin, suggesting that E-cadherin, although not required, enhances membrane fusion efficiency by bringing opposing membranes into close apposition to one another. Additionally, Rho-associated protein kinase inhibition decreases self-contact-induced membrane fusion of epithelial cells, suggesting that this fusion may be mechanically regulated through the actin-myosin network. This self-contact-induced membrane fusion is a key elimination mechanism for unwanted self-junctions and may be a feature of cell self-recognition.seamless tubule | contractility | soft lithography T he formation of intimate cell-cell adhesion is the first critical step in the development of multicellular organisms. Many cell types, particularly epithelial cells, form and maintain cellcell junctions with neighboring cells through homophilic interactions of cell adhesion proteins. When the plasma membranes of neighboring cells physically interact, cell-cell adhesion proteins engage and autonomously and rapidly develop into mature cell junctions. Interestingly, in native environments, epithelial cells extend thin finger-like protrusions along the basal-lateral surface (1, 2). These flexible membrane extensions are capable of touching different regions of their own membrane surface, yet, mature cell junctional complexes have not been observed at self-contacts. The curious absence of cell junctions at selfcontacts suggests that cells are able to either prevent the autonomous assembly of self-junctions or rapidly eliminate nascent self-junctions.The fate of self-contacts is not entirely clear. During neuronal network formation, thin flexible neurites recognize self-contacts through homophilic interactions of surface receptors and repel one another, whereas heterophilic interactions between neighboring cells are more favorable (3,4). This type of self-avoidance is based on surface chemistry defined by the receptors on plasma membrane. Other adhesive cells that rely on homophilic interactions of adhesion proteins need an alternative strategy to avoid or eliminate self-contacts.Such a self-recognition pathway is essential as many other cell types have the capacity to generate self-contacts. For example, dynamic lamellipodia and membrane ruffles often fold onto adjacent membrane re...

Rho GTPases and the Downstream Effectors Actin-related Protein 2/3 (Arp2/3) Complex and Myosin II Induce Membrane Fusion at Self-contacts

“…Microfabrication of Pillar Array-The polydimethylsiloxane micropillar array was fabricated as previously described using standard soft lithography technique (4). The dimensions of individual pillars were 20 m in height and 5 m in diameter and arranged in a series of hexagons with an 18-m pitch along each hexagon side.…”

“…Quantification of Self-contact-induced Membrane FusionThe method to quantify self-contact-induced membrane fusion has been described previously (4). Briefly, cells on self-contactinducing substrates were trypsinized to remove cell adhesion sites including self-junctions, then stained with phalloidin.…”

“…Furthermore, during neuronal development in C. elegans, overlapping neurites undergo auto-fusion to maintain proper dendritic branching, a process mediated by the fusogen EFF-1 (1). In mammalian epithelial and endothelial cells, self-junctions are removed through self-contact-induced membrane fusion (4). Interestingly, membrane fusion of endothelial cells is a key step in the formation of seamless capillaries (5,6).…”

Cell behaviors and dynamics during angiogenesis

“…Interestingly, recent experiments with MDCK cells have shown that mammalian cells tend to eliminate self-contacts (Sumida and Yamada, 2013), a process that might be similar or identical to the EC self-fusion event occurring during pruning. E-cadherin is strictly required for self-contact elimination in MDCK cells, and further studies have shown that Rho GTPases and the downstream effectors Actin-related protein 2/3 (Arp2/3) complex and Myosin II induce membrane fusion at self-contacts of MDCK cells (Sumida and Yamada, 2015).…”

Time to make the doughnuts: Building and shaping seamless tubes

“…Wrapping and auto-fusion also generate some seamless tubes in the zebrafish vasculature [15] and in mammalian Madin-Darby Canine Kidney (MDCK) epithelial cells grown around silicone pegs [66], suggesting this mechanism may be widely used. MDCK auto-fusion requires cytoskeletal and trafficking regulators such as Rac, Rho, Cdc42 and the Arp2/3 complex [67], but the relevant fusogen is unknown.…”