Epsin N-terminal Homology Domain (ENTH) Activity as a Function of Membrane Tension

Abstract

The epsin N-terminal homology domain (ENTH) is a major player in clathrin-mediated endocytosis. To investigate the influence of initial membrane tension on ENTH binding and activity, we established a bilayer system based on adhered giant unilamellar vesicles (GUVs) to be able to control and adjust the membrane tension covering a broad regime. The shape of each individual adhered GUV as well as its adhesion area was monitored by spinning disc confocal laser microscopy. Control of in a range of 0.08-1.02 mN/m was achieved by altering the Mg 2؉ concentration in solution, which changes the surface adhesion energy per unit area of the GUVs. Specific binding of ENTH to phosphatidylinositol 4,5-bisphosphate leads to a substantial increase in adhesion area of the sessile GUV. At low tension (<0.1 mN/m) binding of ENTH can induce tubular structures, whereas at higher membrane tension the ENTH interaction deflates the sessile GUV and thereby increases the adhesion area. The increase in adhesion area is mainly attributed to a decrease in the area compressibility modulus K A . We propose that the insertion of the ENTH helix-0 into the membrane is largely responsible for the observed decrease in K A , which is supported by the observation that the mutant ENTH L6E shows a reduced increase in adhesion area. These results demonstrate that even in the absence of tubule formation, the area compressibility modulus and, as such, the bending rigidity of the membrane is considerably reduced upon ENTH binding. This renders membrane bending and tubule formation energetically less costly.Clathrin-mediated endocytosis is one of the key metabolic pathways for the uptake of macromolecules into eukaryotic cells (1-4). Driven by a chain of remodeling events and an elaborate set of proteins acting in an orchestrated manner, an almost flat patch of plasma membrane is transformed into a closed, cargo-containing vesicle. As plasma membrane shape transformation is associated with significant local bending of the membrane, the process is highly sensitive to lateral membrane tension. Plasma membrane tension originates from two primary sources; that is, hydrostatic pressure across the lipid bilayer and cytoskeleton-membrane adhesion (5). Depending on the cell type, plasma membrane tensions span a range of roughly 0.003-0.45 mN/m (5-8). Even though it had become clear already in the late 1990s that tension plays a role in exoand endocytosis (9, 10), only in recent years has significant evidence been accumulated that membrane tension is of utmost importance for processes that rely on membrane remodeling (11-14). Cells actively maintain and regulate their membrane tension and use it to control exo-and endocytosis (15). In K562 cells it has been reported that endocytosis is completely suppressed under hypoosmotic conditions (16). Generally, high lateral membrane tension suppresses membrane deformation as both stretching the lipid bilayer and opening bonds between the cytoskeleton and the plasma membrane requires a large amount of energy (17).One protein...

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension

“…1f). This stalling and clustering is consistent with in vitro observation of epsin tubulation at low tension in GUVs [16][17][18][19] . Even though clusters of CCSs which are stalled remained in the TIRF field during the acquisition period, they showed dynamic rearrangement of CCS rings including merging and splitting of rings (Sup.…”

“…An alternate hypothesis proposed recently posits the C-terminus intrinsically disordered protein (IDP) domain of epsin initiates membrane bending via steric crowding 16,17 . In vitro studies have shown that insertion of purified ENTH into giant unilamellar vesicles (GUVs) reduces membrane rigidity and area compressibility modulus of the lipid bilayer 18 . Further, recruitment of ENTH softens the bilayer at high tension and initiates tubulation at low tension 19 .…”

Membrane tension increases fusion efficiency of model membranes in the presence of SNAREs

“…Alternatively, we also used adhered giant unilamellar vesicles (GUVs), where the lateral membrane tension was adjusted through the adhesion of the GUVs with a functionalized glass surface. The adhesion area and thus lateral membrane tension of the GUV was increased through an elevated concentration of Mg 2+ -ions in the buffer leading to stronger attractive forces between the bilayer and the substrate 23 . The vesicle fusion efficiency as a function of the corresponding tension adjusted in the supported lipid bilayer (SLB) and GUV-membrane was monitored by fluorescence microscopy.…”

De novo Sequencing, Assembly, and Annotation of the Transcriptome for the Free‐Living Testate Amoeba Arcella intermedia

“…Epsin action results in tubulation or even vesiculation as a result of membrane bending (Gleisner et al. ). Adaptins (AP‐2) are responsible for the recognition of the particles ingested and retention inside the vesicle (Boehm and Bonifacino ).…”