We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell–cell adhesion. The force required to separate E-cadherin–expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin–expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin–based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.

Acute metabolic changes in plasma membrane (PM) lipids, such as those mediating signalling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the extended synaptotagmins (E-Syts), endoplasmic reticulum (ER) proteins that function as PtdIns(4,5)P₂- and Ca²⁺-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro, and this transfer requires Ca²⁺ and their lipid-harbouring SMP domain. Genome-edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but exhibit enhanced and sustained accumulation of PM diacylglycerol following PtdIns(4,5)P₂ hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syt-dependent ER–PM tethers in response to stimuli that cleave PtdIns(4,5)P₂ and elevate Ca²⁺ may help reverse accumulation of diacylglycerol in the PM by transferring it to the ER for metabolic recycling.

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows that Arf1/COPI can bud nano-LDs (∼60 nm diameter) from phospholipid-covered oil/water interfaces in vitro. We show that Arf1/COPI proteins localize to cellular LDs, are sufficient to bud nano-LDs from cellular LDs, and are required for targeting specific TG-synthesis enzymes to LD surfaces. Cells lacking Arf1/COPI function have increased amounts of phospholipids on LDs, resulting in decreased LD surface tension and impairment to form bridges to the ER. Our findings uncover a function for Arf1/COPI proteins at LDs and suggest a model in which Arf1/COPI machinery acts to control ER-LD connections for localization of key enzymes of TG storage and catabolism.

Facilitating Fusion and Release: For neurotransmitters to be released from a neuron, a synaptic vesicle must fuse with the plasma membrane and form a fusion pore. Soluble N -ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes are the core protein machinery involved in fusion; however, there has been debate regarding the mechanism. While in vitro studies have found that a single SNARE can cause fusion of two bilayers, in vivo studies have shown that a minimum of three SNAREs are required for neurotransmitter release. The apparently contradictory results are explained by Shi et al. (p. 1355 ), who monitored fusion of vesicles to membrane nanodiscs. While a single SNARE complex was sufficient to fuse bilayers, a minimum of three SNARE complexes were required to open a pore large enough for neurotransmitter release.