Single-Molecule Manipulation of Macromolecules on Membranes Using High-Resolution Optical Tweezers

Despite their wide applications into soluble macromolecules, optical tweezers have rarely been used to characterize dynamics of membrane proteins, mainly due to lack of model membranes compatible with optical trapping. Here, we found that optical tweezers can stably trap giant unilamellar vesicles (GUVs) containing iodixanol with controlled membrane tension, which can potentially serve as a model membrane to study dynamics of membranes, membrane proteins, or their interactions. We also observed that small unilamellar vesicles (SUVs) are rigid enough to resist large pulling force and offer potential advantages to pull membrane proteins. To demonstrate the use of both model membranes, we pulled membrane tethers from the trapped GUVs and measured the folding or binding dynamics of a single DNA hairpin or synaptotagmin-1 C2 domain attached to the GUV or SUV with high spatiotemporal resolution. Our methodologies facilitate single-molecule manipulation studies of membranes or membrane proteins using optical tweezers.

Junctophilins: Key Membrane Tethers in Muscles and Neurons

Contacts between the endoplasmic reticulum (ER) and plasma membrane (PM) contain specialized tethering proteins that bind both ER and PM membranes. In excitable cells, ER–PM contacts play an important role in calcium signaling and transferring lipids. Junctophilins are a conserved family of ER–PM tethering proteins. They are predominantly expressed in muscles and neurons and known to simultaneously bind both ER- and PM-localized ion channels. Since their discovery two decades ago, functional studies using junctophilin-deficient animals have provided a deep understanding of their roles in muscles and neurons, including excitation-contraction coupling, store-operated calcium entry (SOCE), and afterhyperpolarization (AHP). In this review, we highlight key findings from mouse, fly, and worm that support evolutionary conservation of junctophilins.

Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of membrane tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. In contrast, we reveal here a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or sustained receptor signaling triggers the depletion of cholesterol and associated complex glycosphingolipids from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of bacterial toxins. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites.

Deficiency of GABARAP but Not Its Paralogs Causes Enhanced EGF-Induced EGFR Degradation

The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking.