Analysis of transient membrane protein interactions by single-molecule diffusional mobility shift assay

Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.

A glucose-starvation response regulates the diffusion of macromolecules

The organization and biophysical properties of the cytosol implicitly govern molecular interactions within cells. However, little is known about mechanisms by which cells regulate cytosolic properties and intracellular diffusion rates. Here, we demonstrate that the intracellular environment of budding yeast undertakes a startling transition upon glucose starvation in which macromolecular mobility is dramatically restricted, reducing the movement of both chromatin in the nucleus and mRNPs in the cytoplasm. This confinement cannot be explained by an ATP decrease or the physiological drop in intracellular pH. Rather, our results suggest that the regulation of diffusional mobility is induced by a reduction in cell volume and subsequent increase in molecular crowding which severely alters the biophysical properties of the intracellular environment. A similar response can be observed in fission yeast and bacteria. This reveals a novel mechanism by which cells globally alter their properties to establish a unique homeostasis during starvation.

Weak Solutions for a Sixth Order Cahn-Hilliard Type Equation with Degenerate Mobility

We study an initial-boundary problem for a sixth order Cahn-Hilliard type equation, which arises in oil-water-surfactant mixtures. An existence result for the problem with a concentration dependent diffusional mobility in three space dimensions is presented.

Erlins restrict SREBP activation in the ER and regulate cellular cholesterol homeostasis

Cellular cholesterol levels are controlled by endoplasmic reticulum (ER) sterol sensing proteins, which include Scap and Insig-1. With cholesterol sufficiency, Insig inhibits the activation of sterol regulatory element binding proteins (SREBPs), key transcription factors for cholesterol and fatty acid biosynthetic genes, by associating with Scap–SREBP complexes to promote their ER retention. Here we show that the multimeric ER proteins erlins-1 and -2 are additional SREBP regulators. Depletion of erlins from cells grown with sterol sufficiency led to canonical activation of SREBPs and their target genes. Moreover, SREBPs, Scap, and Insig-1 were physically associated with erlins. Erlins bound cholesterol with specificity and strong cooperativity and responded to ER cholesterol changes with altered diffusional mobility, suggesting that erlins themselves may be regulated by cholesterol. Together, our results define erlins as novel cholesterol-binding proteins that are directly involved in regulating the SREBP machinery. We speculate that erlins promote stability of the SREBP–Scap–Insig complex and may contribute to the highly cooperative control of this system.

Role for Actin in the Polarized Release of Rotavirus

ABSTRACT Rotaviruses are characterized by polarized release from the apical side of infected enterocytes, and the rotavirus VP4 spike protein specifically binds to the actin network at the apical pole of differentiated enterocytic cells. To determine the functional consequences of this VP4-actin interaction, fluorescence recovery after photobleaching experiments were carried out to measure the diffusional mobility of VP4 associated with the microfilaments. Results show that VP4 binds to barbed ends of microfilaments by using actin treadmilling. Actin treadmilling inhibition results in the loss of rotavirus apical preferential release, suggesting a major role for actin in polarized rotavirus release.