Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquidordered raft phases in model membranes. Interestingly, TCR activation domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate the accumulation of specific molecular lipid species with the specific plasma membrane condensation at sites of TCR activation and with early TCR activation responses.

N-WASP is critical for cancer cell invasion through its promotion of the trafficking and capture of MT1-MMP in invasive pseudopods.

WASH coats mature lysosomes and is required for exocytosis of indigestible material.

Highlights d Fast-moving cells in 3D matrix establish low membrane tension at the rear d Caveolae form in response to low membrane tension and recruit the GEF Ect2 d Ect2 activates RhoA to promote F-actin organization and rear retraction d Positive feedback between membrane tension and contractility reinforces retraction

SummaryThe actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for a5b1 integrin recycling. WASH regulates the trafficking of endosomal a5b1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.

Lateral segregation of cell membrane components gives rise to microdomains with a different structure within the membrane. Most prominently, lipid rafts are defined as domains in liquid ordered phase whereas surrounding membranes are more fluid. Here we review a 2-photon fluorescence microscopy approach, which allows the visualization of membrane fluidity. The fluorescent probe Laurdan exhibits a blue shift in emission with increasing membrane condensation caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. The quantification of membrane order is achieved by the Generalized Polarization (GP) values, which are defined as normalized intensity ratios of two emission channels. GP images are therefore not biased by probe concentrations and membrane ruffles. Furthermore, Laurdan reports membrane structure independently from the lipid and protein cargo of the membrane domains. We give examples where Laurdan microscopy was instrumental in quantifying the formation of condensed membrane domains and their cellular requirements. Moreover we discuss how microdomains identified by Laurdan microscopy are consistent with domains identified by other methodologies and put GP images in the context of current raft hypotheses.

Cell migration controls developmental processes (gastrulation and tissue patterning), tissue homeostasis (wound repair and inflammatory responses), and the pathobiology of diseases (cancer metastasis and inflammation). Understanding how cells move in physiologically relevant environments is of major importance, and the molecular machinery behind cell movement has been well studied on 2D substrates, beginning over half a century ago. Studies over the past decade have begun to reveal the mechanisms that control cell motility within 3D microenvironments – some similar to, and some highly divergent from those found in 2D. In this review we focus on migration and invasion of cells powered by actin, including formation of actin-rich protrusions at the leading edge, and the mechanisms that control nuclear movement in cells moving in a 3D matrix.

The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.