Although the highly dynamic and mosaic organization of the plasma membrane is well-recognized, depicting a resolved, global view of this organization remains challenging. We present an analytical single-particle tracking (SPT) method and tool, multiple-target tracing (MTT), that takes advantage of the high spatial resolution provided by single-fluorophore sensitivity. MTT can be used to generate dynamic maps at high densities of tracked particles, thereby providing global representation of molecular dynamics in cell membranes. Deflation by subtracting detected peaks allows detection of lower-intensity peaks. We exhaustively detected particles using MTT, with performance reaching theoretical limits, and then reconnected trajectories integrating the statistical information from past trajectories. We demonstrate the potential of this method by applying it to the epidermal growth factor receptor (EGFR) labeled with quantum dots (Qdots), in the plasma membrane of live cells. We anticipate the use of MTT to explore molecular dynamics and interactions at the cell membrane.

Variations in receptor number at a given synapse are known to contribute to synaptic plasticity, but methods used to establish this idea usually do not allow for the determination of the dynamics of these phenomena. We used single-particle tracking to follow in real time, on the cell surface, movements of the glycine receptor (GlyR) with or without the GlyR stabilizing protein gephyrin. GlyR alternated within seconds between diffusive and confined states. In the absence of gephyrin, GlyR were mostly freely diffusing. Gephyrin induced long confinement periods spatially associated with submembranous clusters of gephyrin. However, even when most receptors were stabilized, they still frequently made transitions through the diffusive state. These data show that receptor number in a cluster results from a dynamic equilibrium between the pools of stabilized and freely mobile receptors. Modification of this equilibrium could be involved in regulation of the number of receptors at synapses.

we explore a new cellular crosstalk between CAFs and tumor cells, based on extracellular vesicles (EVs), and investigate the inhibition of a related candidate therapeutic target as well as detecting it in serum as a diagnostic biomarker to discriminate PDA grade.

Glutamate receptors are clustered at the membrane through interactions with intracellular scaffolding proteins and cytoskeletal elements but can also be found in intracellular compartments or dispersed in the membrane. This distribution results from an equilibrium between the different pools of receptors whose dynamic is poorly known. The group I metabotropic glutamate receptor 5 (mGluR5) is concentrated in an annulus around the postsynaptic density but also found in large amounts in the extrasynaptic membrane. To analyze the dynamic of stabilization of mGluR5, we used single-particle tracking, force measurements, and fluorescence recovery to measure the mobility of mGluR5. We found that receptor activation increases receptor diffusion, whereas the scaffolding protein Homer favors confinement of receptor movements within clusters of Homer-mGluR5. However, this stabilization is reversible, because even in the presence of Homer, receptors still enter and exit from clusters at fast rates. Furthermore, clusters themselves are highly dynamic both in their movements and in their composition, which can vary within tens of seconds. Thus, exchange of receptors between dispersed and clustered states is fast and regulated during physiological processes. These properties may explain certain fast changes in receptor composition observed at postsynaptic densities.

SummarySignalling molecules integrate, codify and transport information in cells. Organisation of these molecules in complexes and clusters improves the efficiency, fidelity and robustness of cellular signalling. Here, we summarise current views on how signalling molecules assemble into macromolecular complexes and clusters and how they use their physical properties to transduce environmental information into a variety of cellular processes. In addition, we discuss recent innovations in live-cell imaging at the sub-micrometer scale and the challenges of object (particle) tracking, both of which help us to observe signalling complexes and clusters and to examine their dynamic character.

Components of the evolutionarily conserved developmental planar cell polarity (PCP) pathway were recently described to play a prominent role in cancer cell dissemination. However, the molecular mechanisms by which PCP molecules drive the spread of cancer cells remain largely unknown. PRICKLE1 encodes a PCP protein bound to the promigratory serine/threonine kinase MINK1. We identify RICTOR, a member of the mTORC2 complex, as a PRICKLE1-binding partner and show that the integrity of the PRICKLE1-MINK1-RICTOR complex is required for activation of AKT, regulation of focal adhesions, and cancer cell migration. Disruption of the PRICKLE1-RICTOR interaction results in a strong impairment of breast cancer cell dissemination in xenograft assays. Finally, we show that upregulation of PRICKLE1 in basal breast cancers, a subtype characterized by high metastatic potential, is associated with poor metastasis-free survival.

αβT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αβT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αβT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αβT cell lineages, notably the induction of Foxp3+ regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αβT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αβT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire−/− mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3+ regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αβT cell lineages to access the thymic medulla.

The thymus ensures the generation of a functional and highly diverse T-cell repertoire. The thymic medulla, which is mainly composed of medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), provides a specialized microenvironment dedicated to the establishment of T-cell tolerance. mTECs play a privileged role in this pivotal process by their unique capacity to express a broad range of peripheral self-antigens that are presented to developing T cells. Reciprocally, developing T cells control mTEC differentiation and organization. These bidirectional interactions are commonly referred to as thymic crosstalk. This review focuses on the relative contributions of mTEC and DC subsets to the deletion of autoreactive T cells and the generation of natural regulatory T cells. We also summarize current knowledge regarding how hematopoietic cells conversely control the composition and complex three-dimensional organization of the thymic medulla.