CD82 and Gangliosides Tune CD81 Membrane Behavior.

Tetraspanins are a family of transmembrane proteins that form a network of protein-protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM.

Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell. Specifically assembly and release of HIV-1 particles require specific cellular components, which are recruited to the egress site by the viral protein Gag. We previously demonstrated that HIV-1 assembly alters both partitioning and dynamics of the tetraspanins CD9 and CD81, which are key players in many infectious processes, forming enriched areas where the virus buds. In this study we correlated super resolution microscopy mapping of tetraspanins with membrane topography delineated by atomic force microscopy (AFM) in Gag-expressing cells. We revealed that CD9 is specifically trapped within the nascent viral particles, especially at buds tips, suggesting that Gag mediates CD9 and CD81 depletion from the plasma membrane. In addition, we showed that CD9 is organized as small membrane assemblies of few tens of nanometers that can coalesce upon Gag expression.

RNA Polymerase Pausing during Initial Transcription.

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

Automatic detection of diffusion modes within biological membranes using back-propagation neural network.

BACKGROUND: Single particle tracking (SPT) is nowadays one of the most popular technique to probe spatio-temporal dynamics of proteins diffusing within the plasma membrane. Indeed membrane components of eukaryotic cells are very dynamic molecules and can diffuse according to different motion modes. Trajectories are often reconstructed frame-by-frame and dynamic properties often evaluated using mean square displacement (MSD) analysis. However, to get statistically significant results in tracking experiments, analysis of a large number of trajectories is required and new methods facilitating this analysis are still needed. RESULTS: In this study we developed a new algorithm based on back-propagation neural network (BPNN) and MSD analysis using a sliding window. The neural network was trained and cross validated with short synthetic trajectories. For simulated and experimental data, the algorithm was shown to accurately discriminate between Brownian, confined and directed diffusion modes within one trajectory, the 3 main of diffusion encountered for proteins diffusing within biological membranes. It does not require a minimum number of observed particle displacements within the trajectory to infer the presence of multiple motion states. The size of the sliding window was small enough to measure local behavior and to detect switches between different diffusion modes for segments as short as 20 frames. It also provides quantitative information from each segment of these trajectories. Besides its ability to detect switches between 3 modes of diffusion, this algorithm is able to analyze simultaneously hundreds of trajectories with a short computational time. CONCLUSION: This new algorithm, implemented in powerful and handy software, provides a new conceptual and versatile tool, to accurately analyze the dynamic behavior of membrane components.

TOM1L1 drives membrane delivery of MT1-MMP to promote ERBB2-induced breast cancer cell invasion.

ERBB2 overexpression in human breast cancer leads to invasive carcinoma but the mechanism is not clearly understood. Here we report that TOM1L1 is co-amplified with ERBB2 and defines a subgroup of HER2(+)/ER(+) tumours with early metastatic relapse. TOM1L1 encodes a GAT domain-containing trafficking protein and is a SRC substrate that negatively regulates tyrosine kinase signalling. We demonstrate that TOM1L1 upregulation enhances the invasiveness of ERBB2-transformed cells. This pro-tumoural function does not involve SRC, but implicates membrane-bound membrane-type 1 MMP (MT1-MMP)-dependent activation of invadopodia, membrane protrusions specialized in extracellular matrix degradation. Mechanistically, ERBB2 elicits the indirect phosphorylation of TOM1L1 on Ser321. The phosphorylation event promotes GAT-dependent association of TOM1L1 with the sorting protein TOLLIP and trafficking of the metalloprotease MT1-MMP from endocytic compartments to invadopodia for tumour cell invasion. Collectively, these results show that TOM1L1 is an important element of an ERBB2-driven proteolytic invasive programme and that TOM1L1 amplification potentially enhances the metastatic progression of ERBB2-positive breast cancers.

TspanC8 tetraspanins differentially regulate the cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane compartmentalization.

The metalloprotease ADAM10 mediates the shedding of the ectodomain of various cell membrane proteins, including APP, the precursor of the amyloid peptide Aß, and Notch receptors following ligand binding. ADAM10 associates with the members of an evolutionary conserved subgroup of tetraspanins, referred to as TspanC8, which regulate its exit from the endoplasmic reticulum. Here we show that 4 of these TspanC8 (Tspan5, Tspan14, Tspan15 and Tspan33) which positively regulate ADAM10 surface expression levels differentially impact ADAM10-dependent Notch activation and the cleavage of several ADAM10 substrates, including APP, N-cadherin and CD44. Sucrose gradient fractionation, single molecule tracking and quantitative mass-spectrometry analysis of the repertoire of molecules co-immunoprecipitated with Tspan5, Tspan15 and ADAM10 show that these two tetraspanins differentially regulate ADAM10 membrane compartmentalization. These data represent a unique example where several tetraspanins differentially regulate the function of a common partner protein through a distinct membrane compartmentalization.

Identification of a region critical for proteolysis of the human growth hormone receptor.

Release of soluble growth hormone binding protein (GHBP) corresponding to the extracellular domain of the GH receptor (GHR) occurs via distinct mechanisms depending on species. In human, proteolysis of full length GHR results in liberation of GHBP into the extracellular medium. A putative protease responsive for GHR cleavage has been identified, however, the residues involved are still unknown. In this study, using the mutational approach to the extracellular domain of the human GHR, we demonstrated that deletion of three residues located close to the transmembrane domain abolishes constitutive GHBP shedding without change in cellular GH binding. Deletion also significantly decreased the phorbol 12-myristate 13-acetate (PMA)-induced release of GHBP and the accumulation of membrane-anchored remnant proteins. Taken together, these results suggest that integrity of the juxtamembrane region of GHR is necessary for its biochemical cleavage and that a common mechanism is involved in constitutive and PMA-induced shedding.