Jean-Paul Chauvin

Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains

T‐cell receptors (TCRs) upon binding to peptide–MHC ligands transduce signals in T lymphocytes. Tyrosine phosphorylations in the cytoplasmic domains of the CD3 (γδϵ) and ζ subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP‐70 kinase and other signaling components. We examined the role of the low‐density detergent‐insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine‐phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM‐associated receptor complexes are highly enriched in the hyperphosphorylated p23 ζ chains, contain most of the TCR/CD3‐associated, phosphorylation‐activated ZAP‐70 kinases and seem to integrate into higher order, multiple tyrosine‐phosphorylated substrate‐containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.

TCR signal initiation machinery is pre‐assembled and activated in a subset of membrane rafts

Recent studies suggest that rafts are involved in numerous cell functions, including membrane traffic and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent‐insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37°C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of ∼70 nm. These findings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR–CD3 complex, Lck and ZAP‐70 kinases, and CD4 co‐receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for efficient TCR signal initiation upon stimulation.

Differential vesicular distribution and trafficking of MMP-2, MMP-9, and their inhibitors in astrocytes.

Astrocytes play an active role in the central nervous system and are critically involved in astrogliosis, a homotypic response of these cells to disease, injury, and associated neuroinflammation. Among the numerous molecules involved in these processes are the matrix metalloproteinases (MMPs), a family of zinc‐dependent endopeptidases, secreted or membrane‐bound, that regulate by proteolytic cleavage the extracellular matrix, cytokines, chemokines, cell adhesion molecules, and plasma membrane receptors. MMP activity is tightly regulated by the tissue inhibitors of MMPs (TIMPs), a family of secreted multifunctional proteins. Astrogliosis in vivo and astrocyte reactivity induced in vitro by proinflammatory cues are associated with modulation of expression and/or activity of members of the MMP/TIMP system. However, nothing is known concerning the intracellular distribution and secretory pathways of MMPs and TIMPs in astrocytes. Using a combination of cell biology, biochemistry, fluorescence and electron microscopy approaches, we investigated in cultured reactive astrocytes the intracellular distribution, transport, and secretion of MMP‐2, MMP‐9, TIMP‐1, and TIMP‐2. MMP‐2 and MMP‐9 demonstrate nuclear localization, differential intracellular vesicular distribution relative to the myosin V and kinesin molecular motors, and LAMP‐2‐labeled lysosomal compartment, and we show vesicular secretion for MMP‐2, MMP‐9, and their inhibitors. Our results suggest that these proteinases and their inhibitors use different pathways for trafficking and secretion for distinct astrocytic functions.

Vesicular trafficking and secretion of matrix metalloproteinases-2, -9 and tissue inhibitor of metalloproteinases-1 in neuronal cells.

Matrix metalloproteinases (MMPs) are endopeptidases that cleave matrix, soluble and membrane-bound proteins and are regulated by their endogenous inhibitors the tissue inhibitors of MMPs (TIMPs). Nothing is known about MMP/TIMP trafficking and secretion in neuronal cells. We focussed our attention on the gelatinases MMP-2 and MMP-9, and their inhibitor TIMP-1. MMPs and TIMP-1 fused to GFP were expressed in N2a neuroblastoma and primary neuronal cells to study trafficking and secretion using real time video-microscopy, imaging, electron microscopy and biochemical approaches. We show that MMPs and TIMP-1 are secreted in 160-200 nm vesicles in a Golgi-dependent pathway. These vesicles distribute along microtubules and microfilaments, co-localise differentially with the molecular motors kinesin and myosin Va and undergo both anterograde and retrograde trafficking. MMP-9 retrograde transport involves the dynein/dynactin molecular motor. In hippocampal neurons, MMP-2 and MMP-9 vesicles are preferentially distributed in the somato-dendritic compartment and are found in dendritic spines. Non-transfected hippocampal neurons also demonstrate vesicular secretion of MMP-2 in both its pro- and active forms and gelatinolytic activity localised within dendritic spines. Our results show differential trafficking of MMP and TIMP-1-containing vesicles in neuronal cells and suggest that these vesicles could play a role in neuronal and synaptic plasticity.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre‐clinical stages of Alzheimers disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimers disease. After self‐proteolysis, the N‐terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three‐dimensional X‐ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple‐helical filament (QHF‐litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

Trafficking and secretion of matrix metalloproteinase-2 in olfactory ensheathing glial cells: A role in cell migration?

Olfactory ensheathing cells (OECs) are unique glia found only in the olfactory system. They retain exceptional plasticity and support olfactory neurogenesis and retargeting across the PNS:CNS boundary in the olfactory system. OECs have been shown to improve functional outcome when transplanted into rodents with spinal cord injury. The growth‐promoting properties of implanted OECs encompass their ability to migrate through the scar tissue and render it more permissive for axonal outgrowth, but the underlying molecular mechanisms remain poorly understood. OECs appear to regulate molecules of the extracellular matrix (ECM) that inhibit axonal growth. Among the proteins that have the potential to promote cell migration, axonal regeneration and remodeling of the ECM are matrix metalloproteinases (MMPs), a family of endopeptidases that cleave matrix, soluble, and membrane‐bound proteins and that are regulated by their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs). Little is known about MMP/TIMP trafficking, secretion, and role in OECs. Using a combination of cell biology, biochemistry, pharmacology, and imaging techniques, we show that MMP‐2 and MMP‐9 are expressed and proteolytically active in the olfactory epithelium and in particular in the OECs of the lamina propria. These proteinases and regulatory proteins such as MT1‐MMP and TIMP‐2 are expressed in cultured OECs. MMPs exhibit nuclear localization and vesicular trafficking and secretion, with distribution along microtubules and microfilaments and co‐localization with the molecular motor protein kinesin. Finally, we show that MMPs are involved in migration of OECs in vitro on different ECM substrates.

PSD95β regulates plasma membrane Ca(2+) pump localization at the photoreceptor synapse

At the presynaptic plasma membrane of the photoreceptor the correct localization of the calcium extruder, plasma membrane Ca2+-ATPase (PMCA), is determined by a unique protein complex. Here, the role of two proteins within the complex; membrane palmitoylated protein 4 (MPP4) and postsynaptic density protein 95 (PSD95) is investigated in more details, using Mpp4 and Psd95 mutant mice. MPP4 deficiency results in the loss of both PMCA and PSD95 from the photoreceptor synapse. Truncation of the C-terminal part of MPP4 leads to a loss of PSD95 and mislocalization of PMCA, while truncation of the C-terminal part of PSD95 did not affect the localization of the complex members. Lentivirus-mediated molecular replacement strategy was used to selectively express either PSD95alpha or PSD95beta in wild type or Mpp4 mutant primary retinal explants. Silencing of the Psd95 gene resulted in the loss of presynaptic MPP4 and PMCA1. The plasma membrane localization of MPP4 and PMCA1 could be restored by the expression of PSD95beta. We conclude that both scaffold proteins PSD95beta and MPP4 are essential for the modulation of PMCA levels at the presynaptic plasma membrane and thereby influence the photoreceptor synaptic calcium handling.

Scanning tunneling microscopy of colloidal gold beads

Abstract Biological molecules have been imaged by scanning tunneling microscopy, although the reliability and reproducibility of such images have been questioned. Colloidal gold beads are good conductors and are of well defined size and shape, hence they provide a convenient positive control for scanning tunneling microscopy (STM) studies of biomolecules. Aggregates of 5 and 20 nm beads can be reproducibly imaged on graphite, provided that the tunneling current be kept low (around 30 pA). Isolated beads are unstable under these conditions, suggesting that even if biomolecules do adequately conduct current, they must be attached securely to the substrate in order to be imaged. The height of 20 nm beads was accurately measured by STM, while the width was overestimated due to tip convolution. Platinum/carbon shadowing appeared to have a stabilizing effect and allowed the imaging of isolated 20 nm beads. Shadowing resulted in a 20% decrease in height measurements, whereas the measured width was increased by 20%.

Scanning tunneling microscopy of proteins of the immunoglobulin super family

Three different members of the immunoglobulin super‐family have been studied. An antibody molecule, prototype of the family, directed against Thy‐1, another member of the family, was seen as a V shaped structure, as expected. The corresponding antigen, Thy‐1, was imaged at different resolutions. Then, the complex of the two molecules was analyzed and distinctive structures corresponding to each of the two partners were observed. The third member of the family, CD4, was seen as a string of four beads, linearly disposed. Further work needed to draw more detailed conclusions on this family is in progress.