31 research outputs found
Respective Contributions of Single and Compound Granule Fusion to Secretion by Activated Platelets
Although granule secretion is pivotal in many platelet responses, the fusion routes of α and δ granule release remain uncertain. We used a 3D reconstruction approach based on electron microscopy to visualize the spatial organization of granules in unstimulated and activated platelets. Two modes of exocytosis were identified: a single mode that leads to release of the contents of individual granules and a compound mode that leads to the formation of granule-to-granule fusion, resulting in the formation of large multigranular compartments. Both modes occur during the course of platelet secretion. Single fusion events are more visible at lower levels of stimulation and early time points, whereas large multigranular compartments are present at higher levels of agonist and at later time points. Although α granules released their contents through both modes of exocytosis, δ granules underwent only single exocytosis. To define the underlying molecular mechanisms, we examined platelets from vesicle-associated membrane protein 8 (VAMP8) null mice. After weak stimulation, compound exocytosis was abolished and single exocytosis decreased in VAMP8 null platelets. Higher concentrations of thrombin bypassed the VAMP8 requirement, indicating that this isoform is a key but not a required factor for single and/or compound exocytosis. Concerning the biological relevance of our findings, compound exocytosis was observed in thrombi formed after severe laser injury of the vessel wall with thrombin generation. After superficial injury without thrombin generation, no multigranular compartments were detected. Our studies suggest that platelets use both modes of membrane fusion to control the extent of agonist-induced exocytosis
Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach
Two different zinc oxide nanoparticles, as well as zinc ions, are used to
study the cellular responses of the RAW 264 macrophage cell line. A proteomic
screen is used to provide a wide view of the molecular effects of zinc, and the
most prominent results are cross-validated by targeted studies. Furthermore,
the alteration of important macrophage functions (e.g. phagocytosis) by zinc is
also investigated. The intracellular dissolution/uptake of zinc is also studied
to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve
readily in the cells, leading to high intracellular zinc concentrations, mostly
as protein-bound zinc. The proteomic screen reveals a rather weak response in
the oxidative stress response pathway, but a strong response both in the
central metabolism and in the proteasomal protein degradation pathway. Targeted
experiments confirm that carbohydrate catabolism and proteasome are critical
determinants of sensitivity to zinc, which also induces DNA damage. Conversely,
glutathione levels and phagocytosis appear unaffected at moderately toxic zinc
concentrations
Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses
The molecular responses of macrophages to copper-based nanoparticles have
been investigated via a combination of proteomic and biochemical approaches,
using the RAW264.7 cell line as a model. Both metallic copper and copper oxide
nanoparticles have been tested, with copper ion and zirconium oxide
nanoparticles used as controls. Proteomic analysis highlighted changes in
proteins implicated in oxidative stress responses (superoxide dismutases and
peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and
mitochondrial proteins (especially oxidative phosphorylation complex subunits).
Validation studies employing functional analyses showed that the increases in
glutathione biosynthesis and in mitochondrial complexes observed in the
proteomic screen were critical to cell survival upon stress with copper-based
nanoparticles; pharmacological inhibition of these two pathways enhanced cell
vulnerability to copper-based nanoparticles, but not to copper ions.
Furthermore, functional analyses using primary macrophages derived from bone
marrow showed a decrease in reduced glutathione levels, a decrease in the
mitochondrial transmembrane potential, and inhibition of phagocytosis and of
lipopolysaccharide-induced nitric oxide production. However, only a fraction of
these effects could be obtained with copper ions. In conclusion, this study
showed that macrophage functions are significantly altered by copper-based
nanoparticles. Also highlighted are the cellular pathways modulated by cells
for survival and the exemplified cross-toxicities that can occur between
copper-based nanoparticles and pharmacological agents
Human monocyte-derived dendritic cells turn into foamy dendritic cells with IL-17A
International audienc
Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.
Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes
Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots
International audiencePrimary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet- dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completelyunderstood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery ofretracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots
Lysosomal-Associated Transmembrane Protein 5 (LAPTM5) Is a Molecular Partner of CD1e
<div><p>The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.</p> </div