Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility (SI) is an important mechanism used in many species to prevent inbreeding, and is controlled by a multi-allelic S locus1,2. “Self” (incompatible) pollen is discriminated from “non-self” (compatible) pollen, by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in pollen inhibition and programmed cell death3-7. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS, from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single copy gene linked to the pistil S gene, PrsS. Sequence analysis indicates that PrsS and PrpS are equally ancient and are likely to have co-evolved. PrpS encodes a novel ~20 kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver SI system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between “self” and “non-self”, which also include histocompatibility systems in primitive chordates and vertebrates.
A dynamic network of polymers, the actin cytoskeleton, co-ordinates numerous fundamental cellular processes. In pollen tubes, organelle movements and cytoplasmic streaming, organization of the tip zone, vesicle trafficking, and tip growth have all been linked to actin-based function. Further, during the self-incompatibility response of Papaver rhoeas, destruction of the cytoskeleton is a primary target implicated in the rapid cessation of pollen tube growth and alterations in actin dynamics are associated with the initiation of programmed cell death. Surprisingly, these diverse cellular processes are accomplished with only a small amount of filamentous actin and a huge pool of polymerizable monomers. These observations hint at incredibly fast and complex actin dynamics in pollen. To understand the molecular mechanisms regulating actin dynamics in plant cells, the abundant actin monomer-binding proteins, a major filament nucleator, a family of bundling and severing proteins, and a modulator of growth at the barbed-end of actin filaments have been characterized biochemically. The activities of these proteins are generally consistent with textbook models for actin turnover. For example, the three monomer-binding proteins, profilin, ADF, and CAP, are thought to function synergistically to enhance turnover and the exchange of subunits between monomer and polymer pools. How individual actin filaments behave in living cells, however, remains largely unexplored. Actin dynamics were examined using variable angle epifluorescence microscopy (VAEM) in expanding hypocotyl epidermal cells. Our observations of single filament behaviour are not consistent with filament turnover by treadmilling, but rather represent a novel property called stochastic dynamics. A new model for the dynamic control of actin filament turnover in plant cells is presented.
Background: CTLA-4 is an essential regulator of T cell immune responses with unusual intracellular trafficking.Results: Endocytosis of CTLA-4 is continuous with subsequent recycling and degradation.Conclusion: Clathrin-mediated endocytosis of CTLA-4 persists in activated T cells.Significance: This alters our understanding of CTLA-4 behavior and, therefore, how it might function.
The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott–Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp−/− mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet–platelet interaction and their absence contributes to the bleeding diathesis of Wiskott–Aldrich syndrome.
Essentials Dimeric high‐affinity collagen receptor glycoprotein VI (GPVI) is present on resting platelets.Spatio‐temporal organization of platelet GPVI‐dimers was evaluated using advanced microscopy.Upon platelet adhesion to collagenous substrates, GPVI‐dimers coalesce to form clusters.Clustering of GPVI‐dimers may increase avidity and facilitate platelet activation SummaryBackgroundPlatelet glycoprotein VI (GPVI) binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen, and occurs constitutively on resting platelets.ObjectiveTo identify higher‐order oligomerization (clustering) of pre‐existing GPVI dimers upon interaction with collagen as a mechanism to initiate GPVI‐mediated signaling.Methods GPVI was located by use of fluorophore‐conjugated GPVI dimer‐specific Fab (antigen‐binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III, GPVI‐specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real‐time interactions, and direct stochastic optical reconstruction microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein Ib, integrin α2β1, and phosphotyrosine.ResultsUpon platelet adhesion to all collagenous substrates, GPVI dimers coalesced to form clusters; notably clusters formed along the fibers of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being the most effective. Clusters on fibrinogen‐adhered platelets were much smaller and more numerous; whether these are pre‐existing oligomers of GPVI dimers or fibrinogen‐induced is not clear. Some GPVI dimer clusters colocalized with areas of phosphotyrosine, indicative of signaling activity. Integrin α2β1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton.ConclusionsPlatelet adhesion to collagen induces GPVI dimer clustering. GPVI clustering increases both avidity for collagen and the proximity of GPVI‐associated signaling molecules, which may be crucial for the initiation and persistence of signaling.
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