The threshold, extent and termination of TCR activation is controlled in part by inhibitory co‐receptors expressed on activated T cells. The lymphocyte activation gene product (LAG‐3), a ligand for MHC class II molecules co‐caps with the CD3/TCR complex and inhibits cell proliferation and cytokine secretion in response to CD3 signaling. We first investigated whether LAG‐3 is localized in activated T cells in detergent‐resistant membrane rafts enriched in glycosphingolipids and cholesterol. We showed that both LAG‐3 and MHC class II are present in the cell fraction of glycosphingolipid‐rich complexes (GSL complexes) before the assembly of the immunological synapse by CD3/TCR complex cross‐linking. Using the LAG‐3 intracytoplasmic region as bait in the yeast two‐hybrid cloning system, we next identified a novel protein termed LAP for LAG‐3‐associated protein. LAP is encoded by a 1.8‐kb RNA message in lymphocytes and encodes a 45‐kDa protein that is expressed in most tissues. We showed that LAP binds specifically in vitro and in vivo to the Glu‐Pro (EP) repeated motif present in the LAG‐3 intracytoplasmic region. LAP also binds to the EP motif of another functionally important receptor, the PDGFR. Thus, LAP is a candidate molecule for a new type of signal transduction and/or coupling of clustered rafts to the microtubule networks that could explain how negative signaling of co‐receptors may occur through molecules devoid of any immunoreceptor tyrosine‐based inhibitory motif consensus sequence.

We have isolated from a subtractive cDNA library of Xenopus laevis a novel transcript, H2A.ZI, which belongs to the H2A.Z variant gene family. Characterization of its expression during oogenesis and development shows significant differences from the expression of the core histone H2A. First, H2A.ZI mRNA is mainly detected only during oogenesis and after the midblastula transition, whereas H2A is constitutively expressed, at much higher levels, throughout embryonic growth. Second, in contrast with H2A, the variant H2A.ZI is polyadenylated during development. Third, expression of H2A.ZI is uncoupled from the S phase after gastrula, whereas synthesis of the core histone H2A mRNA is tightly controlled to DNA replication. Interestingly, H2A.ZI is less charged in the N-terminal tail which is crucial for chromatin-mediated repression. The characteristics of H2A.ZI suggest that its incorporation into nucleosomes would lead to a chromatin structure more competent for gene expression during development.

The Ras protein activates at least three different pathways during early development. Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein. From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral. Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton. To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB. We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton. In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP. We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.

We describe the characterization and a functional analysis in Xenopus development of RalB, a small G protein. RalB RNA and protein are detectable during oogenesis and early development, but the gene is expressed only weakly in adult tissues. The RalB transcripts are processed by poly(A) extension during oocyte maturation and up to the gastrulation stage. Microinjection of wild-type or mutant RalB RNAs was performed in fertilized eggs in order to gain insight into the function of RalB during development. We show that during cleavage stages the activated GTP form of RalB specifically induces a cortical reaction that affects the localization of pigment granules. The use of different drugs suggests that this reaction is dependent on the outer cortical actin array. The relation between F-actin and RalB was shown by confocal analysis. Injection of mRNAs encoding the mutated activated form of RalB leads, at dependent doses, to a blocking of gastrulation or defects in closing of neural folding structures. In contrast, the inactivated form blocks only the closing of neural tube. Altogether, these observations suggest that RalB is part of a regulatory pathway that may affect the blastomere cytoskeleton and take part in early development.