Characterization ofXenopusRalB and Its Involvement in F-Actin Control during Early Development

Abstract

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.

Control of embryonicXenopusmorphogenesis by a Ral-GDS/Xral branch of the Ras signalling pathway

“…Ral proteins are activated by the Ras effector, Ral-GDS (Kikuchi et al, 1994;Wolthuis et al, 1998b) and have been directly implicated in various cellular mechanisms, such as endocytosis (Nakashima et al, 1999) and regulation of the actin cytoskeleton (Ohta et al, 1999). By microinjecting different mutant forms of XralB into Xenopus embryos, we have shown that expression of an inactivate form of XralB, XralB S28N, leads to abnormal gastrulation movements, whereas a constitutively active form, XralB G23V, causes disruption of the actin cytoskeleton (Moreau et al, 1999).…”

“…cellular morphology via modulation of the cytoskeleton. In previous studies, we have characterised XralB, a small G protein of the Ras family, isolated by differential screening of a subtractive cDNA library (Moreau et al, 1999). This protein cycles between an inactive GDP-bound and an active GTPbound conformation.…”

“…Fixation and staining of embryos with QE5 antibody and rhodamine-phalloidin Embryos were prepared for confocal microscopy analysis as described previously (Moreau et al, 1999).…”

“…Total RNA extraction was carried out as previously described (Moreau et al, 1999), except that extracts were treated with 1 U of DNase I RQ1 (Promega) for 30 minutes at 37°C after the LiCl precipitation step. First strand cDNA was prepared from total RNA using the reverse transcriptase M-MLV (Gibco-BRL, ref.…”

Aggregation of maternal pigment granules is induced by the cytosolic discoidin domain of theXenopus Del1 protein

“…In Xenopus oocytes and eggs, pigment granule localization is controlled by actin fibers (Palecek et al, 1978;Campa et al, 1993;Boyle et al, 2001). However, actin fiber disruption is probably incompatible with normal development (Moreau et al, 1999). Hence, we believe that the XDel1-D2 protein displaces the pigment granules from their normal anchorage site.…”