Squid hnRNP protein promotes apical cytoplasmic transport and localization of Drosophila pair-rule transcripts.

Drosophila melanogaster pair-rule segmentation gene transcripts localize apically of nuclei in blastoderm embryos. This might occur by asymmetric (vectorial) export from one side of the nucleus or by transport within the cytoplasm. We have followed fluorescently labeled pair-rule transcripts postinjection into Drosophila embryos. Naked, microinjected fushi tarazu (ftz) transcripts do not localize in blastoderm embryos, indicating that cytoplasmic mechanisms alone are insufficient for apical targeting. However, prior exposure of ftz to Drosophila or human embryonic nuclear extract leads to rapid, specific, microtubule-dependent transport, arguing against vectorial export. We present evidence that ftz transcript localization involves the Squid (Hrp40) hnRNP protein and that the activity of hnRNP proteins in promoting transcript localization is evolutionarily conserved. We propose that cytoplasmic localization machineries recognize transcripts in the context of nuclear partner proteins.

Specific isoforms of squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis.

Heterogeneous nuclear RNA-binding proteins, hnRNPs, have been implicated in nuclear export of mRNAs in organisms from yeast to humans. A germ-line mutation in a Drosophila hnRNP, Squid (Sqd)/hrp40, causes female sterility as a result of mislocalization of gurken (grk) mRNA during oogenesis. Alternative splicing produces three isoforms, SqdA, SqdB, and SqdS. Here we show that these isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in grk mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. We show that this differential nuclear accumulation is correlated with a differential association with the nuclear import protein Transportin. Finally, we provide evidence that grk mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. These results demonstrate the isoform-specific contributions of individual hnRNP proteins in the regulation of a specific mRNA. Moreover, these data suggest a novel role for hnRNPs in localization and translational regulation of mRNAs.

Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells.

Signals transduced through the B cell Ag receptor (BCR) drive B cell development. However, BCR-induced responses are developmentally regulated; immature B cells are tolerized following antigenic exposure while mature B cells are triggered to proliferate and differentiate. This differential responsiveness allows for the negative selection of self-reactive immature B cells while simultaneously allowing for clonal expansion of mature B cells in response to foreign Ags. Intrinsic differences in BCR-induced signal transduction at various stages of development may account for this functional dichotomy. We had previously demonstrated that the BCR-induced proliferation of mature B cells is accompanied by an increase in intracellular calcium levels and polyphosphoinositide bis phosphate (PIP2) hydrolysis. In contrast, immature B cells that undergo BCR-induced apoptosis increase intracellular calcium in the relative absence of PIP2 hydrolysis. Since PIP2 hydrolysis leads to the generation of diacylglycerol, a cofactor for protein kinase C (PKC) activation, these data suggested that an "imbalance" in BCR-induced signal transduction resulting from a relative inability to activate PKC may play a role in the susceptibility of immature B cells to BCR-induced apoptosis. In support of this hypothesis, we demonstrate that PKC activation can rescue immature B cells from BCR-induced apoptosis. Furthermore, the susceptibility of immature B cells to BCR-induced apoptosis is recapitulated in mature B cells that are either PKC depleted or are stimulated in the presence of PKC inhibitors, suggesting that an uncoupling of PKC activation from BCR-induced signaling is responsible for the apoptotic response of immature B cells.

Characterization of anti-single-stranded DNA B cells in a non-autoimmune background.

Anti-DNA Abs are prevalent in the serum of systemic lupus erythematosus (SLE) patients and in the MRL-lpr/lpr mouse model of SLE, but are generally absent in normal individuals. We have studied the regulation of anti-ssDNA B cells in a non-autoimmune (BALB/c) background by using Ig transgenes (Tgs) encoding anti-DNA Abs. In one case, they are present with other non-DNA-binding B cells (the VH3H9 Tg with endogenous light chains); in the other, they are present as an essentially monospecific population (VH3H9/Vkappa8). We have previously observed that serum anti-ssDNA levels in these Tg mice were no higher than those of non-Tg mice, despite the fact that anti-ssDNA B cells dominate the peripheral B cell repertoire. These results suggested that the anti-ssDNA Tg B cells present are functionally inactivated. In this paper, we isolate B cells from VH3H9/Vkappa8 Tg mice to show that this is indeed the case and go on to further define this state. We demonstrate that VH3H9/Vkappa8 Tg B cells have diminished Ig secretion in response to both T-independent and T-dependent stimuli compared with non-Tg controls. VH3H9/Vkappa8 Tg B cells also show suboptimal proliferation in response to anti-IgM F(ab)'2 fragments and LPS, and are phenotypically distinct in expressing decreased total surface Ig. Despite their functional defects, however, VH3H9/Vkappa8 Tg B cells have an in vivo turnover rate comparable to non-Tg B cells, suggesting that they are long lived.

Acquisition of surface IgD fails to protect from tolerance-induction. Both surface IgM- and surface IgD-mediated signals induce apoptosis of immature murine B lymphocytes.

While mature splenic B cells are surface (s)IgM+ and sIgD+, immature, tolerance-susceptible B cells express sIgM and varying levels of sIgD. Differential expression of sIgD on tolerance-susceptible and resistant B cells suggests that sIgM and sIgD may transmit qualitatively different signals. Alternatively, tolerance sensitivity may be associated with intrinsic differences in sIg signaling, regardless of the isotype engaged. Here, we have exploited a stage of B cell development at which immature, tolerance-sensitive B cells co-express sIgD and sIgM. Using these immature stage B cells to evaluate isotypic differences in the ability to induce activation and deletion, we have found that neither ligation of sIgD nor sIgM is capable of inducing proliferation. Moreover, in contrast to mature B cells, both sIgD and sIgM induce apoptosis by immature stage B cells. Importantly, cross-linking sIgD does not protect immature B cells from sIgM-induced apoptosis. Thus, the differences in tolerance susceptibility of immature and mature B cells must be due to intrinsic developmental rather than isotypic differences in Ag receptor signal transduction.

Use of isolated immature-stage B cells to understand negative selection and tolerance induction at the molecular level.

Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.

Engagement of the antigen-receptor on immature murine B lymphocytes results in death by apoptosis.

During their development B lymphocytes pass through a maturational stage in which encounter with Ag leads to tolerance rather than activation. At least four mechanisms for achieving B cell tolerance have been reported: deletion, anergy, receptor editing, and competition for follicular niches. Although turnover rates for immature B cells in the adult mouse bone marrow and several transgenic model systems suggest that a major process contributing to negative selection of B cells is deletion, a detailed study of the negative effect of Ag-receptor engagement on primary, immature B cell survival has never been undertaken. We have utilized an in vitro culture system to determine whether cross-linking sIgM on tolerance-susceptible sIgM+IgD- B cells results in deletion by apoptosis. In contrast to the effect of sIgM cross-linking on mature splenic B cells, treatment of immature, bone marrow-derived B cells results in significant levels of apoptotic death. Ag receptor-mediated apoptosis is detectable by 14 h after sIgM engagement. Moreover, IL-4 and cycloheximide, which have previously been shown to prevent B cell tolerance induction, specifically block the sIgM-induced apoptosis observed in the immature B cells. Similarly, immature B cells from the neonatal spleen are also susceptible to apoptosis after sIgM cross-linking, although they manifest somewhat higher levels of unstimulated apoptosis as compared with bone marrow-derived B cells. These studies are the first detailed demonstration of Ag receptor-mediated apoptosis of primary immature stage B lymphocytes.

Transient transfection of murine B lymphocyte blasts as a method for examining gene regulation in primary B cells.

Studies of the biochemical and genetic processes associated with activation of B lymphocytes have contributed much to the understanding of the regulation of the B cell response to antigen. Primary, non-transformed B cells from the spleen in mice and the tonsils or peripheral blood in humans have proven to be informative models for dissection of the biochemical events leading to B cell activation. In contrast, genetic studies of this process have relied on transformed cell lines grown in culture. The influence of the transformed state on the results obtained using these models may limit their physiological relevance. This report describes a method whereby non-transformed B lymphocytes in primary culture can be transfected for use in studies of gene regulation in response to antigen receptor signals. Transfection was accomplished after only a 72 h exposure to LPS. The cells obtained after LPS treatment were greater than 97% pure, and more importantly, remained responsive to antigen-receptor generated signals. Responsiveness was confirmed by demonstrating induction of mRNA for the primary response gene egr-1, as well as induction of specific transcription factor binding activity in nuclear extracts from these cells. DEAE-dextran-mediated transient transfection was utilized to introduce an egr-1 promoter/reporter construct into these cells. This analysis of promoter activity yielded results which were indistinguishable from the pattern of expression of the endogenous egr-1 gene. Potential applications for dissection of transcriptional regulatory pathways in B lymphocytes are discussed.