The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function

Proper synaptic development is critical for establishing all aspects of neural function including learning, memory, and locomotion. Here, we describe the phenotypic consequences of mutations in the wishful thinking (wit) gene, the Drosophila homolog of the vertebrate BMP type II receptor. Mutations in wit result in pharate lethality that can be rescued by expression of a wit transgene in motor neurons but not in muscles. Mutant larvae exhibit small synapses, severe defects in evoked junctional potentials, a lower frequency of spontaneous vesicle release, and an alteration in the ultrastructure of synaptic active zones. These results reveal a novel role for BMP signaling in regulating Drosophila neuromuscular junction synapse assembly and activity and may indicate that similar pathways could govern vertebrate synapse development.     

Platelet factor 4 selectively inhibits binding of TGF-beta 1 to the type I TGF-beta 1 receptor.

A low molecular weight inhibitor of TGF-beta 1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF-beta 1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF-beta 1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross-linking experiments revealed that these differences in the inhibition of TGF-beta 1 binding by platelet factor 4 were due to differences in the complements of TGF-beta 1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF-beta 1 was cross-linked to a complex which had an apparent molecular weight of 70 kDa. TGF-beta 1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF-beta 1 binding properties, we believe this protein is the type I TGF-beta 1 receptor. Hep 3B cells also had a high-affinity TGF-beta 1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF-beta 1 receptor. TGF-beta 1 binding to this protein was not inhibited by platelet factor 4. TGF-beta 1 was also cross-linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF-beta 1 receptor. In NRK 49F cells, the majority of bound TGF-beta 1 was cross-linked to a high molecular weight complex which probably represented the type III TGF-beta 1 receptor. NRK 49F cells also had type I TGF-beta 1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF-beta 1 binding, however, the overall effects of platelet factor 4 on TGF-beta 1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross-linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF-beta 1 binding by competition for binding to the type I receptor. Modest concentrations of TGF-beta 1 reduced the adherence of Hep 3B cells to tissue culture dishes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mad2 and spindle assembly checkpoint function during meiosis I in mammalian oocytes

During mammalian mitosis, a proofreading network called the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation. An inhibitory SAC signal is deputed to inhibits mitotic cell-cycle progression in response to misaligned chromosomes until such imperfections are rectified thereby ensuring equitable chromosome partitioning to daughter cells. Amongst the cast of SAC proteins, mitotic arrest deficient 2 (Mad2) plays a leading role in transducing the SAC signal. The aneuploidy and cancer predispositions of individuals who harbour genetic mutations in SAC genes emphasise the in vivo significance of this surveillance mechanism. In humans, congenital aneuploidies such as Down's syndrome demonstrate an exponential increase with advancing female age. Although largely the result of female meiosis I errors, the molecular entities that succumb with age in oocytes remain elusive. Declining oocyte SAC function could plausibly contribute to such errors. Until recently however, convincing evidence for a functional SAC in mammalian oocytes during meiosis I was unforthcoming. Here I review the evidence regarding the SAC in female mammalian meiosis I and how our understanding of this system has evolved in recent years. This review will focus on Mad2 as this is the SAC protein that has been most comprehensively investigated.     

Roles of SNARE proteins and synaptotagmin I in synaptic transmission: studies at the Drosophila neuromuscular synapse

The roles of SNARE proteins, i.e. neuronal Synaptobrevin (n-Syb), SNAP-25 and Syntaxin 1A (Syx 1A), and Synaptotagmin I (Syt I) in synaptic transmission have been studied in situ using mutant embryos or larvae that lack these molecules or have alterations in them. Because of the ease of genetic manipulation, the Drosophila neuromuscular synapse is widely used for these studies. The functional properties of synaptic transmission have been studied in mutant embryos using the patch-clamp technique, and in larvae by recording with microelectrodes. A major vesicular membrane protein, n-Syb, is indispensable for nerve-evoked synaptic transmission. Spontaneous synaptic currents (minis), however, are present even in embryos totally lacking n-Syb (N-SYB). Furthermore, Ca(2+)-independent enhancement of mini frequency induced by hypertonic sucrose solutions (hypertonicity response) is totally absent in N-SYB. Embryos that have defects in SNAP-25 (SNAP-25) have similar but milder phenotypes than N-SYB. The phenotype in synaptic transmission was most severe in the synapse lacking Syx 1A. Neither nerve-evoked synaptic currents nor minis occur in embryos lacking Syx 1A (SYX 1A). No hypertonicity response was observed in them. Syt I binds Ca(2+) in vitro and probably serves as a Ca(2+) sensor for nerve-evoked synaptic transmission, since nerve-evoked synaptic currents were greatly reduced in embryos lacking Syt I (SYT I). Also, Syt I has a role in vesicle recycling. Interestingly, the Ca(2+)-independent hypertonicity response is also greatly reduced in SYT I. Minis persist in mutant embryos lacking any of these proteins (n-Syb, SNAP-25 and Syt I), except Syx, suggesting that minis have a distinct fusion mechanism from that for fast and synchronized release. It appears that these SNARE proteins and Syt I are coordinated for fast vesicle fusion. Minis, on the other hand, do not require SNARE complex nor Syt I, but Syx is absolutely required for vesicle fusion. The SNARE complex and Syt I are indispensable for the hypertonicity response. None of these molecules seem to serve for selective docking of synaptic vesicles to the release site. For further studies on synaptic transmission, the Drosophila neuromuscular synapse will continue to be a useful model.     

In the absence of type III receptor, the transforming growth factor (TGF)-beta type II-B receptor requires the type I receptor to bind TGF-beta2

Transforming growth factor beta (TGF-beta) ligands exert their biological effects through type II (TbetaRII) and type I receptors (TbetaRI). Unlike TGF-beta1 and -beta3, TGF-beta2 appears to require the co-receptor betaglycan (type III receptor, TbetaRIII) for high affinity binding and signaling. Recently, the TbetaRIII null mouse was generated and revealed significant non-overlapping phenotypes with the TGF-beta2 null mouse, implying the existence of TbetaRIII independent mechanisms for TGF-beta2 signaling. Because a variant of the type II receptor, the type II-B receptor (TbetaRII-B), has been suggested to mediate TGF-beta2 signaling in the absence of TbetaRIII, we directly tested the ability of TbetaRII-B to bind TGF-beta2. Here we show that the soluble extracellular domain of the type II-B receptor (sTbetaRII-B.Fc) bound TGF-beta1 and TGF-beta3 with high affinity (K(d) values = 31.7 +/- 22.8 and 74.6 +/- 15.8 pm, respectively), but TGF-beta2 binding was undetectable at corresponding doses. Similar results were obtained for the soluble type II receptor (sTbetaRII.Fc). However, sTbetaRII.Fc or sTbetaRII-B.Fc in combination with soluble type I receptor (sTbetaRI.Fc) formed a high affinity complex that bound TGF-beta2, and this complex inhibited TGF-beta2 in a biological inhibition assay. These results show that TGF-beta2 has the potential to signal in the absence of TbetaRIII when sufficient TGF-beta2, TbetaRI, and TbetaRII or TbetaRII-B are present. Our data also support a cooperative model for receptor-ligand interactions, as has been suggested by crystallization studies of TGF-beta receptors and ligands. Our cell-free binding assay system will allow for testing of models of receptor-ligand complexes prior to actual solution of crystal structures.     

Drosophila liprin-alpha and the receptor phosphatase Dlar control synapse morphogenesis

Here, we examine the synaptic function of the receptor protein tyrosine phosphatase (RPTP), Dlar, and an associated intracellular protein, Dliprin-alpha, at the Drosophila larval neuromuscular junction. We show that Dliprin-alpha and Dlar are required for normal synaptic morphology. We also find that synapse complexity is proportional to the amount of Dlar gene product, suggesting that Dlar activity determines synapse size. Ultrastructural analysis reveals that Dliprin-alpha and Dlar are required to define the size and shape of the presynaptic active zone. Accordingly, there is a concomitant decrease in synaptic transmission in both mutants. Finally, epistasis analysis indicates that Dliprin-alpha is required for Dlar's action at the synapse. These data suggest a model where Dliprin-alpha and Dlar cooperate to regulate the formation and/or maintenance of a network of presynaptic proteins.     

Ontogeny and localization of TGF-beta type I receptor expression during lung development

Transforming growth factor (TGF)-beta is a family of multifunctional cytokines controlling cell growth, differentiation, and extracellular matrix deposition in the lung. The biological effects of TGF-beta are mediated by type I (TbetaR-I) and II (TbetaR-II) receptors. Our previous studies show that the expression of TbetaR-II is highly regulated in a spatial and temporal fashion during lung development. In the present studies, we investigated the temporal-spatial pattern and cellular expression of TbetaR-I during lung development. The expression level of TbetaR-I mRNA in rat lung at different embryonic and postnatal stages was analyzed by Northern blotting. TbetaR-I mRNA was expressed in fetal rat lungs in early development and then decreased as development proceeded. The localization of TbetaR-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with an antisense RNA probe. TbetaR-I mRNA was present in the mesenchyme and epithelium of gestational day 14 rat lungs. An intense TbetaR-I signal was observed in the epithelial lining of the developing bronchi. In gestational day 16 lungs, the expression of TbetaR-I mRNA was increased in the mesenchymal tissue. The epithelium in both the distal and proximal bronchioles showed a similar level of TbetaR-I expression. In postnatal lungs, TbetaR-I mRNA was detected in parenchymal tissues and blood vessels. We further studied the expression of TbetaR-I in cultured rat lung cells. TbetaR-I was expressed by cultured rat lung fibroblasts, microvascular endothelial cells, and alveolar epithelial cells. These studies demonstrate a differential regulation and localization of TbetaR-I that is different from that of TbetaR-II during lung development. TbetaR-I, TbetaR-II, and TGF-beta isoforms exhibit distinct but overlapping patterns of expression during lung development. This implies a distinct role for TbetaR-I in mediating TGF-beta signal transduction during lung development.     

Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription

Brinker is a nuclear protein that antagonizes Dpp signalling in Drosophila. Its expression is negatively regulated by Dpp. Here, we show that Brinker represses Ultrabithorax (Ubx) in the embryonic midgut, a HOX gene that activates, and responds to, the localized expression of Dpp during endoderm induction. We find that the functional target for Brinker repression coincides with the Dpp response sequence in the Ubx midgut enhancer, namely a tandem of binding sites for the Dpp effector Mad. We show that Brinker efficiently competes with Mad in vitro, preventing the latter from binding to these sites. Brinker also competes with activated Mad in vivo, blocking the stimulation of the Ubx enhancer in response to simultaneous Dpp signalling. These results indicate how Brinker acts as a dominant repressor of Dpp target genes, and explain why Brinker is a potent antagonist of Dpp.     

Cell adhesion molecules in Drosophila synapse development and function

Synapse is a highly specialized inter-cellular structure between neurons or between a neuron and its target cell that mediates cell-cell communications. Ample results indicate that synaptic adhesion molecules are critically important in modulating the complexity and specificity of the synapse. And disruption of adhesive properties of synapses may lead to neurodevelopmental or neurodegenerative diseases. In this review, we will use the Drosophila NMJ as a model system for glutamatergic synapses to discuss the structure and function of homophilic and heterophilic synaptic adhesion molecules with special focus on recent findings in neurexins and neuroligins in Drosophila.     

From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function

One of the unique characteristics of higher organisms is their ability to learn and adapt to changes in their environment. This plasticity is largely a result of the brain's ability to convert transient stimuli into long-lasting alterations in neuronal structure and function. This process is complex and involves changes in receptor trafficking, local mRNA translation, protein turnover, and new gene synthesis. Here, we review how neuronal activity triggers calcium-dependent gene expression to regulate synapse development, maturation, and refinement. Interestingly, many components of the activity-dependent gene expression program are mutated in human cognitive disorders, which suggest that this program is essential for proper brain development and function.