Mihaela serpe - US grants

BMPs were initially isolated from bone extracts based on their ability to promote bone formation. Today these bone induction activities are sought after in several clinical applications;for example, ectopic applications of recombinant BMPs have increased to some extent the success of dental implants. The clinical applications are however limited by our poor understanding of the mechanisms for localization and concentration of BMP activities. We think that various strategies utilized by the fruit fly to ensure formation of robust/reliable BMP morphogen gradients over relatively long distances may offer exquisite solutions. The Drosophila embryo uses a gradient of Decapentaplegic (Dpp), a homologue of the vertebrate BMP-2/-4, to specify the dorsal structures. In the early embryo, dpp is transcribed uniformly throughout the dorsal domain, yet it forms an activity gradient in which only about 10% cells along the dorsal midline receive high levels of signal and specify the amnioserosa. In the pupal wing, Dpp diffuses from the longitudinal veins into the posterior crossvein competent zone and creates a corridor of peak signaling that is perpendicular to the source of morphogen. In both instances, the formation of the Dpp gradient occurs at a post-transcriptional level and involves modulation by additional secreted gene products. In the early embryo, Dpp is bound in a complex containing Short gastrulation (Sog), a BMP-binding protein secreted from the ventral lateral regions. This complex inhibits binding of Dpp to its receptors in lateral regions but, at the same time, it facilitates long-range ligand diffusion, shuttling Dpp from the lateral domain towards the midline. A critical component that helps create flux and provides directionality is the processing of Sog by Tolloid (Tld), a metalloprotease of the BMP-1 family expressed in the dorsal domain. Tld cleaves Sog when complexed with Dpp and releases the ligand. The net movement of Dpp dorsally is generated by reiterated cycles of complex formation, diffusion and destruction by Tld. Sog plays both positive and negative roles in regulating BMP activity. The negative role comes from blocking access of ligands to receptors. The positive effect comes from its ability to facilitate Dpp diffusion. Without Sog there is no net movement of Dpp dorsally, the peak signaling domain does not form, the amnioserosa is not specified, and the embryos fail to develop and die. Interestingly, Chordin, the vertebrate orthologue of Sog, can only act as an inhibitor when expressed in Drosophila and cannot promote long range Dpp signaling. At the molecular level, the difference between Sog and Chordin is that processing of Sog by Tld requires the BMP ligand as an obligatory co-substrate while Chordin does not. To determine the source of this difference, we modeled the Tld catalytic domain in Drosophila using the crystal structure of the catalytic domain of human Tld. We purified and sequenced the Sog cleavage fragments and derived a consensus cleavage recognition sequence. We used this peptide to study the enzyme-substrate interactions in Sog and compared them with Chordin sequences. From this modeling, we hypothesized that several residues at the processing site might be responsible for making one substrate dependent on BMP binding for processing while the other is not. Our working hypothesis is that Sogs ability to function in a transport process as a long range BMP agonist resides, in molecular terms, in the BMPs co-substrate requirements for Tld mediated Sog degradation. This hypothesis has been supported with computational modeling by our mathematician collaborator. Modeling indicates that the co-substrate requirement for Sog processing by Tld is critical for proper Dpp gradient formation. In computations that relax this constraint and allow for Sog degradation when not complexed with Dpp, Dpp flux towards the dorsal miidline is greatly reduced. To test this hypothesis we first generated Sog variants that are BMP-independent Tld substrates in vitro (Sog-i variants). To study their in vivo effects onto the BMP signaling we created transgenic lines that express Sog under its own endogenous enhancer. Lines expressing wild-type Sog did rescue null sog mutants or trans-heterozygous combinations (sog-/-) to full viability and fertility. In contrast, lines expressing Sog-i variants rescued only partially, and only when present as two or more copies. We then analyzed the profile of the peak BMP signaling domain, by following the activated/phosphorylated effector of the BMP signaling pathway in Drosophila (P-Mad), and the cell fate (amnioserosa cells). In wild-type embryos the P-Mad positive domain is narrow (8-10 cells diameter) and intense. In heterozygous (sog+/-) embryos the P-Mad domain is wider and reduced in relative intensity (by 30%), though it does reach the threshold required to specify amnioserosa cells. Due to the widening of their P-Mad peak domain, sog+/- heterozygous embryos have an average of 314 amnioserosa cells, significantly more than the wild-type embryos (197 cells). The amnioserosa is an extraembryonic membrane that could vary in size (150 to 350 cells) and still perform its function during morphogenetic movements. When we substituted the endogeneous sog for sog-wt transgenes, the profile of the BMP gradient as well as the cell fate were fully rescued, in a concentration dependent manner. More specifically, using several independent transgenes, we found that sog-/- bearing one copy of the sog-wt transgene ressembled sog+/- embryos, while sog-/- bearing two copies of sog-wt had wild-type-like P-Mad profile and amnioserosa. When several independent sog-i transgenes were similarly tested, we found that addition of 2 copies of sog-i in sog-/- background produced a wide, dim and irregularly shaped P-Mad domain, and amnioserosa fields of 294 cells. Our results show that several residues at the processing site are responsible for making Sog (and not Chordin) dependent on BMP binding for its processing and degradation. Mutations of these residues render Sog co-substrate independent for processing by Tld, and alter the in vivo range of the Sog-i-Dpp complexes and consequently the Dpp morphogen gradient profile. Computational modeling predicts that additional copies of sog-i may expand the range of the Sog-i-Dpp complexes and possibly rescue the Dpp gradient profile. We are now in the process of constructing and analyzing sog-/- strains bearing multiple copies of sog-i transgenes.

At the Drosophila NMJ, the subunits that form the glutamate-gated ion channels (iGluRs) are known and relatively well studied. However the mechanisms that control iGluRs clustering and stabilization at the postsynaptic densities, a key step that confers functionality to the nascent synapse, remain a mystery. Several mechanisms have been identified that regulate the subunit compositions and the extent of iGluRs synaptic localization, but no molecules other than the receptors themselves were shown to be absolutely required for clustering of the receptor complexes. In recent studies we have discovered that the neuropillin and tolloid-like protein, Neto, is an essential component for the clustering of the iGluRs at the Drosophila NMJ. Drosophila Neto has two vertebrate homologs called Neto-1 and -2, which have been recently shown to modulate the pharmacological properties of selective kainate-type receptors. Neto1/Neto2 double knockout mice have defects in long term potentiation, and in learning and memory. The neto genes code for transmembrane proteins that contain CUB repeats in their extracellular part. CUB domains are developmentally important protein-protein interaction motifs, which are also present in Tld/BMP-1. To study the function of Drosophila Neto, we generated an allelic series using transposable elements located within the neto locus. Several imprecise excisions were isolated and were molecularly characterized. neto36 is likely a genetic null;neto36 hemizygous embryos do not hatch into the larval stage and die as late embryos that are completely paralyzed, and lack any body wall peristalsis and hatching movements. neto109 lacks the start codon-containing exon and shows 50% lethality. The adult escapers do not fly and have locomotor defects. Neto activity is essential in the striated muscle, since the lethality and the locomotor defects of neto mutant animals can be fully rescued when Neto is expressed in the muscle. Intriguingly, muscle expression of Neto rescued the completely paralyzed neto36 embryos to viable, fertile adults. Neto is present at two distinct locations in the striated muscle on the muscle surface, 1) in a striped pattern reminiscent of the T-tubules, and 2) concentrated at the NMJ. At the NMJ, Neto co-localizes with the ionotropic glutamate receptors (iGluRs) in puncta juxtaposed to sites of neurotransmitter/ glutamate release, the active zones. The iGluRs of the Drosophila NMJ are hetero-tetrameric complexes composed of three essential subunits IIC, IID, IIE, and either IIA or IIB. Embryos without any of the essential subunits, or without IIA and IIB together, are paralyzed and cannot hatch into the larval stages. The essential subunits are not only required for viability, but also for the clustering of the other iGluRs at the NMJ. The similarities in subcellular localizations and loss-of-function phenotypes of Neto and iGluRs raised the possibility that Neto may be important for iGluRs clustering at the NMJ. Through histological and physiological studies we found that Neto is required for clustering of iGluRs at the onset of synaptogenesis, as well as during later developmental stages. iGluRs begin to concentrate at the synaptic cleft soon after contact between the motoneuron growth cone and muscle. However, iGluRs do not cluster in the absence of Neto and remain scattered in small, extrasynaptic aggregates. These defects were not caused by inappropriate axon guidance or lack of adhesion since the pre- and postsynaptic partners appeared properly aligned. The synapse pre-patterning is also normal, as seen by the accumulation of pre-synaptic Bruchpilot (Brp), an active zone protein, and post-synaptic P21 activating kinase (PAK) clusters. The lethality and iGluRs clustering defects in neto null mutants are completely rescued when Neto activity is provided in the striated muscle. The extent of iGluRs clustering at the NMJ was also markedly reduced later in development in third instar larvae for neto hypomorphs. In these larvae, Neto itself forms a drastically reduced number of junctional clusters that co-localized with iGluRs. In neto mutants the level of receptors appeared unchanged by Western analysis, but their immunoreactivity at the NMJ is much decreased and instead shifted to extrajunctional locations. The PAK signals at individual PSDs were also strongly reduced in neto109 larvae. The presence of PAK clusters at the pre-patterning stage in neto null embryos and lack thereof in larval synapses with suboptimal Neto suggest a deficit in the maintenance of mature PSDs in neto mutants. A similar deficit was reported for NMJ synapses developing in the near absence of iGluRs. In contrast, the presynaptic release sites appeared normal at suboptimal levels of Neto or iGluRs. Physiological studies confirmed that neto mutations significantly impair the number and density of postsynaptic iGluRs without an apparent effect on presynaptic release. In collaboration with Bing Zhang at University of Oklahoma, we recorded evoked excitatory junctional potentials (EJPs) and spontaneous miniature potentials (mEJPs) at body-wall muscles of the third instar larvae. Both the frequency and amplitude of miniature synaptic potentials are reduced in neto mutants, but with no apparent defects in presynaptic release. To understand how Neto controls the clustering of iGluRs, we explored the timing of Neto clustering at the NMJ using live imaging of embryos with muscle expressed Neto-eGFP. Neto accumulates and clusters at the NMJ as early as 14 hours after egg laying, at the time when the iGluR receptors begin to accumulate and cluster at the synapse. Most importantly, Neto positive puncta could no longer be detected in gluRIID-/- null embryos. Thus, Neto and iGluRs depend on each other for clustering at the NMJ. Basically, Neto functions as a non-channel, essential subunit of the iGluR complexes. Our data fit best with the model in which Neto and the iGluRs are engaged in targeting each other to the PSDs via a direct interaction. A newly formed PSD grows by a continuous incorporation of iGluRs likely derived from cell-wide plasma membrane pools via lateral diffusion. Several lines of evidence point to a rate-limiting role of iGluR levels in NMJ formation. By controlling the extent of the iGluR clustering, Neto itself is rate limiting and appears to directly impact synapse formation. Thus Netos constitute a family of proteins conserved from flies to humans that influence the function of glutamatergic synapses, and have acquired species- and tissue-specific roles during evolution.

Bone morphogenetic proteins (BMPs) are potent secreted signaling factors that function at long- and short-range to impact critical cellular responses during development and homeostasis. Long-range signaling is key to the formation and function of morphogen gradients. Such gradients control the cell fate and tissue allocation and influence the patterning of early embryos as well as later developmental processes. Shot-range signaling sculpts cellular junctions and has been implicated in the growth, development and homeostasis of synaptic junctions, such as Drosophila neuromuscular junction (NMJ). The fly NMJ is a glutamatergic synapse similar in composition and physiology to mammalian central synapses. The fact that individual NMJs can be reproducibly identified from animal to animal and are easily accessible for electrophysiological and optical analysis makes this genetic model system uniquely suited for in vivo studies on synapse assembly, growth and plasticity. In flies, BMP signaling is critical for NMJ growth and neurotransmitter release, and has been implicated in synapse homeostasis via unknown mechanisms. BMP signals via (i) canonical pathway, which activates transcriptional programs with distinct roles in the structural and functional development of the NMJ in response to accumulation of phosphorylated Smad (pMad) in motor neuron nuclei; and (ii) noncanonical, Mad-independent pathway, which connects synaptic structures to microtubules to regulate synapse stability. Intriguingly, pMad also accumulates at synaptic locations but the biological relevance of this phenomenon remained a mystery for over a decade. In recent work we discovered that synaptic pMad is selectively lost at synapses with reduced levels of postsynaptic ionotropic glutamate receptors (iGluRs). Moreover, mutants that lack a particular receptor subtype, GluRIIA, exhibit complete loss of synaptic pMad signals. In contrast, the pMad-positive signals persist in the motor neuron nuclei of GluRIIA mutant animals, and expression of BMP target genes remains unaffected, indicating a specific impairment in the pMad production/ maintenance at synaptic terminals. More importantly, the accumulation of synaptic pMad followed the activity and not the net levels of GluRIIA-containing iGluR complexes (type-A iGluRs). Thus, synaptic pMad appears to function as a local sensor for NMJ synapse activity. Our findings indicate that synaptic pMad marks a completely novel, noncanonical BMP pathway that is genetically distinguishable from all other known BMP signaling cascades. Unlike the BMP retrograde signaling pathway, this novel pathway does not require the BMP7 ortholog, Glass bottom boat (Gbb), but depends on presynaptic BMP receptors and postsynaptic type-A iGluRs. Super resolution studies revealed that synaptic pMad localizes in large clusters at the active zone, in close proximity to the presynaptic membrane, and in perfect juxtaposition with each postsynaptic density. Since pMad is relatively short lived, synaptic pMad likely represents pMad that is locally generated/ maintained by active BMP/ BMP receptor complexes, protected from endocytosis. Intriguingly, selective disruption of presynaptic pMad reduces the postsynaptic levels of type-A receptors, indicating that synaptic pMad functions to stabilize active type-A receptors at synaptic locations. This positive feedback loop provides a molecular switch controlling which flavor of glutamate receptors will be stabilized at synaptic locations as a function of synapse status. We are currently investigating the mechanisms underlying the ability of synaptic pMad to function as an acute sensor and modulator for postsynaptic activity. Since BMP signaling also controls NMJ growth and stability, BMPs may offer an exquisite means to monitor the status of synapse activity and coordinate NMJ growth with synapse maturation and stabilization.