Role of BMP receptor traffic in synaptic growth defects in an ALS model.

TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to amyotrophic lateral sclerosis (ALS) and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is believed to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it is unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. Here we found that both loss and gain of function of TDP-43 in Drosophila cause a reduction of synaptic growth-promoting bone morphogenic protein (BMP) signaling at the neuromuscular junction (NMJ). Further, we observed a shift of BMP receptors from early to recycling endosomes and increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution and suppressed BMP signaling, synaptic growth, and larval crawling defects. Our results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment.

Quantitative super-resolution imaging with qPAINT.

Counting molecules in complexes is challenging, even with super-resolution microscopy. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative points accumulation in nanoscale topography (qPAINT), works independently of dye photophysics for robust counting with high precision and accuracy over a wide dynamic range. qPAINT was benchmarked on DNA nanostructures and demonstrated for cellular applications by quantifying proteins in situ and the number of single-molecule FISH probes bound to an mRNA target.

The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function.

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. Here, we show that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. Our results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR and uncover new roles for an EHD protein at synapses.

Induction of cyclin D2 in rat granulosa cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from Smad.

Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.

Follicle-stimulating hormone activation of hypoxia-inducible factor-1 by the phosphatidylinositol 3-kinase/AKT/Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) pathway is necessary for induction of select protein markers of follicular differentiation.

We sought to elucidate the role of AKT in follicle-stimulating hormone (FSH)-mediated granulosa cell (GC) differentiation. Our results define a signaling pathway in GCs whereby the inactivating phosphorylation of tuberin downstream of phosphatidylinositol (PI) 3-kinase/AKT activity leads to Rheb (Ras homolog enriched in brain) and subsequent mTOR (mammalian target of rapamycin) activation. mTOR then stimulates translation by phosphorylating p70 S6 kinase and, consequently, the 40 S ribosomal protein S6. Activation of this pathway is required for FSH-mediated induction of several follicular differentiation markers, including luteinizing-hormone receptor (LHR), inhibin-alpha, microtubule-associated protein 2D, and the PKA type IIbeta regulatory subunit. FSH also promotes activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). FSH-stimulated HIF-1 activity is inhibited by the PI 3-kinase inhibitor LY294002, the Rheb inhibitor FTI-277 (farnesyltransferase inhibitor-277), and the mTOR inhibitor rapamycin. Finally, we find that the FSH-mediated up-regulation of reporter activities for LHR, inhibin-alpha, and vascular endothelial growth factor is dependent upon HIF-1 activity, because a dominant negative form of HIF-1alpha interferes with the up-regulation of these genes. These results show that FSH enhances HIF-1 activity downstream of the PI 3-kinase/AKT/Rheb/mTOR pathway in GCs and that HIF-1 activity is necessary for FSH to induce multiple follicular differentiation markers.