RESUMEN
Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/fisiología , Endosomas/metabolismo , Cinesinas/fisiología , Unión Neuromuscular/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Cinesinas/genética , Neuronas Motoras/metabolismo , Terminales Presinápticos/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Sinapsis/metabolismoRESUMEN
Cytoplasmic polyadenylation element binding protein (CPEB) provides temporal and spatial control of protein synthesis required for early development and neuronal synaptic plasticity. CPEB regulates protein expression by inhibiting polyadenylation of selected mRNA transcripts, which prevents binding of the ribosome for protein synthesis. Two RNA recognition motif domains and a C-terminal binuclear zinc-binding domain are required for mRNA binding, but the zinc-binding domain is not required for sequence-specific recognition of the targeted mRNA transcript. The structure and function of the zinc-binding domain of CPEB are unknown. The C-terminal region of CPEB may participate in assembly of the ribonucleoprotein complex that includes the scaffold protein, Symplekin, and the cleavage and polyadenylation specificity factor. Sumoylation of Symplekin is required for polyadenylation, and both cleavage and polyadenylation specificity factor and poly(A) polymerase are sumoylated. The foreshortened poly(A) tail is maintained by poly(A) ribonuclease, which associates with CPEB. While zinc-binding domains are renowned for nucleic acid recognition, binuclear zinc-binding structural motifs, such as LIM (Lin-11, Isl-1, Mec-3), RING (really interesting new gene), PHD (plant homeodomain) and ZZ (ZZ-type zinc finger) domains, participate in protein-protein interactions. Here, we report the solution structure of the C-terminal zinc-binding domain of CPEB1 (CPEB1-ZZ), which has a cross-braced zinc binding topology. The structural similarity to other ZZ domains suggests that the CPEB1-ZZ domain recruits sumoylated proteins during assembly of the ribonucleoprotein complex prior to mRNA export from the nucleus.
Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Zinc/metabolismoRESUMEN
Homeostatic mechanisms operate to stabilize synaptic function; however, we know little about how they are regulated. Exploiting Drosophila genetics, we have uncovered a critical role for the target of rapamycin (TOR) in the regulation of synaptic homeostasis at the Drosophila larval neuromuscular junction. Loss of postsynaptic TOR disrupts a retrograde compensatory enhancement in neurotransmitter release that is normally triggered by a reduction in postsynaptic glutamate receptor activity. Moreover, postsynaptic overexpression of TOR or a phosphomimetic form of S6 ribosomal protein kinase, a common target of TOR, can trigger a strong retrograde increase in neurotransmitter release. Interestingly, heterozygosity for eIF4E, a critical component of the cap-binding protein complex, blocks the retrograde signal in all these cases. Our findings suggest that cap-dependent translation under the control of TOR plays a critical role in establishing the activity dependent homeostatic response at the NMJ.
Asunto(s)
Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/fisiología , Homeostasis/fisiología , Unión Neuromuscular/metabolismo , Proteínas Quinasas/metabolismo , Transmisión Sináptica/fisiología , Animales , Drosophila , Proteínas de Drosophila/genética , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/fisiología , Exocitosis/fisiología , Larva/metabolismo , Neuronas Motoras/metabolismo , Mutación , Proteínas Quinasas/genética , Transporte de Proteínas/fisiología , Receptores de Glutamato/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TORRESUMEN
Emerging data implicate microRNAs (miRNAs) in the regulation of synaptic structure and function, but we know little about their role in the regulation of neurotransmission in presynaptic neurons. Here, we demonstrate that the miR-310-313 cluster is required for normal synaptic transmission at the Drosophila larval neuromuscular junction. Loss of miR-310-313 cluster leads to a significant enhancement of neurotransmitter release, which can be rescued with temporally restricted expression of mir-310-313 in larval presynaptic neurons. Kinesin family member, Khc-73 is a functional target for miR-310-313 as its expression is increased in mir-310-313 mutants and reducing it restores normal synaptic function. Cluster mutants show an increase in the active zone protein Bruchpilot accompanied by an increase in electron dense T bars. Finally, we show that repression of Khc-73 by miR-310-313 cluster influences the establishment of normal synaptic homeostasis. Our findings establish a role for miRNAs in the regulation of neurotransmitter release.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , MicroARNs/genética , Unión Neuromuscular/genética , Transmisión Sináptica/genética , Animales , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Cinesinas/metabolismo , Larva/genética , Larva/metabolismo , Neuronas Motoras/metabolismo , Familia de Multigenes/genética , Unión Neuromuscular/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Retrograde signaling is essential for coordinating the growth of synaptic structures; however, it is not clear how it can lead to modulation of cytoskeletal dynamics and structural changes at presynaptic terminals. We show that loss of retrograde bone morphogenic protein (BMP) signaling at the Drosophila larval neuromuscular junction (NMJ) leads to a significant reduction in levels of Rac GEF Trio and a diminution of transcription at the trio locus. We further find that Trio is required in motor neurons for normal structural growth. Finally, we show that transgenic expression of Trio in motor neurons can partially restore NMJ defects in larvae mutant for BMP signaling. Based on our findings, we propose a model in which a retrograde BMP signal from the muscle modulates GTPase activity through transcriptional regulation of Rac GEF trio, thereby regulating the homeostasis of synaptic growth at the NMJ.