Tight control of synapse formation ensures that neurons connect to appropriate targets. In this issue of Neuron, Klassen et al. identify ARL-8 GTPase as a regulator of presynaptic assembly. Without ARL-8, presynaptic material aggregates en route to its destination, suggesting that ARL-8 acts like a dispersant to prevent premature synaptic assembly in the axon.
Importin proteins act both at the nuclear pore to promote substrate entry and in the cytosol during signal trafficking. Here, we describe mutations in the Drosophila gene importin-β11, which has not previously been analyzed genetically. Mutants of importin-β11 died as late pupae from neuronal defects, and neuronal importin-β11 was present not only at nuclear pores but also in the cytosol and at synapses. Neurons lacking importin-β11 were viable and properly differentiated but exhibited discrete defects. Synaptic transmission was defective in adult photoreceptors and at larval neuromuscular junctions (NMJs). Mutant photoreceptor axons formed grossly normal projections and synaptic terminals in the brain, but synaptic arbors on larval muscles were smaller while still containing appropriate synaptic components. Bone morphogenic protein (BMP) signaling was the apparent cause of the observed NMJ defects. Importin-β11 interacted genetically with the BMP pathway, and at mutant synaptic boutons, a key component of this pathway, phosphorylated mothers against decapentaplegic (pMAD), was reduced. Neuronal expression of an importin-β11 transgene rescued this phenotype as well as the other observed neuromuscular phenotypes. Despite the loss of synaptic pMAD, pMAD persisted in motor neuron nuclei, suggesting a specific impairment in the local function of pMAD. Restoring levels of pMAD to mutant terminals via expression of constitutively active type I BMP receptors or by reducing retrograde transport in motor neurons also restored synaptic strength and morphology. Thus, importin-β11 function interacts with the BMP pathway to regulate a pool of pMAD that must be present at the presynapse for its proper development and function.
Cellularization of the Drosophila embryo is the process by which a syncytium of ∼6000 nuclei is subdivided into discrete cells. In order to individualize the cells, massive membrane addition needs to occur by a process that is not fully understood. The exocyst complex is required for some, but not all, forms of exocytosis and plays a role in directing vesicles to appropriate domains of the plasma membrane. Sec5 is a central component of this complex and we here report the isolation of a new allele of sec5 that has a temperature-sensitive phenotype. Using this allele, we investigated whether the exocyst complex is required for cellularization. Embryos from germline clones of the sec5ts1 allele progress normally through cycle 13. At cellularization, however, cleavage furrows do not invaginate between nuclei and consequently cells do not form. A zygotically translated membrane protein, Neurotactin, is not inserted into the plasma membrane and instead accumulates in cytoplasmic puncta. During cellularization, Sec5 becomes concentrated at the apical end of the lateral membranes, which is likely to be the major site of membrane addition. Subsequently, Sec5 concentrates at the sub-apical complex, indicating a role for Sec5 in the polarized epithelium. Thus, the exocyst is necessary for, and is likely to direct, the polarized addition of new membrane during this form of cytokinesis.
Nuclear import is required for communication between the cytoplasm and the nucleus and to enact lasting changes in gene transcription following stimuli. Binding to an Importin-α molecule in the cytoplasm is often required to mediate nuclear entry of a signaling protein. As multiple isoforms of Importin-α exist, some may be responsible for the entry of distinct cargoes rather than general nuclear import. Indeed, in neuronal systems, Importin-α isoforms can mediate very specific processes such as axonal tiling and communication of an injury signal. To study nuclear import during development, we examined the expression and function of Importin-α2 in Drosophila melanogaster. We found that Importin-α2 was expressed in the nervous system where it was required for normal active zone density at the NMJ and axonal commissure formation in the central nervous system. Other aspects of synaptic morphology at the NMJ and the localization of other synaptic markers appeared normal in importin-α2 mutants. Importin-α2 also functioned in development of the body wall musculature. Mutants in importin-α2 exhibited errors in muscle patterning and organization that could be alleviated by restoring muscle expression of Importin-α2. Thus, Importin-α2 is needed for some processes in the development of both the nervous system and the larval musculature.
Summary Mitochondria are mobile organelles and cells regulate mitochondrial movement in order to meet the changing energy needs of each cellular region. Ca2+ signaling, which halts both anterograde and retrograde mitochondrial motion, serves as one regulatory input. Anterograde mitochondrial movement is generated by kinesin-1, which interacts with the mitochondrial protein Miro through an adaptor protein, milton. We show that kinesin is present on all axonal mitochondria, including those that are stationary or moving retrograde. We also show that the EF-hand motifs of Miro mediate Ca2+-dependent arrest of mitochondria and elucidate the regulatory mechanism. Rather than dissociating kinesin-1 from mitochondria, Ca2+-binding permits Miro to interact directly with the motor domain of kinesin-1, preventing motor/microtubule interactions. Thus, kinesin-1 switches from an active state in which it is bound to Miro only via milton, to an inactive state in which direct binding to Miro prevents its interaction with microtubules. Disrupting Ca2+-dependent regulation diminishes neuronal resistance to excitotoxicity.
The mechanisms for delivering components to nerve terminals are diverse and highly regulated. The diversity of kinesin motors alone is insufficient to account for the specificity of delivery. Additional specificity and control are contributed by adaptor proteins and associated regulatory molecules. The interaction of cargos with these complexes can confer distinct behaviors on the transport of synaptic organelles. The rich regulatory mechanisms of transport that are only now emerging as the cargo–motor complexes are defined and subsequent local events that regulate their dynamic relationship are examined. Here we review recent studies of kinesin-related axonal transport of three crucial synaptic components, Piccolo-bassoon Transport Vesicles (PTVs), Synaptic Vesicle Precursors (SVPs), and mitochondria, and the mechanisms that modulate their transport.
