Far from home: the role of glial mRNA localization in synaptic plasticity.

Neurons and glia are highly polarized cells, whose distal cytoplasmic functional subdomains require specific proteins. Neurons have axonal and dendritic cytoplasmic extensions containing synapses whose plasticity is regulated efficiently by mRNA transport and localized translation. The principles behind these mechanisms are equally attractive for explaining rapid local regulation of distal glial cytoplasmic projections, independent of their cell nucleus. However, in contrast to neurons, mRNA localization has received little experimental attention in glia. Nevertheless, there are many functionally diverse glial subtypes containing extensive networks of long cytoplasmic projections with likely localized regulation that influence neurons and their synapses. Moreover, glia have many other neuron-like properties, including electrical activity, secretion of gliotransmitters and calcium signaling, influencing, for example, synaptic transmission, plasticity and axon pruning. Here, we review previous studies concerning glial transcripts with important roles in influencing synaptic plasticity, focusing on a few cases involving localized translation. We discuss a variety of important questions about mRNA transport and localized translation in glia that remain to be addressed, using cutting-edge tools already available for neurons.

Current methods to analyze lysosome morphology, positioning, motility and function.

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

Peripheral axonal ensheathment is regulated by RalA GTPase and the exocyst complex.

Axon ensheathment is fundamental for fast impulse conduction and the normal physiological functioning of the nervous system. Defects in axonal insulation lead to debilitating conditions, but, despite its importance, the molecular players responsible are poorly defined. Here, we identify RalA GTPase as a key player in axon ensheathment in Drosophila larval peripheral nerves. We demonstrate through genetic analysis that RalA action through the exocyst complex is required in wrapping glial cells to regulate their growth and development. We suggest that the RalA-exocyst pathway controls the targeting of secretory vesicles for membrane growth or for the secretion of a wrapping glia-derived factor that itself regulates growth. In summary, our findings provide a new molecular understanding of the process by which axons are ensheathed in vivo, a process that is crucial for normal neuronal function.

Ral mediates activity-dependent growth of postsynaptic membranes via recruitment of the exocyst.

Remodelling neuronal connections by synaptic activity requires membrane trafficking. We present evidence for a signalling pathway by which synaptic activity and its consequent Ca(2+) influx activate the small GTPase Ral and thereby recruit exocyst proteins to postsynaptic zones. In accord with the ability of the exocyst to direct delivery of post-Golgi vesicles, constitutively active Ral expressed in Drosophila muscle causes the exocyst to be concentrated in the region surrounding synaptic boutons and consequently enlarges the membrane folds of the postsynaptic plasma membrane (the subsynaptic reticulum, SSR). SSR growth requires Ral and the exocyst component Sec5 and Ral-induced enlargement of these membrane folds does not occur in sec5(-/-) muscles. Chronic changes in synaptic activity influence the plastic growth of this membrane in a manner consistent with activity-dependent activation of Ral. Thus, Ral regulation of the exocyst represents a control point for postsynaptic plasticity. This pathway may also function in mammals as expression of activated RalA in hippocampal neurons increases dendritic spine density in an exocyst-dependent manner and increases Sec5 in spines.

Sec5, a member of the exocyst complex, mediates Drosophila embryo cellularization.

Cellularization of the Drosophila embryo is the process by which a syncytium of approximately 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 sec5(ts1) 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.

Drosophila motor neuron boutons remodel through membrane blebbing coupled with muscle contraction.

Wired neurons form new presynaptic boutons in response to increased synaptic activity, however the mechanism(s) by which this occurs remains uncertain. Drosophila motor neurons (MNs) have clearly discernible boutons that display robust structural plasticity, being therefore an ideal system in which to study activity-dependent bouton genesis. Here, we show that in response to depolarization and in resting conditions, MNs form new boutons by membrane blebbing, a pressure-driven mechanism that occurs in 3-D cell migration, but to our knowledge not previously described to occur in neurons. Accordingly, F-actin is decreased in boutons during outgrowth, and non-muscle myosin-II is dynamically recruited to newly formed boutons. Furthermore, muscle contraction plays a mechanical role, which we hypothesize promotes bouton addition by increasing MN confinement. Overall, we identified a mechanism by which established circuits form new boutons allowing their structural expansion and plasticity, using trans-synaptic physical forces as the main driving force.

Nitric oxide-induced suspended animation promotes survival during hypoxia.

Oxygen plays a key role in energy metabolism. However, there are organisms that survive severe shortfalls in oxygen. Drosophila embryos rapidly arrest development upon severe hypoxia and recover upon restoration of oxygen, even days later. Stabilization of the normally unstable engrailed RNA and protein preserved the localized striped pattern of this embryonic patterning gene during 3 days in hypoxia. Severe hypoxia blocked expression of a heat-shock-inducible lacZ transgene. Cyanide, a metabolic poison, did not immediately block gene expression or turnover, arguing against a passive response to energy limitation. In contrast, nitric oxide, a putative hypoxia signal, induced a reversible arrest of development, gene expression and turnover. Reciprocally, a nitric oxide scavenger allowed continued gene expression and turnover during hypoxia, but it reduced hypoxia tolerance. We suggest that hypoxia-induced stasis preserves the status quo of embryonic processes and promotes survival. Our data implicate nitric oxide as a mediator of this response and provide a system in which to investigate its action.