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Funding: We thank Aino Järvelin and Jeffrey Y. Lee for help with the conceptualization and discussions of this review. This work was funded by a Wellcome Investigator Award 209412/Z/17/Z and Wellcome Strategic Awards (Micron Oxford) 091911/B/10/Z and 107457/Z/ 15/Z to I.D. D.S.G. is funded by Medial Sciences Graduate Studentships, University of Oxford. R.O.T. is funded by iNOVA4Health—UIDB/04462/2020 and EXPL/BIA-CEL/1484/ 2021. 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. publishersversion published

Funding: We thank Liliana Bento for valuable assistance in formatting the manuscript. This article was supported by the LYSOCIL project. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 811087. CGA has funding from Maratona da Saúde 2016 and Fundaçao para a Ciência e a Tecnologia ˜ , I.P. (CEECIND/00410/2017). FMP is a Wellcome Investigator in Science and a Royal Society Wilson merit award holder. 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 revealed 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 analyse 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. publishersversion published

Funder: Maratona da Saúde, Funder: Royal Society Wolfson, Funder: Wellcome; Id: http://dx.doi.org/10.13039/100010269, 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.

The complexity of the nervous system demands and relies on a diversity of molecular processes essential for its correct development and functioning. Coordinated growth and communication between neurons and glia is crucial and indispensable for the physiological functions of the nervous system. Balanced neuronal and glial interaction and communication is decisive, for instance, to ensure suitable axonal insulation and fasciculation. Axonal ensheathment and insulation are critical for fast and reliable impulse conduction and disturbances in these processes often result in highly debilitating disorders, such as multiple sclerosis. Despite the obvious importance, our knowledge regarding the molecular players responsible for controlling the development, growth and communication between neurons and glia is still somewhat limited. In my thesis, we identified RalA GTPase and the exocyst complex as key regulators of axonal ensheathment in Drosophila melanogaster larval peripheral nerves. Detailed genetic analysis showed that RalA is required for wrapping glia growth and development, acting together with its effector exocyst. Our data suggests that this mechanism is regulated through the activation of the EGF receptor and downstream signaling, with RalA and the exocyst likely controlling the secretion of the activating EGFR ligand and/or regulating EGFR endocytic pathway. In summary, we established RalA GTPase and the exocyst as a novel regulators of wrapping glia development providing a new perspective of the molecular pathways governing glia growth and development in the peripheral nervous system. With this, we expect to contribute for the identification of potential new points of intervention for potential improvement or development of novel therapies to mitigate symptoms caused by neurological disorders in which axonal ensheathment and/or Ral GTPase pathway are impaired. Resumo A complexidade do sistema nervoso exige e depende de uma diversidade de processos moleculares que são essenciais para o seu normal desenvolvimento e funcionamento. Um crescimento e comunicação coordenados entre neurónios e glia é crucial e indispensável para a função fisiológica do sistema nervoso. Uma interação e comunicação equilibrada entre neurónios e células da glia são decisivas, por exemplo, para garantir um adequado isolamento axonal e fasciculação. O isolamento axonal é critico para garantir que a condução de impulsos nervosos ocorre de forma rápida e fiável. Qualquer perturbação que comprometa o isolamento dos axónios resulta na sua maioria em condições extremamente debilitantes, como é o caso da esclerose múltipla. Apesar da óbvia importância, o conhecimento sobre os mecanismos responsáveis pela regulação e controlo do desenvolvimento, crescimento e comunicação entre neurónios e células da glia continua, de certa forma, limitado. Nos estudos desta tese, identificámos RalA GTPase e o complexo exocisto como reguladores cruciais para o isolamento axonal dos nervos periféricos de larvas de Drosophila melanogaster. Através de uma análise genética detalhada, mostrámos que RalA é necessária para o crescimento e desenvolvimento das células wrapping glia, agindo em conjunto com o seu efetor exocisto. Os nossos resultados sugerem que este mecanismo é regulado através da ativação do recetor EGF, com RalA e o complexo exocisto provavelmente a controlarem a secreção do ligando ativador do recetor EGFR ou através da regulação da via endocítica deste mesmo recetor. Em suma, estabelecemos RalA GTPase e o complexo exocisto como novos reguladores do desenvolvimento das células wrapping glia, gerando uma nova perspectiva das vias moleculares que governam o crescimento e desenvolvimento das células glia no sistema nervoso periférico. Deste modo, o nosso estudo contribui para a identificação de possíveis novos pontos de intervenção para um potencial melhoramento ou desenvolvimento de novas terapias para mitigar sintomas resultantes de doenças neurológicas, as quais sejam consequência de defeitos no isolamento axonal ou de falhas na via da Ral GTPase.