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Ellipsoid Localisation Microscopy
Multilayered protein coats are crucial to the dormancy, robustness, and germination of bacterial spores. In Bacillus subtilis spores, the coat contains over 70 distinct proteins. Identifying which proteins reside in each layer may provide insight into their distinct functions. We present image analysis methods that determine the order and geometry of concentric protein layers by fitting a model description for a spheroidal fluorescent shell image to optical micrographs of spores incorporating fluorescent fusion proteins. The radius of a spherical protein shell can be determined with <10聽nm error by fitting an equation to widefield fluorescence micrographs. Ellipsoidal shell axes can be fitted with comparable precision. The layer orders inferred for B. subtilis and B. megaterium are consistent with measurements in the literature. The aspect ratio of elongated spores and the tendency of some proteins to localize near their poles can be quantified, enabling measurement of structural anisotropy.We gratefully acknowledge support from MedImmune through the Beacon collaboration, the EPSRC Centre for Doctoral Training in Sensor Technologies and Applications (EP/L015889/1), and thank Clemens Kaminski, Romain Laine and Jose Casas-Finet for inspiring discussions.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.bpj.2015.09.02
The SAC1 domain in synaptojanin is required for autophagosome maturation at presynaptic terminals
Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P2, but this function appears normal in SynaptojaninRQ knock-in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P2, two lipids found in autophagosomal membranes. Using advanced imaging, we show that SynaptojaninRQ mutants accumulate the PI(3)P/PI(3,5)P2-binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell-derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in SynaptojaninRQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic-specific autophagy defects to Parkinson's disease