|dc.description.abstracteng||An important anatomical feature of neuronal synapses is the presynaptic bouton, a structure that isolates synaptic vesicle recycling from constitutive membrane trafficking pathways. However, in some neurotransmitter-releasing cells this structure is not present and instead they develop active zones directly located at the cell soma (somatic active zones). This is the case for the auditory inner hair cells (IHCs), polarized cells responsible for sound encoding in mammals, with somatic active zones located at their basal pole. As most sensory synapses, IHCs present particularly high rates of synaptic vesicle release, which need to be compensated by equally efficient membrane retrieval mechanisms. Up to now, two models of synaptic vesicle recycling have been proposed in IHCs: 1) apical membrane retrieval that involves organelles of constitutive pathways in vesicle reformation (e.g. endoplasmic reticulum and Golgi apparatus), and 2) local basal recycling, in proximity to the vesicle release sites. Establishing which of these models is correct has been difficult, since conventional endocytosis markers have failed to accurately report membrane uptake events in these cells.
In this study a new membrane-binding probe, called mCLING (membrane-binding fluorophore-Cysteine-Lysine-Palmitoyl Group), was developed to study membrane uptake and trafficking in IHCs, under high-resolution Stimulated Emission Depletion (STED) microscopy. mCLING is not toxic and does not affect membrane trafficking physiology. Moreover, mCLING can be fixed and combined with immunostaining, in order to establish the molecular composition of recycling organelles. mCLING uptake combined with immunostaining against vesicular markers confirmed that synaptic vesicle recycling in IHCs exclusively localizes at the cell base. Synaptic vesicles seem to reform from endocytic intermediates, such as membrane infoldings and cisterns that arise in the vicinity of synaptic active zones. mCLING labeling also revealed that constitutive recycling pathways take place at the top and nuclear IHC levels, in the form of large tubulo-cisternal structures related to recycling endosomes. These results indicate that IHCs functionally and spatially separate synaptic vesicle recycling from constitutive membrane traffic. Moreover, they evidence the importance of keeping synaptic vesicle recycling as a separate trafficking pathway, especially in the absence of a synaptic bouton.
The applicability of mCLING to other biological preparations was further explored. In hippocampal cultured neurons, mCLING allowed to answer still open questions on synaptic function and protein organization: 1) are the same synaptic vesicles undergoing active and spontaneous release? mCLING labeling combined with immunostaining revealed that actively and spontaneously released vesicles differ in molecular composition, being the latter more related to constitutive endosomal traffic. 2) What is the fraction of synaptic vesicle proteins that remains stranded on the plasma membrane as a potential readily retrievable pool of vesicles? This quantification has been difficult, since it has been estimated mainly by overexpression of different proteins fused with the pH sensor pHluorin. Surface labeling with mCLING combined with immunolabeling of endogenous synaptic proteins allowed to establish that ~12 to 22% of them remain stranded on the plasma membrane. 3) What is the organization of SNAP-25 and syntaxin 1 on intracellular organelles? So far clusters of these proteins have only been studied on the plasma membrane. Using mCLING as a surface marker, it was possible to establish that SNAP-25 forms clusters of similar size on the plasma membrane and in intracellular organelles. In contrast, Syntaxin 1 forms larger clusters on the plasma membrane.
Additionally, mCLING labeling and endocytosis were compatible with immunolabeling in COS7 cells, the Drosophila larva neuromuscular junction and yeast cells.
I conclude that mCLING is the first fixable endocytosis marker that can be successfully combined with immunolabeling techniques, and is also compatible with a high-resolution microscopy technique. mCLING helped to answer long-standing questions in a conventional and a sensory synapse, and has a strong potential in the study of membrane traffic in any biological preparation, from cultured cells to complex tissues. ||de