Oral Presentation 2019 Hunter Cell Biology Meeting

Super-resolving presynaptic endocytic pathway dynamics: from plasma membrane binding to internalization in synaptic vesicles (#2)

Merja Joensuu 1 2 3 , Vanessa Lanoue 1 2 , Pranesh Padmanabhan 1 2 , Mahdie Mollazade1,2, 1 2 , Ailisa Blum 1 2 , Christopher Small 1 2 , Stefan Mahrhold 4 , Nadja Krez 4 , Andreas Rummel 4 , Frédéric A Meunier 1 2
  1. Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Australia
  2. Queensland Brain Institute, The University of Queensland, Brisbane, Australia
  3. Minerva Foundation Institute for Medical Research, Helsinki, Finland
  4. Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany

The capacity of neurons to communicate and store information in the brain critically depends on neurotransmission, a process which relies on the release of chemicals called neurotransmitters stored in small synaptic vesicles at the presynapse. Following their fusion with the presynaptic plasma membrane, synaptic vesicles are rapidly reformed by a process called endocytosis. Neurotoxins, such as Botulinum Neurotoxin type-A (BoNT/A) take advantage of this endocytic pathway to gain access into motor nerve terminals as part of its intoxication strategy to incapacitate nerve-muscle communication. The investigation of the endocytic pathway dynamics is severely restricted by the diffraction limit of light and, therefore, the recycling of synaptic vesicles, which are 45 nm in diameter, has been primarily studied with electrophysiology, low resolution fluorescence-based techniques and electron microscopy. To address this, we implemented a novel super-resolution technique called the subdiffractional tracking of internalized molecules (sdTIM), which is a pulse-chase based method for studying the single molecule mobility of endocytic vesicles. With 30-40 nm localization precision, we revealed that, once internalized, VAMP2-positive synaptic vesicles exhibit a markedly lower mobility than on the plasma membrane (imaged by uPAINT), an effect that was reversed upon restimulation in presynapses. Using HMM-Bayes modelling, we found that synaptic vesicles constantly oscillate between diffusive states and a combination of diffusive and transport states with opposite directionality. The plasma membrane binding of BoNT/A revealed a different mobility pattern than that of VAMP2 and upon internalization BoNT/A internalized into a distinct synaptic vesicle pool different from that of VAMP2-positive vesicles. Genetic inactivation of the BoNT/A ganglioside (GT1b) and co-receptor SV2 binding sites affects the plasma membrane binding, internalization and long range retrograde transport of the toxin. I will also discuss an sdTIM dual-colour imaging of recycling and signalling endosomes containing cholera toxin subunit-B.

  1. Joensuu M, Martínez-Mármol R, Padmanabhan P, Glass NR, Durisic N, Pelekanos M, Mollazade M, Balistreri G, Amor R, Cooper-White JJ, Goodhill GJ and Meunier FA (2017): Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules. Nature Protocols, 12, 2590-2622.
  2. Joensuu M, Padmanabhan P, Durisic N, Bademosi AT, Cooper-Williams E, Morrow IC, Harper CB, Jung W, Parton RG, Goodhill GJ, Papadopulos A and Meunier FA (2016) Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles. The Journal of Cell Biology, 215, 277-292.
  3. Joensuu M, Martínez-Mármol R, Padmanabhan P, Mollazade M and Meunier FA. Single molecule imaging of recycling synaptic vesicles in living nerve terminals by subdiffractional tracking of internalized molecules. Single Molecular Microscopy Neuromethods Series 2019. In review.