Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.
Our understanding of endocytic pathway dynamics is severely restricted by the diffraction limit of light microscopy. To address this, we implemented a novel technique based on the subdiffractional tracking of internalized molecules (sdTIM). This allowed us to image anti-green fluorescent protein Atto647N-tagged nanobodies trapped in synaptic vesicles (SVs) from live hippocampal nerve terminals expressing vesicle-associated membrane protein 2 (VAMP2)-pHluorin with 36-nm localization precision. Our results showed that, once internalized, VAMP2-pHluorin/Atto647N-tagged nanobodies exhibited a markedly lower mobility than on the plasma membrane, an effect that was reversed upon restimulation in presynapses but not in neighboring axons. Using Bayesian model selection applied to hidden Markov modeling, we found that SVs oscillated between diffusive states or a combination of diffusive and transport states with opposite directionality. Importantly, SVs exhibiting diffusive motion were relatively less likely to switch to the transport motion. These results highlight the potential of the sdTIM technique to provide new insights into the dynamics of endocytic pathways in a wide variety of cellular settings.
Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Queensland 4072, Australia.|Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Merja
http://orcid.org/0000-0002-4533-0455
M
Joensuu
Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Pranesh
http://orcid.org/0000-0001-5569-8731
P
Padmanabhan
Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Nela
http://orcid.org/0000-0002-7668-1039
N
Durisic
Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Queensland 4072, Australia.|Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Adekunle T D
http://orcid.org/0000-0001-5157-8572
AT
Bademosi
Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Elizabeth
http://orcid.org/0000-0002-4219-9447
E
Cooper-Williams
Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Queensland 4072, Australia.|Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Isabel C
IC
Morrow
Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Queensland 4072, Australia.|Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Callista B
CB
Harper
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.|Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Queensland 4072, Australia.
WooRam
http://orcid.org/0000-0001-5748-5473
W
Jung
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.|Centre for Microscopy and Microanalysis, The University of Queensland, Brisbane, Queensland 4072, Australia.
Robert G
http://orcid.org/0000-0002-7494-5248
RG
Parton
Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.|School of Mathematics and Physics, The University of Queensland, Brisbane, Queensland 4072, Australia.
Geoffrey J
http://orcid.org/0000-0001-9789-9355
GJ
Goodhill
Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Queensland 4072, Australia f.meunier@uq.edu.au a.papadopulos@outlook.com.|Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Andreas
http://orcid.org/0000-0002-3376-1979
A
Papadopulos
Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, Queensland 4072, Australia f.meunier@uq.edu.au a.papadopulos@outlook.com.|Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia.
Frédéric A
http://orcid.org/0000-0001-6400-1107
FA
Meunier
D000818:Animals; D001369:Axons / Q000378:metabolism; D001499:Bayes Theorem; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D021621:Imaging, Three-Dimensional; D008390:Markov Chains; D009038:Motion; D009474:Neurons / Q000378:metabolism; D055620:Optical Phenomena; D017207:Rats, Sprague-Dawley; D061905:Single-Domain Antibodies / Q000378:metabolism; D013269:Stochastic Processes; D013569:Synapses / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
C485187:PHluorin; D061905:Single-Domain Antibodies; D050988:Vesicle-Associated Membrane Protein 2; D049452:Green Fluorescent Proteins