TY - JOUR
T1 - Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules
AU - Joensuu, Merja
AU - Martínez-Mármol, Ramon
AU - Padmanabhan, Pranesh
AU - Glass, Nick R.
AU - Durisic, Nela
AU - Pelekanos, Matthew
AU - Mollazade, Mahdie
AU - Balistreri, Giuseppe
AU - Amor, Rumelo
AU - Cooper-White, Justin J.
AU - Goodhill, Geoffrey J.
AU - Meunier, Frédéric A.
N1 - Funding Information:
acknoWleDGMents The super-resolution imaging was carried out at the Queensland Brain Institute’s (QBI’s) Advanced Microimaging and Analysis Facility with the help of A. Chien. We thank R.G. Parton (Institute for Molecular Bioscience, The University of Queensland, Australia) for helpful comments, N. Valmas (QBI) for the photography and the schematic illustration in Figure 1, J. Carroll and his team (QBI) for research computing infrastructure support, data management and processing, and R. Tweedale (QBI) for critical appraisal of the manuscript. We also thank J.B. Sibarita (Centre National de la Recherche Scientifique, Interdisciplinary Institute for Neuroscience, University of Bordeaux. France) for helpful comments and help with the PalmTracer software. We thank T. Wang and A. Papadopoulos for performing the original studies. This work was supported by an Australian Research Council Discovery Project grant (DP150100539) an Australian Research Council Linkage Infrastructure, Equipment, and Facilities grant (LE130100078) and a National Health and Medical Research Council (NHMRC) grant (APP1120381) to F.A.M. M.J. is supported by an Academy of Finland Postdoctoral Research Fellowship (298124), G.B. by the Finnish Cancer Foundation, P.P. and N.D. by University of Queensland Postdoctoral Research Fellowships and N.D. by a UQ Early Career Researcher Grant (UQECR1718789). R.M.-M. is funded by the Clem Jones Centre for Ageing Dementia Research. F.A.M. is a Senior Research Fellow of the National Health and Medical Research Council (1060075).
Publisher Copyright:
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
PY - 2017/12/1
Y1 - 2017/12/1
N2 - O ur understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulsechase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulsechase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take 1 week.
AB - O ur understanding of endocytic pathway dynamics is restricted by the diffraction limit of light microscopy. Although super-resolution techniques can overcome this issue, highly crowded cellular environments, such as nerve terminals, can also dramatically limit the tracking of multiple endocytic vesicles such as synaptic vesicles (SVs), which in turn restricts the analytical dissection of their discrete diffusional and transport states. We recently introduced a pulsechase technique for subdiffractional tracking of internalized molecules (sdTIM) that allows the visualization of fluorescently tagged molecules trapped in individual signaling endosomes and SVs in presynapses or axons with 30- to 50-nm localization precision. We originally developed this approach for tracking single molecules of botulinum neurotoxin type A, which undergoes activity-dependent internalization and retrograde transport in autophagosomes. This method was then adapted to localize the signaling endosomes containing cholera toxin subunit-B that undergo retrograde transport in axons and to track SVs in the crowded environment of hippocampal presynapses. We describe (i) the construction of a custom-made microfluidic device that enables control over neuronal orientation; (ii) the 3D printing of a perfusion system for sdTIM experiments performed on glass-bottom dishes; (iii) the dissection, culturing and transfection of hippocampal neurons in microfluidic devices; and (iv) guidance on how to perform the pulsechase experiments and data analysis. In addition, we describe the use of single-molecule-tracking analytical tools to reveal the average and the heterogeneous single-molecule mobility behaviors. We also discuss alternative reagents and equipment that can, in principle, be used for sdTIM experiments and describe how to adapt sdTIM to image nanocluster formation and/or tubulation in early endosomes during sorting events. The procedures described in this protocol take 1 week.
UR - http://www.scopus.com/inward/record.url?scp=85036672352&partnerID=8YFLogxK
U2 - 10.1038/nprot.2017.116
DO - 10.1038/nprot.2017.116
M3 - Article
C2 - 29189775
AN - SCOPUS:85036672352
SN - 1754-2189
VL - 12
SP - 2590
EP - 2622
JO - Nature Protocols
JF - Nature Protocols
IS - 12
ER -