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Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.

ABSTRACT: 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.

SUBMITTER: Joensuu M

PROVIDER: S-EPMC5080683 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.

Joensuu Merja M Padmanabhan Pranesh P Durisic Nela N Bademosi Adekunle T D AT Cooper-Williams Elizabeth E Morrow Isabel C IC Harper Callista B CB Jung WooRam W Parton Robert G RG Goodhill Geoffrey J GJ Papadopulos Andreas A Meunier Frédéric A FA

The Journal of cell biology 20161024 2

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. Ou ...[more]

PMID: 27810917

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Project description:IntroductionOptical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs.MethodsNTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values.ResultsThe following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364-1.375 and 1.398-1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV.DiscussionUsing NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37-1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles.ConclusionThis method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.

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Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.

Publications

Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles.

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