Rapid recycling of glutamate transporters on the astroglial surface
- UCL Queen Square Institute of Neurology, University College London, United Kingdom
- BRAINCITY, Laboratory of Neurobiology, Nencki Institute of Experimental Biology PAS, Poland
- School of Biotechnology and National Institute for Cellular Biotechnology (NICB), Dublin City University, Ireland
Figures
Figure 1 with 3 supplements
Super-Ecliptic pHluorin-tagged GLT1 (GLT1-SEP) enables monitoring of cell membrane and cytosolic fractions of glial glutamate transporters.
(A) Diagram illustrating molecular composition of GLT1-SEP. (B) Functional probing of GLT1-SEP expressed in HEK cells shows a prominent current response to glutamate application, similar to that …
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Figure 1—source data 1
Original data readout for Figure 1D–F, Figure 1—figure supplement 2B,D and Figure 1—figure supplement 3B,C.
- https://cdn.elifesciences.org/articles/64714/elife-64714-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Testing glutamate transport function and the membrane/cytosol fraction ratio for GLT1-SEP.
(A) Summary of transporter current recordings in wild-type GLT1 (black) and GLT1-SEP (red) expressing HEK cells, as indicated; absolute current values are shown. (B) Diagram illustrating the …
Figure 1—figure supplement 2
Expression of GLT1, GLT1-SEP, and GLT1ΔC-SEP in HEK cells.
(A) Examples of immunostaining of GLT1 in HEK 293 T cells co-transfected with plasmids coding GLT1 or GLT1-SEP under CMV promoter together with mRFP1 under β-actin promoter (control reference) in a …
Figure 1—figure supplement 3
Relative expression GLT1, GLT1-SEP, and GLT1ΔC-SEP astrocytes.
(A) Examples of immunostaining for GFP in astroglia transfected with GLT1-SEP or GLT1ΔC-SEP, and total GLT1 (using an antibody which recognises C-terminus of both endogenous GLT1 and transfected …
Figure 2 with 1 supplement
Whole-cell FRAP reveals the kinetics of the GLT1-SEP membrane surface turnover.
(A) One-cell example illustrating FRAP protocol; upper left, DIC + SEP channel image; serial images, GLT1-SEP channel at different time points (indicated) after a photobleaching pulse (t = 0 s); …
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Figure 2—source data 1
Original data readout for Figure 2B,C.
- https://cdn.elifesciences.org/articles/64714/elife-64714-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Establishing the kinetics of whole-cell FRAP for GLT1-SEP molecules in astrocytes.
FRAP reactions diagram reflects exchange (turnover) between membrane cytosol fractions of non-bleached GLT1-SEP molecules, with and standing for their relative concentrations, respectively, …
Figure 3 with 1 supplement
Distribution of GLT-1 species in relation to postsynaptic densities in the astroglial membrane: A super-resolution SMLM analysis.
(A) Wide-field fluorescent images (examples) illustrating antibody labelled GLT1 species (green channel) and postsynaptic density protein PSD95 (magenta), as indicated, in mixed astroglia-neuron …
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Figure 3—source data 1
Original data readout for Figure 3C and Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/64714/elife-64714-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Distribution of GLT-1 species in the astroglial membrane: macroscopic wide-field view and super-resolution SMLM analysis.
(A) Wide-field fluorescent images (examples) displaying antibody labelled GLT1 species (green channel) and postsynaptic density protein PSD95 (magenta), as indicated, in mixed astroglia-neuron …
Figure 4 with 1 supplement
Microscopic-ROI FRAP probes lateral membrane mobility of GLT1-SEP in cultured astroglia.
(A) One-cell example as seen in GLT1-SEP + DIC channel (left), with a selected area (dotted rectangle) illustrating a circular, 2.06 µm wide FRAP spot (dotted circle, right). (B) Diagram, the …
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Figure 4—source data 1
Original data readout for Figure 4B,D–H.
- https://cdn.elifesciences.org/articles/64714/elife-64714-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Microscopic-ROI FRAP probes lateral membrane mobility of GLT1-SEP in cultured astroglia.
(A) One-cell example of FRAP kinetics at four trials (two paired-trial FRAP stages); time-lapse images of a cell fragment with a FRAP spot (dotted red circle), at selected time points before and …
Figure 5 with 1 supplement
Microscopic-ROI FRAP probes lateral membrane mobility of astroglial GLT1-SEP in organotypic hippocampal slices.
(A) Example of astroglia in an organotypic slice, seen in GLT1-SEP, tdTomato, and merge +DIC channel, as indicated. (B) One-slice example of boosted excitatory activity (field potential recording, …
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Figure 5—source data 1
Original data readout for Figure 5C–H and Figure 5—figure supplement 1A,B.
- https://cdn.elifesciences.org/articles/64714/elife-64714-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Control tests for microscopic-ROI FRAP probing of lateral membrane mobility of GLT1-SEP in organotypic hippocampal slices.
(A) Average time course of the truncated transmembrane protein (C-terminal transmembrane anchoring domain of platelet-derived growth factor receptor) in fusion with SEP (dots and shade: mean ± 95% …
