Journal: The Journal of Cell Biology

Article Title: Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

doi: 10.1083/jcb.201603108

Figure Lengend Snippet: The diffusion of membrane molecules is restricted by DIV5 in hippocampal neurons. (A) Experimental design for the developmental time course in cultured primary hippocampal neurons. (1) Neurons were maintained in gridded glass-bottom Petri dishes and (2) transfected with the membrane-probe GPI-GFP. (3) Individual neurons were relocated on DIV3, 5, 7, and 10, respectively, based on their position on the grid. (4) Single-particle tracking (SPT) experiments were conducted by sparsely labeling transfected neurons with anti-GFP nanobodies conjugated to fluorescent organic dyes. (5) After SPT, neurons were fixed and immunostained for cytoskeletal and or AIS marker. The soma (i) and the proximal axon with the AIS (ii), identified by AnkG staining (inset micrograph), are outlined. (B) Results from the developmental SPT time course on a typical neuron. The AIS was identified by live immunolabeling of neurofascin. (left box) The proximal axon was neurofascin negative on DIV3 and neurofascin positive from DIV5 onwards. (middle box) Plot of trajectories of mobile particles tracked in SPT experiments color-coded for instantaneous diffusion coefficients D . (right box) Histogram of D on the proximal axon where the AIS assembles (white dashed line). Number of trajectories: DIV3, n = 787; DIV5, n = 1,815; DIV7, n = 2005; and DIV10, n = 1,067. (C) Plots of the cumulative D in the AIS (left, shades of green) and a portion of the distal axon (right, shades of blue) of the neuron shown in B. (D) Graph of the median D for all neurons ( n = 4) between DIV3 and DIV10. Statistical analysis of median D by paired t test; *, P < 0.01; ns, not significant. Bars, 5 µm.

Article Snippet: SPT using Atto647N-coupled anti-GFP nanobodies (ChromoTek) was done using the uPAINT method.

Techniques: Diffusion-based Assay, Cell Culture, Transfection, Single-particle Tracking, Labeling, Marker, Staining, Immunolabeling

Journal: The Journal of Cell Biology

Article Title: Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

doi: 10.1083/jcb.201603108

Figure Lengend Snippet: High-speed SPT shows that diffusing GPI-GFP molecules revisit small areas in the AIS. (A) Merged fluorescence micrograph of a DIV4 hippocampal neuron expressing GPI-GFP (green) after PFA fixation with fiduciary markers (white) for drift correction and image correlation. (B) Plot of SPT trajectories of anti-GFP nanobody-coupled quantum dots ( n = 3,375) on GPI-GFP. Trajectories are color-coded according to the instantaneous diffusion coefficient. The AIS was identified by live immunolabeling of neurofascin (box). Not all areas were covered homogeneously by trajectories (arrowheads). (C) Plot of selected long trajectories (>500 steps, orange, green, and blue) along a segment with reduced lateral mobility (white box in B). The trajectories cover the axon inhomogeneously and show local zones, which are revisited by individual QDs (black dashed lines). (D) Same plot of area as in C, with all trajectories >20 steps plotted ( n = 231) and color-coded according to the instantaneous diffusion coefficient as in B. Arrowheads emphasize a pattern emerging from the distribution of the trajectories. (E) Plot of trajectories of anti-GFP nanobody-coupled QDs on the AIS of a DIV11 neuron expressing GPI-GFP ( n = 501). Trajectories are color coded as in B. Arrowheads emphasize an emergent pattern similar to that in D. Bars: (A and B) 5 µm; (C–E) 200 nm.

Article Snippet: SPT using Atto647N-coupled anti-GFP nanobodies (ChromoTek) was done using the uPAINT method.

Techniques: Fluorescence, Expressing, Diffusion-based Assay, Immunolabeling