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doi: 10.1242/jcs.00854

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Kinesin dependent, rapid, bi-directional transport of ER sub-compartment in dendrites of hippocampal neurons

¹ Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
² Division of Molecular Neurobiology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
³ Department of Pediatrics, Tokyo Women's Medical University, Tokyo 162-8666, Japan
⁴ Calcium Oscillation Project, ICORP, Japan Science and Technology Corporation (JST), Tokyo 108-0071, Japan

View larger version (59K): [in a new window]   Fig. 1. Expression of GFP-tagged ER marker proteins was induced in a pattern similar to that of the endogenous ER proteins in the dendrites. (A-C) Dense reticular structures and spotty patterns of the GFP-tagged ER markers in living neurons: (A) GFP-KDEL, (B) GFP-SERCA2a, and (C) GFP-Ins(1,4,5)P3R1, which are quite similar to those of endogenous ER proteins (D,E). Untransfected neurons were fixed, permeabilized and stained with anti-Ins(1,4,5)P3R1 monoclonal antibody 18A10 (D) or anti-calnexin polyclonal antibody (E). All the images were taken with a confocal microscope. Arrowheads indicate spotty shapes. Scale bar: 10 µm.

View larger version (45K): [in a new window]   Fig. 2. Movements of the large spots (arrowheads) labeled with GFP-tagged ER markers: (A) GFP-KDEL, (B) GFP-SERCA2a, and (C) GFPIns(1,4,5)P3R1 in dendrites. Asterisks indicate positions of the spots at 0 seconds. Arrows indicate the proximal directions (toward the cell body). Scale bars: 2 µm. (D-F) Representative movement patterns of the spots labeled with GFP-KDEL, GFP-SERCA2a and GFPIns(1,4,5)P3R1, respectively. The net movement of each vesicle (µm) was plotted against time (seconds). `Anterograde' (positive direction) is the movement from the cell body toward the periphery, and `retrograde' (negative direction) is the movement in the opposite direction. The interval between the time-lapse image frames was 1.5 seconds. (G,H) The photobleaching experiment revealed that the vesicular sub-compartment of ER is separate from the bulk ER. Part of the dendrites of neurons expressing GFP-SERCA2a (G) and GFP-Ins(1,4,5)P3R1 (H) were photobleached by continuous laser illumination. The left panels show the images before the photobleaching. All the areas indicated in this image were subjected to photobleaching, and the rectangles indicate the areas shown as time-lapse images. The right panels are time-lapse images taken after the photobleaching reached a steady level during continuous laser illumination, showing that vesicles (arrowheads) moved into photobleached areas. Relative time from the first time-lapse image is indicated below the time-lapse images. In order to visualize the moving vesicles clearly, the images of the first frame (0 seconds) are subtracted from the subsequent images. Scale bars of left images (before photobleaching), 10 µm, and in the time-lapse images, 2 µm. The raw data for G and H without image subtraction are presented as Movies 1 and 2, respectively. http://jcs.biologists.org/supplemental/). All fluorescent images are reversed for a clearer view.

View larger version (119K): [in a new window]   Fig. 3. ER markers did not overlap with markers of endosomes, lysosomes, and mitochondria. (A-C) Double labeling with Texas Red-dextran (red) and GFP-tagged ER markers (green): (A) GFPKDEL, (B) GFP-SERCA2a and (C) GFP-Ins(1,4,5)P3R1. (D-F) Double labeling with MitoTracker Red CMXRos (red) and (D) GFP-KDEL, (E) GFP-SERCA2a, and (F) GFP-Ins(1,4,5)P3R1. The upper images are high power micrographs of the soma and the lower images are those of the dendrites. Note that most of the lysosomal and mitochondrial signals that appear to be outside of the transfected cells arise from surrounding cells. All images were taken with a confocal microscope. Scale bars: 10 µm.

View larger version (51K): [in a new window]   Fig. 4. The RFP-KDEL protein was not localized on the reticular ER, but was only seen in the vesicular sub-compartment of ER. (A) Expression of RFP-KDEL in a hippocampal neuron observed with a CCD camera. Arrowheads indicate branch points of the dendrites. (B) Double-labeling with RFP-KDEL and GFP-KDEL. In contrast to GFP-KDEL that labeled both reticular ER and vesicles, RFP-KDEL fluorescence was found only on vesicles. RFP-KDEL and GFP-KDEL co-localized on the same vesicles (arrowheads). (C) Representative movements of vesicles labeled with RFP-KDEL. The net movement of each vesicle (µm) was plotted against time (seconds). (D) Double-labeling with RFP-KDEL and endogenous Ins(1,4,5)P3R1 using monoclonal antibody 18A10. Endogenous Ins(1,4,5)P3R1 was also found on the RFP-KDEL-labeled vesicles (arrowheads). (E,F) Double-labeling with RFP-KDEL (red) and fluorescein-dextran (E; green), or MitoTracker Green FM (F; green). The upper images are high power micrographs of the soma and the lower images are those of the dendrites. The lysosomal and mitochondrial signals that appear to be outside the transfected cells arise form surrounding cells. RFP-KDEL did not co-localize with these markers. B,D-F are confocal micrographs. Scale bars: 10 µm.

View larger version (26K): [in a new window]   Fig. 5. Luminal Ca2+ imaging in cultured hippocampal neurons loaded with mag-fura-2 AM (A) and fluo-5N AM (B) and permeabilized with ß-escin. (Top left) RFP-KDEL-labeled vesicles, (bottom left) Ca2+ indicators. Scale bar, 10 µm. (Top right) Time-course plots of changes in fluorescence intensity of the Ca2+ indicators, (right bottom) time-course plots of changes in the ratio (F340/F380 for mag-fura-2 and F/F0 for fluo-5N). The ratio plots are indicated after high-cut filtering with a Gaussian filter in order to reduce the noise. The neurons were subjected to Ca2+ uptake (1 mM MgATP), washout of MgATP and Ca2+ release (20 µM Ins(1,4,5)P3), as indicated by the horizontal boxes above the traces.

View larger version (22K): [in a new window]   Fig. 6. Velocity profile of vesicles labeled with the ER marker proteins. N indicates the number of events, i.e., consecutive mono-directional movement. Positive velocity corresponds to anterograde movement and negative velocity corresponds to retrograde movement. Average velocity of the vesicle movement is summarized in Table 1.

View larger version (21K): [in a new window]   Fig. 7. The ratio of the numbers of moving vesicles vs. total vesicles after drug treatments, overexpression of GFP-tagged dominant-negative kinesin (GFP-DNKHC) and antisense oligonucleotide treatment against kinesin. ER vesicles were labeled with RFP-KDEL. The proportion of moving vesicles was significantly decreased by nocodazole treatment (10 µg/ml), but not by latrunculin A treatment (1 µg/ml). Overexpression of GFP-DNKHC and antisense oligonucleotide treatment against kinesin also decreased the proportion of the moving vesicles. Numbers in parentheses indicate the number of neurons examined.

View larger version (36K): [in a new window]   Fig. 8. Velocity profile of vesicles labeled with RFP-KDEL after drug treatments, overexpression of GFP-DNKHC and antisense oligonucleotide treatment against kinesin. N indicates the number of events, i.e., consecutive mono-directional movement. Positive velocity corresponds to anterograde movement, and negative velocity to retrograde movement. Average velocity of the vesicle movement is summarized in Table 2.

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