The Journal of Neuroscience, July 27, 2005, ():
A Mechanism for Ca2+/Calmodulin-Dependent Protein Kinase II Clustering at Synaptic and Nonsynaptic Sites Based on Self-Association
J. Neurosci. Hudmon et al.
25: 6971
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Figure 2-movie1.mov: Intracellular self-association of CaMKII. Time-lapse imaging of HEK293 cells transfected with GFP-aCaMKII and stimulated with ionomycin/Ca2+ in regular HBSS external solution as described in Fig. 2A. Note that clusters of fluorescence begin to appear in some cells after 5 min of stimulation. Also note the random-like mobility of many of the clusters. Frames were collected every 20 sec (time shown in lower left corner) and are displayed at 6 frames per sec.
- supplemental material
-
Figure 2-movie2.mov: Dynamic translocation of CaMKII to NR2B. Time-lapse imaging of HEK293 cells transfected with GFP-aCaMKII and NR2B, and stimulated with ionomycin/Ca2+ in regular HBSS external solution as described in Fig. 2B. Note the redistribution of the fluorescence, beginning after the first min of stimulation, into elongated clusters primarily around the nucleus, where overexpressed NR2B is localized (Bayer et al., 2001; Fukaya et al., 2003). Frames were collected every 20 sec (time shown in lower right corner) and are displayed at 6 frames per sec.
- supplemental material
-
Figure 3-movie3.mov: Role of pHi in CaMKII self-association. Time-lapse imaging of HEK293 cells transfected with GFP-aCaMKII and stimulated with ionomycin/Ca2+ in nigericin-based external solution as described in the Methods. The pHi was changed to 6.5 at t = 120s. Note that clusters begin to form immediately after the pH drop. Frames were collected every 20 sec (time shown in lower left corner) and are displayed at 6 frames per sec.
- supplemental material
-
Figure 4-movie4.mov: Reversibility of CaMKII self-association in zero Ca2+. HEK293 cells were transfected with GFP-aCaMKII and stimulated with ionomycin/Ca2+ in nigericin-based external solution as described in the methods. The pHi was changed to 6.5 at t = 120s, leading to CaMKII self-association, which was reversed when Ca2+ was replaced with 5 mM BAPTA. Frames were collected every 20 sec (time shown in lower right corner) and are displayed at 6 frames per sec.
- supplemental material
-
Supplemental Figure 1: GFP-CaMKII translocation in HEK293 cells does not repetitively occur at the same intracellular sites. Cells were imaged using the standard Ca2+/ionomycin-pH drop protocol followed by BAPTA treatment (as in Fig. 4 A) except that it was repeated multiple times. Panels (A) and (C) show the cells during the BAPTA treatment, whereas panels (B) and (D) show the cells during the peak of GFP-CaMKII clustering. E) The location of the fluorescent puncta was compared for two of these cycles by overlaying panels B (pseudocolored in green) and D (pseudocolored in red). This color panel shows little colocalization of the puncta, suggesting that they formed in different subcellular locations.?Scale bar: 6 mm.
- supplemental material
-
Supplemental Figure 2: Role of CaMKII self-association in its activity-dependent translocation in hippocampal neurons. (A) Semi-quantitative assessment of post-synaptic translocation of WT and mutant GFP-aCaMKII, plotted as a cumulative percentage (n=106 to 195 neurons per condition, 3 experiments). An observer, blind to the condition, evaluated the fluorescence contrast between synaptic areas (PSD95 positive) and the rest of the dendrites on a scale of “–“ to “++++”. Representative examples of the scoring applied are shown above. Scale bar: 3mm. (B) Mean percentage (± SEM, n=same as in A) of neurons that contained non-synaptic fluorescent clusters in their cell body, as seen in Fig. 1B, following 1 or 5 min stimulation, assessed by an observer, blind to the condition. For statistical analysis in (B), data were entered into a contingency table comparing each mutant with WT. Significance for the chi-squared analyses was set at 0.0125, which accounts for the number of comparisons made (alpha = 0.05, divided by 4 comparisons, according to the Bonferroni method). Statistical analyses were conducted using Epi Info 2002, Version 2 and revealed that every mutant differed from WT (p < 0.001).
- supplemental material
-
Supplemental Figure 3: Potential effect of CaMKII self-association in the organization of PSD. Self-associated CaMKII holoenzymes may form a scaffold embedded with multiple PSD anchoring proteins, cytoskeletal elements and signaling molecules. The multivalent architecture of CaMKII would allow the lattice of holoenzymes to retain the capacity to bind and phosphorylate multiple substrates (brown shapes) important for PSD structure and function.
