Article Figures & Data
Figures
- Figure 1.
Effect of PIP3 levels on spinule formation in spines induced by sLTP. A–C, The effect of PIP3 levels on spinule formation during sLTP using glutamate uncaging (Ctrl 0.4% DMSO; A), a PTEN inhibitor–1 μm BpV(HOpic; B), and a PI3K inhibitor, 30 μm LY294002 (C). Each drug was added over 30 min before induction of sLTP. Red spots on baseline images indicate the points that were subjected to glutamate uncaging. Spinules are indicated by arrows. Scale bar, 1 μm. D, Time course of the enlargement of GFP-expressing spines following the induction of sLTP. The red bar indicates the time period of glutamate uncaging. First and second black bars indicate early (0–7 min) and (10–30 min) late phases, respectively. E, Time course of the formation of spinule-positive spines; n = the number of spines subjected to glutamate uncaging. F, The occurrence of spinule-positive spines in early and late phases in the presence of each drug. Asterisks denote a statistically significant difference (p < 0.05) from the value in the spine that was subjected to sLTP. G, H, The effect of PH domain on spinule formation. mCherry-PH and mCherry-PH (R284C) were overexpressed with mEGFP as a volume marker.
- Figure 2.
Characterization of FLIMPA3 in living cells. A, Principle of FLIMPA3 for visualizing PIP3. PIP3 production induces a conformational change in FLIMPA3 through the binding of the PH domain to PIP3, leading to an increase in FRET and a decrease in fluorescence lifetime. B, Fluorescence lifetime image of FLIMPA3 in PDGFR-expressed CHO cells. A color gradient was used to represent PIP3 levels, with a warmer color indicating a shorter fluorescence lifetime and higher PIP3 levels. The images before (0 min), and 5.5, 10.5, 15.5, and 20. 5 min after addition of 50 ng/ml PDGF were shown. Scale bar, 1 μm. C, Time course of the fluorescence lifetime change of FLIMPA3 in CHO cells after the addition of HBSS, or 50 ng/ml PDGF with or without pre-incubation with 100 μm LY294002. D, Time course analysis of the fluorescence lifetime change of FLIMPA3 mutant in CHO cells after administration of HBSS or 50 ng/ml PDGF. E, F, Effect of FLIMPA3 on PIP3 signaling assessed by PDGF-induced Akt phosphorylation. Akt phosphorylation at serine residue 473 was detected with Alexa 555-conjugated anti-phospho 473 antibody. Bottom, Indicates immunostaining signal taken from white lines in top.
- Figure 3.
PIP3 accumulation in spines at static state. A, Fluorescence lifetime imaging of FLIMPA3 and FLIMPA3 mutant (R284CK343A), where PIP3 binding was abolished. A color gradient was used to represent PIP3 levels with a warmer color indicating a shorter fluorescence lifetime and higher PIP3 levels. Scale bar, 1 μm. B, Asterisk denotes a statistically significant difference between the fluorescence lifetime of spines and dendritic shafts of FLIMPA3 (p < 0.05); n means the number of spines. The number of neurons observed is 25 and 12 in FLIMPA3 and FLIMPA3 mutant, respectively. C, D, Fluorescence lifetime imaging of PIP3 after addition of PTEN inhibitor, BpV(HOpic), and PI3K inhibitor, LY294002. Drugs were administered during the period indicated by the red line. Scale bar, 1 μm. E–H, Time course of the fluorescence lifetime change of FLIMPA3 in spines and dendritic shafts after addition of 1 μm BpV(HOpic), 30 μm LY294002, 0.4% DMSO (Ctrl), or 1 μm BpV(HOpic) and 30 μm LY294002. I, The fluorescence lifetime change averaged >40–60 min. N.S., Not significant. Asterisks denote a statistically significant difference (p < 0.05). J, Time course of the change in spine size based on FLIMPA3 intensity after the addition of each inhibitor.
- Figure 4.
Spatiotemporal dynamics of PIP3 in spines subjected to sLTP. A, Fluorescence lifetime imaging of FLIMPA3 during sLTP of a single spine induced by two-photon glutamate uncaging. The red spot in the −2 min image indicates the location of the uncaging laser point. White scale bar, 1 μm. The red line indicates the time period of glutamate uncaging. B, Time course of fluorescence lifetime change of FLIMPA3 in a spine subjected to glutamate uncaging, a neighboring spine (within 5 μm of the stimulated spine), and a region of the dendritic shaft next to the stimulated spine. The time course of FLIMPA3 mutant was also shown; n indicates the number of spines. The red line indicates the time of glutamate uncaging. Time course of FLIMPA3 intensity change in spines is shown on the right y-axis. C, Relationship between PIP3 concentration at the 2 min time point (y-axis) and basal PIP3 enrichment at basal state. The linear regression line is shown.
- Figure 5.
Subspine PIP3 imaging following sLTP stimulation. A–C, Subspine distribution of PIP3 by fluorescence lifetime imaging during sLTP (Ctrl 0.4% DMSO (A), 1 μm BpV(HOpic) (B), and 30 μm LY294002 (C), each drug was added >30 min before glutamate uncaging). Red spots on baseline images show the points that were subjected to glutamate uncaging. Arrows show PIP3 accumulation in spinules. Scale bar, 1 μm. D, Time course of PIP3 change in spinules; n = number of spines. The red bar indicates the time period of glutamate uncaging. In the case of LY294002, 7–10, 15–20, and 25–30 min, were averaged out due to the smaller dataset. E, Comparison between fluorescence lifetime change in FLIMPA3 (black line) and spinule length (red line) after sLTP in spines pre-incubated with BpV(HOpic). F, Fluorescence lifetime of spinules and spines that exhibit spinules during sLTP was averaged during the early phase and the late phase. Asterisks denote a statistically significant difference (p < 0.05).
