Fig. 4

Reintroducing GFP-labeled synaptopodin into synaptopodin-deficient neurons increases SHP lifetimes. a Examples of small regions of dendrites from SP-KO (top two rows) and GFP/SP (bottom two rows) slices exposed to 60 min of either control or TTX-containing medium (as indicated). Arrows denote SHPs. Time is in minutes in top-left corner, and 10 min was the starting time point. Scale bar, 2 μm. b Quantification of mean lifetime of all SHPs (from both control and TTX experiments) from SP-KO and GFP/SP slices. Mean lifetime of SHPs from GFP/SP dendrites is longer (21.50 ± 4.22 min, n = 14 SHPs from 11 of 18 slices that formed SHPs) than SHPs formed on SP-KO dendrites (7.40 ± 1.21 min, n = 20 SHPs from 12 of 20 slices that formed SHPs). *P < 0.05, Mann-Whitney test. c Cumulative distribution of all SHP lifetimes from SP-KO (gray line) and GFP/SP (black line) slices. d Two examples of SHPs formed on spines containing GFP-labeled synaptopodin. Left, example 1 is the same dendrite from the GFP/SP TTX-treated slice in a (last row), just prior to the beginning (Pre) and following the end (Post) of a 60 min imaging session. Right, another example (example 2) of a SHP formed on a synaptopodin-positive spine. Arrowheads show synaptopodin (green), the neuron is labeled in red (tdTomato) and is rendered semi-transparent and the arrows point to the SHPs. Scale bar, 2 μm. e Quantification of mean lifetime of all SHPs (from both GFP/SP and SP-KO slices and control and TTX experiments) from SP-negative and SP-positive spines. Mean lifetime of SHPs on SP-positive spines is longer (26.75 ± 5.61 min, n = 8 SHPs from 8 of 10 slices) than SHPs formed on SP-negative spines (9.04 ± 1.64 min, n = 26 SHPs from 18 of 38 slices). *P < 0.01, Mann-Whitney test. In b and d gray circles are individual lifetimes of SHPs, and black circles are mean lifetimes ± SEMs. f Cumulative distribution of all SHP lifetimes from SP-negative (gray line) and SP-positive (black line) spines