Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization
Spine head contains polymerization hot spots. (a) Example of spines from neurons in culture with astrocytes present, transfected with GFP-actin. The white line highlights the area selected for FRAP, employing a line scanning mode. In this mode, the scanned area is limited to the drawn line (around 200–300 nm wide). The spine is labeled with an arrow and the dendrite with an asterisk. Scale bar: 5 μm. (b) Section of a recovery image from the initial postbleach period, obtained with the lineal acquisition mode. The -axis corresponds to the acquisition time in ms (each line, 1 ms) and the -axis corresponds to the localization in microns along the line. Spine and dendrite are marked with an arrow and an asterisk, respectively. Spine length was arbitrarily divided into two sections (highlighted by the yellow dashed box), corresponding to the distal and proximal part of the spine (notice how the proximal part includes the neck of the spine and a portion of the head). Scale bar: 2 μm. (c) Example of normalized fluorescence recovery curve of the spine in (b). The distal part of the spine (red) and the proximal area (black) were analyzed and plotted independently. The blue line shows the fluorescence recovery curve of the entire yellow box area (sum of distal and proximal areas). In this particular example, at the times when the distal area recovery rate drops, a proportional increase at the distal area is found. Therefore, the total average fluorescence recovery does not change (blue line). Out of the 25 spines analyzed, 13 (52%) showed differences in the recovery rate between the distal and proximal areas of the spine, as shown in the example. (d) Normalized fluorescence recovery curve after addition to the culture media of Latrunculin A (200 nM), an organic compound that blocks actin polymerization by sequestering actin monomers. Notice the reduction in mobile fraction value and the absence of distributed polymerization.