Investigating sub-spine actin dynamics in rat hippocampal neurons with super-resolution optical imaging

Abstract

Morphological changes in dendritic spines represent an important mechanism for synaptic plasticity which is postulated to underlie the vital cognitive phenomena of learning and memory. These morphological changes are driven by the dynamic actin cytoskeleton that is present in dendritic spines. The study of actin dynamics in these spines traditionally has been hindered by the small size of the spine. In this study, we utilize a photo-activation localization microscopy (PALM)-based single-molecule tracking technique to analyze F-actin movements with approximately 30-nm resolution in cultured hippocampal neurons. We were able to observe the kinematic (physical motion of actin filaments, i.e., retrograde flow) and kinetic (F-actin turn-over) dynamics of F-actin at the single-filament level in dendritic spines. We found that F-actin in dendritic spines exhibits highly heterogeneous kinematic dynamics at the individual filament level, with simultaneous actin flows in both retrograde and anterograde directions. At the ensemble level, movements of filaments integrate into a net retrograde flow of approximately 138 nm/min. These results suggest a weakly polarized F-actin network that consists of mostly short filaments in dendritic spines.

Figure 1. F-Actin cytoskeleton dynamic in the lamellipodia of a Xenopus fibroblast cell visualized with single-molecule tracking.

A. The fluorescence image of the lamellipodia region showing images of single EosFP-actin molecules. B. kymographs (white rectangle area of the left image) of the same cell showing retrograde flow of single EosFP-Actin molecules from the leading edge. Red arrow heads tracked one molecule moving away from the cells leading edge. Molecules deeper inside the cell had a much slower retrograde flow rate. Scale bar represented 2 µm. C. The average actin retrograde flow rate versus the distance of the molecule from the edge of the cell (The right-side gray-line in panel A).

Figure 2. Localization of EosFP-actin in neuron.

Figure 3. Heterogeneous F-Actin kinematic flow in dendritic spines.

A. Fluorescence images of dendrites (23DIV) showing mature dendritic spines. B. Time-lapse image sequences of single EosFP-actin molecules representing molecules that are moving in retrograde direction (B1), stationary (B2), moving in anterograde direction (B3) and moving randomly (B4). All scale bars are 2 µm.

Figure 4. Quantification of the F-actin kinematics in dendritic spines.

(A) 2D scatter plot of the Sv and MSR values from molecules in neuron after treatment with jasplakinolide. (B) 2D scatter plot of the Sv and MSR values from molecules in dendritic spines. Each dot represents one actin molecule. The open circles represent molecules with a net motion in anterograde direction. (C) 2D scatter plot of the Sv and MSR values from molecules in the lamellipodia region of the Xenopus fibroblast. Note the different scale in the y-axis. Shaded area denotes molecules that showed little or no vectorial movements (Sv <7.5 nm/sec).

Figure 5. F-actin redistribution in the dendritic spine modeled on single-molecule kinematics measurements.

(A) The redistribution of a pool F-actin initially at a spine tip over one minute time span. The initial spread of the molecules is assumed to be diffraction-limited (full-width at half-maximum (FWHM)  = 300 nm). The intensity profile evolves over time, calculated according to the distribution of Sv values from single-molecule measurements. (B) Calculated net retrograde flow of F-actin based on the simulated data in A. (C) The redistribution of a pool F-actin initially at the center of a spine over one minute time span. (D) Calculated net retrograde flow of F-actin based on the simulated data in C. See text for the details.

Figure 6. Kinetic dynamics of F-actin in dendritic spines.

(A) Distributions of the lengths of single-molecule tracks from dendritic spines. The solid line is the single exponential fit. The dashed line is the expected distribution from only photo-bleaching. The distribution for the subpopulation excluding stationary filaments is also plotted as comparison. (B) FRAP measurements in dendritic spines. Top images show bleaching and recovery of a spine. The bottom graph shows the average normalized fluorescence intensity of 10 spines measured. Errorbars represent standard deviation. The solid line is the fit with single exponential recovery.

Figure 7. Models of actin organization in dendritic spines.

(A) Highly polariozed actin cytoskeleton. Actin forms long filaments with the barbed ends pointing towards the tip of the spine, where the polymerization rate is high. This model is inconsistent with the experimental results. (B) Weakly polarized actin cytoskeleton. Most actin filaments are short and not well aligned. Barbed ends are distributed all over the spine. Polymerization and filament growth could happen everywhere. This model is more consistent with the experimental results. See text for the details.