Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie 2 - Movie 2: FRAP experiment After acquiring a 20 sec baseline, a single filopodium from 10 DIV neurons expressing actin-GFP was photo-bleached with a 488 nm laser pulse (white dot appearing). Fluorescence was then recorded for an additional 200 sec. False-color coding, from blue (low intensity) to red (high intensity).
- Movie 9 - Movie 9: Example of a dendritic spine pulling on an axon. Neurons electroporated with either RFP or actin-GFP were plated together, and imaged 10 days later by dual-color time lapse microscopy. The dendrite is expressing actin-GFP and the axon is expressing RFP. Note that the spine in the center of the image is pulling on the axon, which bends upon the tension applied in a 10 min time period. This movie illustrates the trans-synaptic mechanical connection between the spine and the axon.
- Movie 7 - Movie 7: Mimicking the connection to N-cadherin beads This movie shows a vertical dendritic filopodium of dimensions 0.4 �m x 1 �m connected to a piece of dendritic shaft of dimensions 1 �m x 2 �m. The whole movie represents 450 s. The actin flow rate V was decreased from 0.02 �m/s to 0.002 �m/s at time 150 s, to mimic the connection to N-cadherin coated beads presented at the tips of filopodia. Note a slower turnover and the gradual accumulation of actin at the tip of spines.
- Movie 6 - Movie 6: PAF simulation This movie shows a vertical dendritic filopodium of dimensions 0.4 μm × 1 μm connected to a piece of dendritic shaft of dimensions 1 μm × 2 μm. Actin fluorescence is activated in a 0.4 × 0.4 μm region at the tip of the filopodium, at time 50 s after the start of the simulation. Note rearward motion of the spot, driven by actin retrograde flow (V = 0.0025 μm/s), and the progressive decline in fluorescence due to actin disassembly at the base of the filopodium. The whole sequence lasts 300 s.
- Movie 1 - Movie 1: Spine and filopodium motility Morphing of dendritic spines and filopodia in a 21 DIV neuron expressing actin-GFP. The movie lasts 5 min (1 image every 5 sec). Note actin enrichment in spines, and the absence of enrichment in actively moving filododia.
- Movie 4 - Movie 4: Optical trap experiment An N-cadherin coated bead was placed at the tip of an actively moving dendritic filopodium at time zero, and the redistribution of actin-GFP was followed over time. Note actin enrichment at the tip of the filopodium where the Ncad-Fc coated bead is placed. The movie corresponds to 15 min.
- Movie 8 - Movie 8: Simulation of Latrunculin A treatment This movie shows a vertical dendritic filopodium of dimensions 0.4 �m x 1 �m connected to a piece of dendritic shaft of dimensions 1 �m x 2 �m. The whole movie represents 450 s. The parameter kc was decreased from 0.02 s-1 to 0 s-1 at time 150 s, to mimic the addition of Latrunculin A which sequesters actin monomers, thereby preventing their polymerization at the tips of filopodia. This results in a rapid loss of actin filaments in the spines.
- Movie 5 - Movie 5: FRAP simulation The movie shows a piece of dendritic shaft of dimensions 1 μm × 6 μm, connected to two perpendicular filopodia of 0.4 μm × 1 μm. Each actin monomer is treated as a single molecule associated with a Gaussian intensity distribution. Notice the Brownian motion in the shaft. The actin flow rate if 0.002 μm/s, inducing steady state actin enrichment at the tip of filopodia. The whole sequence lasts 450 s. The spine on the right is photobleached at time 150 s. Note the progressive fluorescence recovery. The intensity is color coded.
- Movie 3 - Movie 3: Calyculin A experiment The movie shows a dendritic spine expressing actin-GFP, which was treated with 1 μM calyculin A at the beginning of the recording. The whole movie lasts 10 min. Images were thresholded to highlight an actin-GFP cluster at the tip of the spine (red), and merged with the spine contour obtained at lower threshold (green). Note the displacement of the actin cluster from the tip to the base of the spine over time.