Journal: Nature Communications

Article Title: Live cell imaging reveals 3′-UTR dependent mRNA sorting to synapses

doi: 10.1038/s41467-019-11123-x

Figure Lengend Snippet: Rgs4 3′-UTR mediates mRNA recruitment to synapses dependent on neuronal activity. a Representative dual-color kymograph showing MS2+Rgs4 3′-UTR reporter mRNA (green) and PSD-95-tagRFPt (magenta) from a dendrite of a rat hippocampal neuron. First and last frames are shown at top and bottom. Scale bar 5 µm. Extracted track (right) of an mRNA granule docking at a PSD-95 positive area indicated by arrowheads. Distance of MS2 only or MS2+Rgs4 3′-UTR reporter mRNA docking events to closest PSD-95 positive cluster in neurons displayed as dot plot ( b ) and density plot ( c ). d Distribution of MS2 only or MS2+Rgs4 reporter mRNA-positive (estimated RNA number ≥1 at t = 0 s) and -negative (RNA = 0 at t = 0 s) PSD-95-tagRFPt clusters in soma and dendrites. P-values of Chi tests against control are indicated. e Integrated frequency of reporter docking and undocking events at dendritic synapses. Number of observations and population means are indicated. f Average net change of MS2 only or MS2+Rgs4 mRNA content at mRNA reporter-positive or -negative synapses per min, calculated from estimated reporter molecules docking or undocking at synapses per event. Numbers indicate mean value of net RNA level change. Error bars represent 95% confidence intervals. ** indicates significant ( α = 0.01) difference compared to zero (null hypothesis, two sided one-sample t -test). Distance of MS2+Rgs4 3′-UTR reporter mRNA docking events to closest PSD-95 positive cluster under untreated, vehicle treated (DMSO) or silenced (100 µM CNQX, 50 µM AP5, 1 µM TTX) conditions displayed as dot plot ( g ) and density plot ( h ). Data represent mean ± standard deviation of three independent experiments (individual experiments shown as gray dots; b , g ). Dashed lines represent mean values of single data points ( c , h ). Asterisks represent p -values obtained by Student’s t test ( b ), Mann Whitney U test ( e ) or Tukey’s test post-hoc to one-way ANOVA analysis ( g ) (** p < 0.01, *** p < 0.001). Data were obtained from 40-µm dendritic segments at a minimal distance of 20 µm from the cell body. At least 10 dendrites/condition/experiment ( a – c , g , h ) or 12 neurons/condition ( d – f ) from 3 independent biological replicates were analyzed. Total number of dendrites ( n d ), events ( n e ) and synapses ( n ) analyzed per condition are indicated

Article Snippet: The following antibodies were used: (i) polyclonal antibodies, i.e. selfmade rabbit anti-Stau2 , guinea pig anti-VGLUT1 (Synaptic Systems, 419005); (ii) secondary antibodies, i.e. donkey anti-rabbit and goat anti-guinea pig Alexa555 or Alexa647 conjugated (Life Technologies, A31572, A31573, A21450).

Techniques: Activity Assay, Control, Standard Deviation, MANN-WHITNEY