Bestman JE and Cline HT (2009), The Relationship between Dendritic Branch Dynam...

XB-ART-40379

Front Neural Circuits 2009 Sep 01;3:10. doi: 10.3389/neuro.04.010.2009.

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The Relationship between Dendritic Branch Dynamics and CPEB-Labeled RNP Granules Captured in Vivo.

Bestman JE , Cline HT .

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Cytoplasmic Polyadenylation Element Binding protein (CPEB) is an RNA binding protein involved in dendritic delivery of mRNA and activity-dependent, polyadenylation-induced translation of mRNAs in the dendritic arbor. CPEB affects learning and memory and impacts neuronal morphological and synaptic plasticity. In neurons, CPEB is concentrated in ribonucleoprotein (RNP) granules that distribute throughout the dendritic arbor and localize near synapses, suggesting that the trafficking of RNP granules is important for CPEB function. We tagged full-length CPEB and an inactive mutant CPEB with fluorescent proteins, then imaged rapid dendritic branch dynamics and RNP distribution using two-photon time-lapse microscopy of neurons in the optic tectum of living Xenopus laevis tadpoles. Though the inactive CPEB mutant transports mRNA in the dendritic arbor, its expression interferes with CPEB-dependent translation because it is incapable of activity-triggered mRNA polyadenylation. In dendrites, the distributions of the active and inactive CPEB-containing RNP granules do not differ; the RNP granules are dense and their positions do not correlate with sites of rapid dendritic branch dynamics or the eventual fate of the dendritic branches. Because CPEB's sensitivity to activity-dependent signaling does not alter its dendritic distribution, it indicates that active sites in the dendritic arbor are not targeted for RNP granule localization. Nevertheless, inactive CPEB accumulates in granules in terminal dendritic branches, supporting the hypothesis that upon activation CPEB and its mRNA cargo are released from granules and are then available for dendritic translation.

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Species referenced: Xenopus laevis
Genes referenced: cfp cpeb1 fubp1

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	Figure 1. Distribution of CPEB- and delCPEB-containing RNP granules in optic tectal neurons. Optic tectal neurons labeled with cytosolic YFP (magenta) and CPEB–CFP (A) or delCPEB–CFP (B). CFP-tagged RNP granules are green puncta that when overlapping with magenta, appear white. Labeled RNP granules are distributed throughout the dendritic arbor of all cells imaged, whereas axons in 25% of the cells contain RNP granules. Examples of puncta in the locally branching axon (axons indicated with asterisks): arrows point to puncta within the axon arbor and arrowhead points to punctum at an axon tip. Scale bars = 10 μm.
	Figure 2. Time-lapse of CPEB–CFP/YFP expressing tectal neuron. One minute interval time-lapse frames made of the dendrites of the CPEB–CFP/YFP-expressing cell in Figure 1A. The time-lapse series focuses on the boxed area of the dendrites. Arrows indicate the progressive history of locations of one punctum over the 12 min, with the current position in yellow, past positions in white. Smaller insert illustrates the rapid fusing and splitting of puncta. Scale bar = 10 μm. See also Supplementary Video 1.
	Figure 3. Time-lapse of CPEB–CFP/YFP-expressing tectal neuron. One minute interval time-lapse frames made of the dendrites of a CPEB–CFP/YFP-expressing optic tectal neuron (A), with the dendritic branch boxed in yellow shown in (B). Arrow indicates the formation of a punctum and dotted lines follow puncta between time points and indicate puncta that fuse together and shift position. Scale bar = 10 μm. See also Supplementary Video 2.
	Figure 4. Time-lapse of delCPEB–CFP/YFP expressing tectal neuron. One minute interval time-lapse frames made of the dendrites of a delCPEB–CFP/YFP-expressing optic tectal neuron (A), with the dendritic branch boxed in yellow shown in (B). Arrows indicate the formation of a punctum and dotted lines follow puncta between time points and indicate puncta that fuse together and shift position. Scale bar = 10 μm. See also Supplementary Video 3.
	Figure 5. Dynamic branch behaviors of tectal neurons expressing YFP, CPEB, and delCPEB. Example of dendrites from a control cell expressing YFP alone (A), full-length CPEB–CFP and YFP (B) and delCPEB–CFP and YFP (C). Each cell was imaged seven times, once every 10 min. Below each photomicrograph is the projection of the 3D reconstruction of the dendritic arbor with the branches color coded to reflect their behaviors (illustrated in D) over the course of the 60 min. (B,C) CFP-labeled RNP granules appear as white puncta when colocalized with the YFP (in magenta). (D) Diagram illustrating the four categories of branch behaviors. Maintained branches appeared in all of the time points. The remaining branches fall into three groups: gained, branches that were absent initially and present in the last time point; transient, branches that were both added and completely retracted within the imaging session; and lost, branches that were present in the first time point and absent by the last time point. Axons are marked with asterisks, and arrows indicate puncta that are within the axon. Scale bars = 10 μm. See also Supplementary Videos 4A–C.
	Figure 6. Quantification of branching behaviors. (A,B) Control YFP-, full-length CPEB-, or delCPEB-expressing cells have similar proportions of branches with different dynamic branch behaviors (A) and with similar increases or decreases in branch length over the course of the 60-min experiment (B). (C) Proportion of branches with different durations in control, CPEB- and delCPEB-expressing neurons. delCPEB-expressing cells have fewer branches that were present for a single time point of the experiment compared to control cells (Chi-square, p = 0.01). (D) Durations of the dynamic branches of the control YFP-, CPEB-, and delCPEB-expressing neurons. The gained and lost branches of delCPEB-expressing neurons were present for significantly longer periods than control cells (unpaired t-test, p = 0.02 and 0.03, respectively). Values were measured from branches in their last appearance in the time-lapse. See Tables 1 and 2 for values.
	Figure 7. Dendritic branching is not predicted from RNP puncta positions. (A,B) Example of a tectal neuron expressing CPEB–CFP/YFP imaged once every 10 min over 1 h at the 0-min (A) and 60-min (B) time point. (C,D) Projection of the 3D reconstruction made from the 0-min (C) and 60-min (D) time point. The six side branches (in pink) that appear on the green branch are stable for the duration of the 60-min experiment. (E) The green branch from (C) and (D) was digitally linearized for each of the seven time points, and the CFP fluorescence along the branch is shown with the branch positioned with the branch tip to the right. The arrows indicate the branch point sites of the six side branches. (F,G) The green branch in this representation of the 0-min time point (F) and 60-min time point (G) loses its 5th side branch and gains three side branches (11, 12, and 13) over the 1 h time-lapse period. (H) The CFP content of the green branch from (F) and (G) as described in (E). Scale bar = 10 μm.
	Figure 8. delCPEB puncta have greater fluorescence intensity values than CPEB puncta. (A) The distributions of intensities of the puncta from delCPEB-expressing neurons, or the mean puncta intensity (inset) are significantly higher than those of the CPEB-expressing cells (Kolmogorov–Smirnov test, p < 0.0001 and unpaired t-test, p = 0.04). (B) Together, the delCPEB-expressing cells have a higher fluorescence intensity of the puncta nearest the branch points than CPEB-expressing cells (unpaired t-test, p = 0.002). The lost and maintained delCPEB-expressing neuronal branches have higher branch point puncta intensity levels compared to transient branches or the branch point puncta intensity values for lost and maintained branches of the CPEB-expressing neurons. (C,D) The mean intensity values of the branch point puncta (C) or a sum of all the puncta within a branch (D) show that compared to CPEB-expressing neurons, delCPEB-expressing cells have a significant increase in punctum intensities over the branches duration in the time-lapse, reaching values that are significantly greater than cells expressing CPEB. See Tables 3 and 4 for values.

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