The Journal of Neuroscience, June 14, 2006, ():
Dendritic Localization of the Translational Repressor Pumilio 2 and Its Contribution to Dendritic Stress Granules
J. Neurosci. Vessey et al.
26: 6496
Supplemental data
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Supplemental Table
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Supplementary Figure 1. A, Pum2 antibody competition test. Rat embryonic brain lysate was prepared, electrophoresed and immunoblotted with anti-Pum2 antibody (Ab) or anti-Pum2 antibody pre-absorbed (for 2h at 4°C with 5-fold by weight excess) with full length GST-Pum2 (Ab + P). The arrow indicates Pum2. M; molecular weight marker. B, Fluorescence intensity line scans of distal dendrites in neurons stained for or transfected with components of SGs and P-bodies after 1 hour of exposure to a stress stimulus. The top left panel is a line scan from Figure 5A and represents fluorescence intensity for antibodies detecting Pum2 (red) and TIAR (green). The distribution of the signal for both antibodies demonstrates significant overlap. The top right panel is an antibody stain for TIAR (green) and FISH for polyadenylated mRNA (red). Again, a similar distribution of intensities is apparent. The lower left line scan of a dendrite from Figure 4B represents the signal distribution of Pum2 (green) and mRNA (red) also showing significant signal overlap. The lower right panel is of a dendrite from a neuron in Figure 7A (lower panels; + arsenite) that was transfected with Dcp1-EGFP, a marker for P-bodies, and stained for Pum2 (red). The patterns of fluorescence intensity show little similarity, representing no significant co-localization of the two proteins. C, Time-course of SG formation in HeLa cells exposed to arsenite. HeLa expressing Pum2FL-EYFP were exposed to arsenite (at time point 0) and imaged over a period of 48 minutes. SG formation is detected approximately 16 minutes following the addition of arsenite in the cell displaying the highest levels of Pum2 FL-EYFP over-expression (left). In the two cells on the right, showing lower levels of Pum2 FL-EYFP expression, SG formation occurs approximately 24 minutes following the addition of arsenite. The phase contrast image in the bottom right-hand corner shows the HeLa cells at the end of the time-lapse video 48 minutes after the addition of arsenite (note the presence of two untransfected HeLa cells not visible in the fluorescence images). Videos are available upon request.
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Supplementary Figure 2. A, The amino acid sequence of the Pum2 N-terminus showing the Q (glutamine) rich region (QRR). The glutamine repeats have been highlighted in black. B, Over-expression of full length Pum2-EGFP leads to the formation of SGs even in the absence of a stress stimuli that recruit other RNA-binding proteins such as Btz and Stau1 (see also glial cell image). Scale bar = 10 μm.
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Supplementary Figure 3. A, Over-expression of Maskin-CFP and CPEB1-EGFP followed by treatment with 0.5 mM arsenite for one hour leads to the formation of Pum2-positive SGs in the soma and distal dendrites. Unlike Stau1 and Btz, Pum2 does not reside in the population of granules that contain Maskin and CPEB1. B, PABP is a marker for neuronal SGs. Immunostaining for TIAR and PABP was performed in hippocampal neurons after exposure of arsenite. Significant co-localization of the two proteins is evident. C, Co-transfection of COS cells with Pum2-myc and either si-Pum2-2, mis-Pum2-2, the empty pSuperior vector or untransfected control cells. A Western blot stained with an anti-myc antibody demonstrates significant loss of the myc signal in the presence of si-Pum2-2. D, Transfection of the si-Pum2-2 or mis-Pum2-2 plasmids followed by immunostaining for Pum2 (red). si-Pum2-2 transfected cells displayed little or no fluorescence signal representing loss of the Pum2 protein. Neurons transfected with the mis-Pum2-2 plasmid did not undergo changes in Pum2 fluorescence intensity. Scale bar = 10 μm.
