Neuronal mRNAs travel singly into dendrites - PubMed (original) (raw)
Neuronal mRNAs travel singly into dendrites
Mona Batish et al. Proc Natl Acad Sci U S A. 2012.
Abstract
RNA transport granules deliver translationally repressed mRNAs to synaptic sites in dendrites, where synaptic activity promotes their localized translation. Although the identity of many proteins that make up the neuronal granules is known, the stoichiometry of their core component, the mRNA, is poorly understood. By imaging nine different dendritically localized mRNA species with single-molecule sensitivity and subdiffraction-limit resolution in cultured hippocampal neurons, we show that two molecules of the same or different mRNA species do not assemble in common structures. Even mRNA species with a common dendritic localization element, the sequence that is believed to mediate the incorporation of these mRNAs into common complexes, do not colocalize. These results suggest that mRNA molecules traffic to the distal reaches of dendrites singly and independently of others, a model that permits a finer control of mRNA content within a synapse for synaptic plasticity.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Individual molecules of different mRNA species do not colocalize with each other. Single-molecule FISH was performed on cultured hippocampal neurons in pairwise combinations of indicated mRNA targets. Gray-scale fluorescence images in the two Left column panels are merged images (_z_-stacks) obtained from each fluorescence channel. The color images in the third column were obtained by coding the images in the first column green, the images in the second column red, and then merging them. The _z_-stacks in both channels were analyzed using an algorithm that finds spot-like signals in each image, determines their 3D coordinates, and then looks for spots in one channel that have a counterpart in the other channel that lies within a distance of 250 nm; and if a pair of spots is found to meet that criterion, the algorithm classifies them as being colocalized. The locations of these colocalized molecules (displayed as yellow circles) along with the locations of solitary molecules (displayed as red or green circles) are overlaid onto DIC images of the neurons in the Right column panels. Although all 28 pairwise combinations were imaged (Table 1), only 4 of the 28 pairwise combinations are shown here, as these pairs provide representative results for each of the eight mRNAs that were studied. (Scale bars, 5 μm.)
Fig. 2.
Determination of empirical distances between centers of spots produced by two probe sets bound to different regions of the same target mRNA. (A) Two adjacent regions of MAP2 mRNA were probed with two probe sets labeled with distinct fluorophores. (B) Merged raw images of MAP2 mRNA molecules from two fluorescence channels were created while coding the image from one channel with green color and the other with red color. (Scale bar, 5 μm.) (C) Distribution of distances from the centers of the green spots to their nearest-neighbor red spots. (D) Classification of spots in B based on the colocalization distance limit of 250 nm. We found that 73% of all molecules were colocalized, indicating that each probe set may be able to detect 85% of the molecules (
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Fig. 3.
The number of molecules of MAP2 mRNA per neuron obtained by counting spots in the FISH images is very similar to the number obtained by real-time PCR performed on RNA extracted from the neurons. (A) Merged _z_-stacks of neurons imaged after hybridizing with probes for MAP2 mRNA. (Scale bar, 5 μm.) (B) Identified spots are overlaid onto the image shown in A to demonstrate that almost all MAP2 mRNA molecules are counted by our algorithm. (C) Quantification of mRNA copy number by real-time RT-PCR. A standard curve obtained from serial dilutions of full-length in vitro-transcribed MAP2 RNA is shown. The cyan dot identifies the result obtained from a reaction initiated by RNA isolated from resting neurons, and the pink dot identifies the result obtained from a reaction initiated by RNA isolated from the same number of neurons that had been exposed to bicuculline. (D) Comparison of the mRNA molecules per neuron determined by single-molecule FISH with the mRNA copy number per neuron determined by real-time PCR. The error bars represent 95% confidence intervals.
Fig. 4.
The maximum intensities of spots produced by three different dendritic mRNAs follow unimodal distributions. (A_–_C) Upper panels show the data obtained with a single set of 48 probes against native mRNAs, and Lower panels are different controls designed to show how the distributions would have looked if the population contained a significant proportion of two or more mRNA molecules within a single granule. To the right of each histogram is a representative image of one of the cells from the population. (A) MAP2 mRNA either bound to one set of probes that is specific for one region of each mRNA molecule (Upper) or bound to two sets of probes that are each specific for different regions of the same mRNA molecule (Lower). (B) A set of probes that bind specifically to natural β-actin mRNA was used to image uninfected neurons (Upper), and the same set of probes was used to image β-actin mRNA in neurons that had been infected with a lentiviral construct that expresses a tandem dimer of β-actin mRNA that was fused to a sequence encoding GFP (Lower). Approximately half of the spots in the images of neurons that contain mRNA molecules possessing the β-actin dimer were twice as intense as the other spots (which arise from endogenous β-actin mRNAs). (C) Two different sets of probes were bound to naturally occurring isoforms of BDNF mRNA in neurons. One set of probes bound to a region of the BDNF mRNA that is common to both isoforms, and the other set of probes bound to a region of the BDNF mRNA that occurs only in the longer isoform. Histogram and images resulting from the first set of probes and from both sets of probes. The images to the right of B and C are overlaid with circles that identify spots of unit intensity (red) and of double intensity (green). The intensities of all of the spots were fitted to a unimodal Gaussian distribution (blue curves) and to a mixture of two Gaussian distributions (red curves). The number of spots analyzed ranged from 446 to 2,306. (Scale bar, 5 μm.)
References
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