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Correlated Levels of mRNA and Soma Size in Single Identified Neurons: Evidence for Compartment-specific Regulation of Gene Expression.

Ransdell JL, Faust TB, Schulz DJ - Front Mol Neurosci (2010)

Bottom Line: Levels of mRNA for the K+ channel shal, which is localized exclusively to the soma, are negatively correlated with soma size, suggesting that gene expression does not simply track positively with compartment size.Conversely, levels of beta-actin and beta-tubulin mRNA, which are major cytoskeletal proteins of neuronal processes, do not correlate with soma size, but are strongly correlated with one another.These results suggest that steady-state transcript levels are differentially regulated based on the subcellular compartment within which a given gene product primarily acts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Missouri Columbia Columbia, MO, USA.

ABSTRACT
In addition to the overall complexity of transcriptional regulation, cells also must take into account the subcellular distribution of these gene products. This is particularly challenging for morphologically complex cells such as neurons. Yet the interaction between cellular morphology and gene expression is poorly understood. Here we provide some of the first evidence for a relationship between neuronal compartment size and maintenance of mRNA levels in neurons. We find that single-cell transcript levels of 18S rRNA, GAPDH, and EF1-alpha, all gene products with primary functions in the cell soma, are strongly correlated to soma size in multiple distinct neuronal types. Levels of mRNA for the K+ channel shal, which is localized exclusively to the soma, are negatively correlated with soma size, suggesting that gene expression does not simply track positively with compartment size. Conversely, levels of beta-actin and beta-tubulin mRNA, which are major cytoskeletal proteins of neuronal processes, do not correlate with soma size, but are strongly correlated with one another. Additionally, actin/tubulin expression levels correlate with voltage-gated ion channels that are uniquely localized to axons. These results suggest that steady-state transcript levels are differentially regulated based on the subcellular compartment within which a given gene product primarily acts.

No MeSH data available.


Representative images demonstrating the measurements of soma size in STG neurons. (A) The STG consists of cell somata surrounding an area of central neuropil. (B) Individual neurons can be identified, and their somata filled with dye. (C) Following dye fill, the somata areas are digitized and quantified with Metamorph software. (B,C) From the same animal, (A) is a different individual. Scale bars as indicated.
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Figure 1: Representative images demonstrating the measurements of soma size in STG neurons. (A) The STG consists of cell somata surrounding an area of central neuropil. (B) Individual neurons can be identified, and their somata filled with dye. (C) Following dye fill, the somata areas are digitized and quantified with Metamorph software. (B,C) From the same animal, (A) is a different individual. Scale bars as indicated.

Mentions: Following dye fills, the STG was dissected out of the STNS with small pieces of surrounding nerves still attached. The STG was then pinned on a thin piece of Sylgard (Dow Corning, Midland, MI, USA), inverted and placed on a microscope slide in physiological saline. Preparations remained un-fixed to avoid any change in soma volume as a result of fixation or dehydration. 10× fluorescent images of the STG were taken with an Olympus IX70 microscope and ORCA-AG camera at the University of Missouri-Columbia Cytology Core (Figure 1B). These images were analyzed with Metamorph software (Molecular Devices, Sunnyvale, CA, USA). The image was calibrated to a 10× objective and the somata were isolated from the neurite using a threshold algorithm (Figure 1C). Area measures of the isolated cell soma were taken and normalized to a PD cell's soma area within the same preparation to avoid any variability due to mounting conditions and/or focal length. The total time elapsed from dye fill to imaging of the soma was between 1 and 2 h. Area measurements have been shown to correlate with cell volume across multiple species (Satoh et. al., 1996). Figure 1 displays an example of cell soma measurements in a single STG.


Correlated Levels of mRNA and Soma Size in Single Identified Neurons: Evidence for Compartment-specific Regulation of Gene Expression.

Ransdell JL, Faust TB, Schulz DJ - Front Mol Neurosci (2010)

Representative images demonstrating the measurements of soma size in STG neurons. (A) The STG consists of cell somata surrounding an area of central neuropil. (B) Individual neurons can be identified, and their somata filled with dye. (C) Following dye fill, the somata areas are digitized and quantified with Metamorph software. (B,C) From the same animal, (A) is a different individual. Scale bars as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2991126&req=5

Figure 1: Representative images demonstrating the measurements of soma size in STG neurons. (A) The STG consists of cell somata surrounding an area of central neuropil. (B) Individual neurons can be identified, and their somata filled with dye. (C) Following dye fill, the somata areas are digitized and quantified with Metamorph software. (B,C) From the same animal, (A) is a different individual. Scale bars as indicated.
Mentions: Following dye fills, the STG was dissected out of the STNS with small pieces of surrounding nerves still attached. The STG was then pinned on a thin piece of Sylgard (Dow Corning, Midland, MI, USA), inverted and placed on a microscope slide in physiological saline. Preparations remained un-fixed to avoid any change in soma volume as a result of fixation or dehydration. 10× fluorescent images of the STG were taken with an Olympus IX70 microscope and ORCA-AG camera at the University of Missouri-Columbia Cytology Core (Figure 1B). These images were analyzed with Metamorph software (Molecular Devices, Sunnyvale, CA, USA). The image was calibrated to a 10× objective and the somata were isolated from the neurite using a threshold algorithm (Figure 1C). Area measures of the isolated cell soma were taken and normalized to a PD cell's soma area within the same preparation to avoid any variability due to mounting conditions and/or focal length. The total time elapsed from dye fill to imaging of the soma was between 1 and 2 h. Area measurements have been shown to correlate with cell volume across multiple species (Satoh et. al., 1996). Figure 1 displays an example of cell soma measurements in a single STG.

Bottom Line: Levels of mRNA for the K+ channel shal, which is localized exclusively to the soma, are negatively correlated with soma size, suggesting that gene expression does not simply track positively with compartment size.Conversely, levels of beta-actin and beta-tubulin mRNA, which are major cytoskeletal proteins of neuronal processes, do not correlate with soma size, but are strongly correlated with one another.These results suggest that steady-state transcript levels are differentially regulated based on the subcellular compartment within which a given gene product primarily acts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Missouri Columbia Columbia, MO, USA.

ABSTRACT
In addition to the overall complexity of transcriptional regulation, cells also must take into account the subcellular distribution of these gene products. This is particularly challenging for morphologically complex cells such as neurons. Yet the interaction between cellular morphology and gene expression is poorly understood. Here we provide some of the first evidence for a relationship between neuronal compartment size and maintenance of mRNA levels in neurons. We find that single-cell transcript levels of 18S rRNA, GAPDH, and EF1-alpha, all gene products with primary functions in the cell soma, are strongly correlated to soma size in multiple distinct neuronal types. Levels of mRNA for the K+ channel shal, which is localized exclusively to the soma, are negatively correlated with soma size, suggesting that gene expression does not simply track positively with compartment size. Conversely, levels of beta-actin and beta-tubulin mRNA, which are major cytoskeletal proteins of neuronal processes, do not correlate with soma size, but are strongly correlated with one another. Additionally, actin/tubulin expression levels correlate with voltage-gated ion channels that are uniquely localized to axons. These results suggest that steady-state transcript levels are differentially regulated based on the subcellular compartment within which a given gene product primarily acts.

No MeSH data available.