Krishan L. Taneja

Sorting of β-Actin mRNA and Protein to Neurites and Growth Cones in Culture

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes for in situ hybridization, we observed distinct localization patterns for β- and γ-actin within cultured cerebrocortical neurons. β-Actin protein was highly enriched within growth cones and filopodia, in contrast to γ-actin protein, which was distributed uniformly throughout the cell. β-Actin protein also was shown to be peripherally localized after transfection of β-actin cDNA bearing an epitope tag. β-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike γ-actin mRNAs, which were restricted to the cell body. The rapid localization of β-actin mRNA, but not γ-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolution in situ hybridization and image-processing methods, we showed that the distribution of β-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. β-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of β-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.

Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridization

The expression of muscle-specific mRNAs was analyzed directly within individual cells by in situ hybridization to chicken skeletal myoblasts undergoing differentiation in vitro. The probes detected mRNAs for sarcomeric myosin heavy chain (MHC) or the skeletal, cardiac, and beta isoforms of actin. Precise information as to the expression of these genes in individual cells was obtained and correlated directly with analyses of cell morphology and interactions, cell cycle stage, and immunofluorescence detection of the corresponding proteins. Results demonstrate that mRNAs for the two major muscle-specific proteins, myosin and actin, are not synchronously activated at the time of cell fusion. The mRNA for alpha-cardiac actin (CAct), known to be the predominant embryonic actin isoform in muscle, is expressed prior to cell fusion and prior to the expression of any isoform of muscle MHC mRNA. MHC mRNA accumulates rapidly immediately after fusion, whereas skeletal actin mRNA is expressed only in larger myofibers. Single cells expressing CAct mRNA have a characteristic short bipolar morphology, are in terminal G1, and do not contain detectable levels of the corresponding protein. In a pattern of expression reciprocal to that of CAct mRNA, beta-actin mRNA diminishes to low or undetectable levels in myofibers and in cells of the morphotype which expresses CAct mRNA. Finally, the intracellular distribution of mRNAs for different actin isoforms was compared using nonisotopic detection of isoform-specific oligonucleotide probes. This work illustrates a generally valuable approach to the analysis of cell differentiation and gene expression which directly integrates molecular, morphological, biochemical, and cell cycle information on individual cells.

Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA.

We have employed an analytical approach for the development of an in situ hybridization methodology using synthetic oligodeoxynucleotide probes for actin messenger RNA detection in cultures of chicken fibroblasts and myoblasts. The methodology developed shows that oligonucleotides can complement the use of nick-translated probes in specific situations. Since they can be made to specific nucleic acid regions independent of restriction enzyme sites, they may be the most convenient approach for analysis of gene families among which sequences are highly conserved. However, it was found that oligonucleotides synthesized to different regions of a messenger RNA behave in situ with differing efficiencies, indicating that not all target sequences are equivalent. Therefore it was necessary to screen several oligonucleotide probes to a target molecule to find the optimal one. The convenience of using synthetic DNA probes makes it worthwhile to explore some of these characteristic properties so as to increase the sensitivity of this approach beyond its application to targets in high abundance.

Actin Filaments and the Spatial Positioning of mRNAS

Filamentous actin has been shown to play a major role in the control of mRNA expression. Previous work emphasized RNA-cytoskeletal interactions using biochemical fractionation. More recently, in situ hybridization at the light microscopic and ultrastructural levels has shown that actin, in particular, is directly associated with mRNAs. It is proposed that these interactions play a major regulatory role in how the mRNA is spatially sequestered within the cytoplasm and provide a mechanism for its regulation (Singer, 1992).