Development and Application of Ultrastructural in Situ Hybridization to Visualize the Spatial Organization of mRNA: a Dissertation
AuthorsBassell, Gary J.
Faculty AdvisorDr. Robert H. Singer
Academic ProgramCell Biology
UMass Chan AffiliationsCell Biology
Document TypeDoctoral Dissertation
Amino Acids, Peptides, and Proteins
Nucleic Acids, Nucleotides, and Nucleosides
MetadataShow full item record
AbstractIt has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/31451
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