Poly(A) RNA Codistribution with Microfilaments: Evaluation by in situ Hybridization and Quantitative Digital Imaging Microscopy

• Published in: The Journal of cell biology 1992-12, Vol.119 (5), p.1245-1260
• Main Authors: Taneja, Krishan L., Lifshitz, Lawrence M., Fay, Fredric S., Singer, Robert H.
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - drug effects; Actin Cytoskeleton - ultrastructure; Actins; Actins - isolation & purification; Biological and medical sciences; Cells; Cellular biology; Chi-Square Distribution; Cytochalasin D - pharmacology; Cytochalasins; Cytoplasm; Cytoplasm - ultrastructure; Cytoskeleton; Diploidy; Diverse techniques; Dose-Response Relationship, Drug; Fibroblasts - drug effects; Fibroblasts - ultrastructure; Fundamental and applied biological sciences. Psychology; HeLa cells; Histocytochemistry; Humans; Image Processing, Computer-Assisted; In Situ Hybridization; Intracellular Membranes - ultrastructure; Messenger RNA; Microfilaments; Microscopy, Fluorescence; Molecular and cellular biology; Poly A - isolation & purification; Polyethylene Glycols - pharmacology; Puromycin - pharmacology; Ribonuclease, Pancreatic - pharmacology; Ribonucleic acid; Ribosomes - ultrastructure; RNA; RNA, Messenger - drug effects; RNA, Messenger - isolation & purification; Time Factors
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.119.5.1245

The distribution of poly(A) RNA has been visualized in single cells using high-resolution fluorescent in situ hybridization. Digital imaging microscopy was used to quantitate the signal in various cellular compartments. Most of the poly(A) signal remained associated with the cellular filament systems after solubilization of membranes with Triton, dissociation of ribosomes with puromycin, and digestion of nonpoly(A) RNA with ribonuclease A and T1. The actin filaments were shown to be the predominant cellular structural elements associating with the poly(A) because low doses of cytochalasin released about two-thirds of the poly(A). An approach to assess the extent of colocalization of two images was devised using in situ hybridization to poly(A) in combination with probes for ribosomes, membranes, or F-actin. Digital imaging microscopy showed that most poly(A) spatially distributes most significantly with ribosomes, slightly less with F-actin, and least of all with membranes. The results suggest a mechanism for anchoring (and perhaps moving) much of the cellular mRNA utilizing the interaction between actin filaments and poly(A).