Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy.
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 non-poly(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).
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
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Taneja
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Lifshitz
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Fay
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Singer
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure*; D000199:Actins / Q000302:isolation & purification; D016009:Chi-Square Distribution; D015638:Cytochalasin D / Q000494:pharmacology; D003593:Cytoplasm / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000648:ultrastructure*; D006651:Histocytochemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization; D007425:Intracellular Membranes / Q000648:ultrastructure; D008856:Microscopy, Fluorescence; D011061:Poly A / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology; D012270:Ribosomes / Q000648:ultrastructure; D013997:Time Factors
D000199:Actins; D012333:RNA, Messenger; D015638:Cytochalasin D; D011061:Poly A; D011092:Polyethylene Glycols; D011691:Puromycin; D012259:Ribonuclease, Pancreatic