This protocol describes a method for observing and measuring the movement of RNA molecules in the nucleus of living mammalian cells. Caged fluorescein-labeled DNA oligonucleotides are introduced into living mammalian cells, where they demonstrably hybridize to complementary RNA. After site-specific photoactivation at desired sites within the cell, the RNA movements away from those sites are followed and digitally recorded using a rapid acquisition microscopy system.
"Cells were either plated into two-well dishes (Nalge Nunc, Naperville, IL) or onto 25-mm coverslips and transfected with oligo(dT) or oligo(dA) as described previously (Politz et al., 2004) except that the concentration of oligo was 0.125 M. After a 1-h incubation without oligo in DMEM (with serum), cells on coverslips were mounted in a holder and then maintained at 37 or 22°C as described in DMEM buffered with 25 mM HEPES (10% FBS, no phenol red) (Politz et al., 2004). Cells containing fluorescein-labeled oligo(dT) growing in two-well dishes were imaged using a Quantix 57 charge-coupled device camera (Roper Scientific Photometrics, Tucson, AZ) coupled to a Leica DMIRB microscope equipped with a 100ϫ objective (numerical aperture 1.4) as described previously (Politz et al., 2000). "
[Show abstract][Hide abstract] ABSTRACT: Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.
Molecular Biology of the Cell 04/2006; 17(3):1239-49. DOI:10.1091/mbc.E05-10-0952 · 4.47 Impact Factor
"A rapid wide-field epifluorescence imaging system previously described (Rizzuto et al., 1998; see also Politz et al., 2003) was used to photolytically uncage and follow the movement of the oligo-tagged 28S rRNA as previously described for poly(A) RNA tracking experiments (Politz et al., 1999). Briefly, the caged-fl oligos taken up by living cells were uncaged by a 65-msec exposure to an argon laser beam ( ϭ 360 nm) directed through a pinhole inserted into the epifluorescence optical path and focused to a 1–2-m diameter spot in either the nucleolus or the nucleoplasm. "
[Show abstract][Hide abstract] ABSTRACT: Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus.
Molecular Biology of the Cell 01/2004; 14(12):4805-12. DOI:10.1091/mbc.E03-06-0395 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When cells are observed by phase contrast microscopy, nucleoli are among the most conspicuous structures. The nucleolus was formally described between 1835 and 1839, but it was another century before it was discovered to be associated with a specific chromosomal locus, thus defining it as a cytogenetic entity. Nucleoli were first isolated in the 1950s, from starfish oocytes. Then, in the early 1960s, a boomlet of studies led to one of the epochal discoveries in the modern era of genetics and cell biology: that the nucleolus is the site of ribosomal RNA synthesis and nascent ribosome assembly. This epistemologically repositioned the nucleolus as not merely an aspect of nuclear anatomy but rather as a cytological manifestation of gene action-a major heuristic advance. Indeed, the finding that the nucleolus is the seat of ribosome production constitutes one of the most vivid confluences of form and function in the history of cell biology. This account presents the nucleolus in both historical and contemporary perspectives. The modern era has brought the unanticipated discovery that the nucleolus is plurifunctional, constituting a paradigm shift.
Cold Spring Harbor perspectives in biology 11/2010; 3(3). DOI:10.1101/cshperspect.a000638 · 8.68 Impact Factor
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