We present a depth-resolved Image Mapping Spectrometer (IMS) which is capable of acquiring 4D (x, y, z, λ) datacubes. Optical sectioning is implemented by structured illumination. The device's spectral imaging performance is demonstrated in a multispectral microsphere and mouse kidney tissue fluorescence imaging experiment. We also compare quantitatively the depth-resolved IMS with a hyperspectral confocal microscope (HCM) in a standard fluorescent bead imaging experiment. The comparison results show that despite the use of a light source with four orders of magnitude lower intensity in the IMS than that in the HCM, the image signal-to-noise ratio acquired by the IMS is 2.6 times higher than that achieved by the equivalent confocal approach. OCIS codes: (110.4234) Multispectral and hyperspectral imaging; (180.2520) Fluorescence microscopy; (170.6280) Spectroscopy, fluorescence and luminescence.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(T)) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 alpha-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E(app)) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on E(app) measurements for two donor/acceptor pairs, Ste2p-CFP/Ste2p-YFP and Ste2p-GFP/Ste2p-YFP. From E(app) and E we determined alpha(T) approximately 1 and n approximately 2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization.

RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro and in vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show that RPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of ⌬rpb9 cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of ⌬rpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of ⌬rpb9 cells revealed only a few changes, predominantly in genes related to metabolism.RNA polymerase II comprises 12 subunits in yeast (1). Four of the subunits, Rpb1, Rpb2, Rpb3, and Rpb11, form a catalytic core that is homologous in structure and function to the prokaryotic core RNA polymerase (2, 3). The other eight eukaryotic subunits are less well characterized. Five of these subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are found in all three eukaryotic RNA polymerases (4 -6). The other three, Rpb4, Rpb7, and Rpb9, are unique to RNA polymerase II, although both Rpb7 and Rpb9 have sequence homologues in RNA polymerases I and III (7). The gene for Rpb9 is not essential for yeast cell viability, but is essential in Drosophila (8).Rpb9 has roles both in transcription initiation and in transcription elongation. In the initiation reaction, Rpb9 modulates the selection of the transcription start site. In cells lacking Rpb9 and in reconstituted transcription reactions lacking Rpb9, the population of start sites is shifted upstream at a variety of promoters (9 -11). In the elongation reaction, Rpb9 is required, along with TFIIS, to effect transcription through blocks to elongation encoded by the DNA template (12). A role in the modulation of initiation and elongation is consistent with the localization of Rpb9 in the three-dimensional structure of yeast RNA polymerase II. Rpb9 is located at the tip of the so-called "jaws" of the enzyme, which is thought to function by clamping the DNA downstream of the active site (3, 13, 14). The Rpb9 homologue in RNA polymerase III, C11, also has been implicated in regulating RNA chain elongation (15).Rpb9 comprises two zinc ribbon domains joined by a 30-amino acid l...

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M 2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M 2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M 2 receptor to enhanced green fluorescent protein-M 2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n ‫؍‬ 2, 0.495 ؎ 0.019; n ‫؍‬ 4, 0.202 ؎ 0.010; n ‫؍‬ 6, 0.128 ؎ 0.006; n ‫؍‬ 8, 0.093 ؎ 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 -0.24, identifying the M 2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.Much evidence now indicates that G protein-coupled receptors can exist as oligomers (1, 2), a development that has implications for all aspects of GPCR 4 -mediated signaling. Among the many questions prompted by the emergence of such structures is that of oligomeric size. Although commonly referred to as dimers, oligomers of GPCRs have been detected most often by means of coimmunoprecipitation or resonance energy transfer (3). As typically applied, neither technique can distinguish dimers from larger oligomers. The latter have been identified on the basis of their electrophoretic mobility (reviewed in Ref. 4), but the composition of the bands may be unclear, and the size under the conditions of electrophoresis may have little in common with that in the membrane. Larger oligomers also have been identified by approaches in which detection requires the colocalization of three or four proteins, each bearing a different tag (5-11), but such procedures place only a lower limit on the possible size of the array.There have been comparatively few attempts to examine the oligomeric status of a GPCR in a more quantitative and explicit manner. Measurements of BRET at different ratios of acceptor to donor have pointed to dimers of the melatonin receptor (12), the ␤ 1 -and ␤ 2 -adrenergic receptors (13), the M 1 , M 2 , and M 3 muscarinic receptors (14), and the neurotensin receptor (15)....