The structure of many proteins entering the secretory pathway is dependent on stabilization by disulfide bonds. To support disulfide-linked folding, the endoplasmic reticulum (ER) must maintain a strongly oxidizing environment compared to the highly reduced environment of the cytosol. We report here the identification and characterization of Ero1p, a novel and essential ER-resident protein. Mutations in Ero1p cause extreme sensitivity to the reducing agent DTT, whereas overexpression confers DTT resistance. Strikingly, compromised Ero1p function results in ER retention of disulfide-stabilized proteins in a reduced, nonnative form, while not affecting structural maturation of a disulfide-free protein. We conclude that there exists a specific cellular redox machinery required for disulfide-linked protein folding in the ER and that Ero1p is an essential component of this machinery.

Four-dimensional fluorescence microscopy-which records 3D image information as a function of time-provides an unbiased way of tracking dynamic behavior of subcellular components in living samples and capturing key events in complex macromolecular processes. Unfortunately, the combination of phototoxicity and photobleaching can severely limit the density or duration of sampling, thereby limiting the biological information that can be obtained. Although widefield microscopy provides a very light-efficient way of imaging, obtaining high-quality reconstructions requires deconvolution to remove optical aberrations. Unfortunately, most deconvolution methods perform very poorly at low signal-to-noise ratios, thereby requiring moderate photon doses to obtain acceptable resolution. We present a unique deconvolution method that combines an entropy-based regularization function with kernels that can exploit general spatial characteristics of the fluorescence image to push the required dose to extreme low levels, resulting in an enabling technology for high-resolution in vivo biological imaging.4D microscopy | low dose microscopy | noise-suppressing regularization T he study of dynamic processes is an important facet of cell biology research. Fluorescently tagged proteins combined with four-dimensional fluorescence microscopy, which records 3D image information as a function of time, provide a powerful framework for studying the dynamics of molecular processes in vivo. One of the most crucial challenges in 4D fluorescence microscopy is to ensure that normal biological function is not significantly perturbed as a result of the high doses of illumination (phototoxicity) incurred during 4D imaging. Recent work indicates that the maximal photon dose that avoids biological perturbation is 100-to 1,000-fold lower than that typically used for in vivo imaging (1). Dose limitations are even more challenging, given the desire to densely sample in time or to record over extended periods, especially in the context of analyzing multiple subcellular components via multiwavelength imaging.Under normal imaging conditions, widefield microscopy combined with image restoration using deconvolution methods provides an excellent modality for multiwavelength 4D imaging as it makes very efficient use of the illuminating photons. However, its effectiveness, in particular its ability to resolve subcellular detail sufficiently in the presence of noise, is limited by the performance of the deconvolution method. Such limitations can seriously degrade image quality at the low signal levels required for unperturbed in vivo imaging. The noise behavior of the deconvolution algorithm is determined by the efficiency of the noise stabilization term, known as the regularization functional. In particular, the functional's ability to discriminate the noise-related high frequencies from weak high frequencies in the signal ultimately determines the final resolution of the deconvolution. Currently used noise-stabilization techniques are largely based on ad hoc form...

The modified nucleoside 2-thiocytidine (s 2 C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and Bacteria. In the bacteria Escherichia coli and Salmonella enterica serovar

Tracking biological events in living cells provides kinetic information about biological processes that can be missed in more traditional methods using fixed samples at designated time intervals. Here we describe a methodology for in vivo fluorescence microscopy of yeast cells undergoing meiosis. This method allows tracking of individual cells over extended periods of time through every stage of the meiotic transformation while minimizing phototoxicity and sustaining conditions that support meiotic growth.

The synaptonemal complex (SC) is a dynamic structure formed between chromosomes during meiosis which stabilizes and supports many essential meiotic processes such as pairing and recombination. In budding yeast, Zip1 is a functionally conserved element of the SC that is important for synapsis. Here, we directly measure the kinetics of Zip1-GFP assembly and disassembly in live cells of the yeast S. cerevisiae. The imaging of SC assembly in yeast is challenging due to the large number of chromosomes packed into a small nucleus. We employ a zip3Δ mutant in which only a few chromosomes undergo synapsis at any given time, initiating from a single site on each chromosome, thus allowing the assembly and disassembly kinetics of single SCs to be accurately monitored in living cells. SC assembly occurs with both monophasic and biphasic kinetics, in contrast to the strictly monophasic assembly seen in C. elegans. In wild-type cells, once maximal synapsis is achieved, programmed final disassembly rapidly follows, as Zip1 protein is actively degraded. In zip3Δ, this period is extended and final disassembly is prolonged. Besides final disassembly, we found novel disassembly events involving mostly short SCs that disappeared in advance of programmed final disassembly, which we termed “abortive disassembly.” Abortive disassembly is distinct from final disassembly in that it occurs when Zip1 protein levels are still high, and exhibits a much slower rate of disassembly, suggesting a different mechanism for removal in the two types of disassembly. We speculate that abortive disassembly events represent defective or stalled SCs, possibly representing SC formation between non-homologs, that is then targeted for dissolution. These results reveal novel aspects of SC assembly and disassembly, potentially providing evidence of additional regulatory pathways controlling not just the assembly, but also the disassembly, of this complex cellular structure.