No abstract

Fluorescence microscopy offers a minimally perturbative approach to probe biology in vivo. However, available techniques are limited both in sensitivity and temporal resolution for commonly used fluorescent proteins. Here we present a new imaging system with a diagnostic toolkit that caters for the detection and quantification of fluorescent proteins for use in fast functional imaging at the single-molecule level. It utilizes customized microscopy with a mode of illumination we call "slimfield" suitable for rapid (approximately millisecond) temporal resolution on a range of common fluorescent proteins. Slimfield is cheap and simple, allowing excitation intensities approximately 100 times greater than those of widefield imaging, permitting single-molecule detection at high speed. We demonstrate its application on several purified fluorescent proteins in standard use as genetically-encoded reporter molecules. Controlled in vitro experiments indicate single protein molecules over a field of view of approximately 30 microm(2) area, large enough to encapsulate complete prokaryotic and small eukaryotic cells. Using a novel diagnostic toolkit we demonstrate automated detection and quantification of single molecules with maximum imaging rates for a 128 x 128 pixel array of approximately 500 frames per second with a localization precision for these photophysically poor fluorophores to within 50 nm. We report for the first time the imaging of the dim enhanced cyan fluorescent protein (ECFP) and CyPet at the single-molecule level. Applying modifications, we performed simultaneous dual-colour slimfield imaging for use in co-localization and FRET. We present preliminary in vivo imaging on bacterial cells and demonstrate approximately millisecond timescale functional imaging at the single-molecule level with negligible photodamage.

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not timely repaired. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including topoisomerase-1 trapped in covalent crosslinks (Top1ccs). Here we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S-phase dependent manner to assist eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitize cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among potential Ddi1 targets we found the core component of RNAP II and show that its genotoxin-induced degradation is impaired in ddi1. Together, we propose that the Ddi1 protease contributes to DPC proteolysis.

Caloric restriction, a reduction in calorie intake without malnutrition, retards age-related degeneration and extends lifespan in several organisms. CR induces multiple changes, yet its underlying mechanisms remain poorly understood. In this work, we first performed a meta-analysis of microarray CR studies in mammals and identified genes and processes robustly altered due to CR. Our results reveal a complex array of CR-induced changes and we re-identified several genes and processes previously associated with CR, such as growth hormone signalling, lipid metabolism and immune response. Moreover, our results highlight novel associations with CR, such as retinol metabolism and copper ion detoxification, as well as hint of a strong effect of CR on circadian rhythms that in turn may contribute to metabolic changes. Analyses of our signatures by integrating co-expression data, information on genetic mutants, and transcription factor binding site analysis revealed candidate regulators of transcriptional modules in CR. Our results hint at a transcriptional module involved in sterol metabolism regulated by Srebf1. A putative regulatory role of Ppara was also identified. Overall, our conserved molecular signatures of CR provide a comprehensive picture of CR-induced changes and help understand its regulatory mechanisms.

Protein arginine methylation is a PTM involved in various cellular processes in eukaryotes. Recent discoveries led to a vast expansion of known sites in higher organisms, indicating that this modification is more widely spread across the proteome than previously assumed. An increased knowledge of sites in lower eukaryotes may facilitate the elucidation of its functions. In this study, we present the discovery of arginine mono-methylation sites in Saccharomyces cerevisiae by a combination of immunoaffinity enrichment and MS/MS. As detection of methylation is prone to yield false positives, we demonstrate the need for stringent measures to avoid elevated false discovery rates. To this end, we employed MethylSILAC in combination with a multistep data analysis strategy. We report 41 unambiguous methylation sites on 13 proteins. Our results indicate that, while substantially less abundant, arginine methylation follows similar patterns as in higher eukaryotes in terms of sequence context and functions of methylated proteins. The majority of sites occur on RNA-binding proteins participating in processes from transcription and splicing to translation and RNA degradation. Additionally, our data suggest a bias for localization of arginine methylation in unstructured regions of proteins, which frequently involves Arg-Gly-Gly motifs or Asn-rich contexts.

TOR and PKA signaling are the major growth-regulatory nutrient-sensing pathways in S. cerevisiae. A number of experimental findings demonstrated a close relationship between these pathways: Both are responsive to glucose availability. Both regulate ribosome production on the transcriptional level and repress autophagy and the cellular stress response. Sch9, a major downstream effector of TORC1 presumably shares its kinase consensus motif with PKA, and genetic rescue and synthetic defects between PKA and Sch9 have been known for a long time. Further, studies in the first decade of this century have suggested direct regulation of PKA by TORC1. Nonetheless, the contribution of a potential direct cross-talk vs. potential sharing of targets between the pathways has still not been completely resolved. What is more, other findings have in contrast highlighted an antagonistic relationship between the two pathways. In this review, I explore the association between TOR and PKA signaling, mainly by focusing on proteins that are commonly referred to as shared TOR and PKA targets. Most of these proteins are transcription factors which to a large part explain the major transcriptional responses elicited by TOR and PKA upon nutrient shifts. I examine the evidence that these proteins are indeed direct targets of both pathways and which aspects of their regulation are targeted by TOR and PKA. I further explore if they are phosphorylated on shared sites by PKA and Sch9 or when experimental findings point towards regulation via the PP2ASit4/PP2A branch downstream of TORC1. Finally, I critically review data suggesting direct cross-talk between the pathways and its potential mechanism.

Inherited retinal diseases are mainly caused by mutations in genes that are highly expressed in photoreceptors of the retina. The majority of these genes is under the control of the transcription factor Cone rod homeobox (Crx), that acts as a master transcription factor in photoreceptors. Using a genome-wide chromatin immunoprecipitation dataset that highlights all potential in vivo targets of Crx, we have identified a novel sterile alpha motif (SAM) domain containing protein, Samd7. mRNA Expression of Samd7 was confined to the late postnatal and adult mouse retina as well as the pineal gland. Using immunohistochemistry and Western blot, we could detect Samd7 protein in the outer nuclear layer of adult mouse retina. Ectopic over-expression in HEK293 cells demonstrated that Samd7 resides in the cytoplasm as well as the nucleus. In vitro electroporation of fluorescent reporters into living mouse retinal cultures revealed that transcription of the Samd7 gene depends on evolutionary conserved Crx motifs located in the first intron enhancer. Moreover, Crx knock-down with shRNA strongly reduced Samd7 reporter activity and endogenous Samd7 protein, indicating that Crx is required for retinal expression of Samd7. Finally, using co-transfections in luciferase reporter assays we found that Samd7 interferes with Crx-dependent transcription. Samd7 suppressed luciferase activity from a reporter plasmid with five Crx consensus repeats in a dose dependent manner and reduced Crx-mediated transactivation of regulatory sequences in the retinoschisin gene and the Samd7 gene itself. Taken together, we have identified a novel retinal SAM domain protein, Samd7, which could act as a transcriptional repressor involved in fine-tuning of Crx-regulated gene expression.

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not timely repaired. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including topoisomerase-1 trapped in covalent crosslinks (Top1ccs). Here we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S-phase dependent manner to assist eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitize cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among potential Ddi1 targets we found the core component of RNAP II and show that its genotoxin-induced degradation is impaired in ddi1. Together, we propose that the Ddi1 protease contributes to DPC proteolysis.