Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging

The Compact Linear Collider (CLIC) - 2018 Summary Report

The Compact Linear Collider (CLIC) is a TeV-scale high-luminosity linear $e^+e^-$ collider under development at CERN. Following the CLIC conceptual design published in 2012, this report provides an overview of the CLIC project, its current status, and future developments. It presents the CLIC physics potential and reports on design, technology, and implementation aspects of the accelerator and the detector. CLIC is foreseen to be built and operated in stages, at centre-of-mass energies of 380 GeV, 1.5 TeV and 3 TeV, respectively. CLIC uses a two-beam acceleration scheme, in which 12 GHz accelerating structures are powered via a high-current drive beam. For the first stage, an alternative with X-band klystron powering is also considered. CLIC accelerator optimisation, technical developments and system tests have resulted in an increased energy efficiency (power around 170 MW) for the 380 GeV stage, together with a reduced cost estimate at the level of 6 billion CHF. The detector concept has been refined using improved software tools. Significant progress has been made on detector technology developments for the tracking and calorimetry systems. A wide range of CLIC physics studies has been conducted, both through full detector simulations and parametric studies, together providing a broad overview of the CLIC physics potential. Each of the three energy stages adds cornerstones of the full CLIC physics programme, such as Higgs width and couplings, top-quark properties, Higgs self-coupling, direct searches, and many precision electroweak measurements. The interpretation of the combined results gives crucial and accurate insight into new physics, largely complementary to LHC and HL-LHC. The construction of the first CLIC energy stage could start by 2026. First beams would be available by 2035, marking the beginning of a broad CLIC physics programme spanning 25-30 years

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging

Single-cell sperm transcriptomes and variants from fathers of children with and without autism spectrum disorder

The human sperm is one of the smallest cells in the body, but also one of the most important, as it serves as the entire paternal genetic contribution to a child. Investigating RNA and mutations in sperm is especially relevant for diseases such as autism spectrum disorders (ASD), which have been correlated with advanced paternal age. Historically, studies have focused on the assessment of bulk sperm, wherein millions of individual sperm are present and only high-frequency variants can be detected. Using 10× Chromium single-cell sequencing technology, we assessed the transcriptome from >65,000 single spermatozoa across six sperm donors (scSperm-RNA-seq), including two who fathered multiple children with ASD and four fathers of neurotypical children. Using RNA-seq methods for differential expression and variant analysis, we found clusters of sperm mutations in each donor that are indicative of the sperm being produced by different stem cell pools. Finally, we have shown that genetic variations can be found in single sperm

C-terminal tagging of Cit1p with mTFP1 does not disrupt localization or function of the protein.

<p>A) BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, or Cit1-mCherry were stained with 1 μg/ml DAPI for 10 min as described in Materials and Methods. DAPI-stained cells were imaged on a wide-field microscope. Z-series were collected through the entire cell at 0.5 μm intervals using a metal halide lamp and appropriate filters at 216 gain and 200 ms exposure time. Cell outlines were drawn over phase images. Scale bar = 1 μm. n = nucleus. mtDNA = mitochondrial DNA. B) BY4741 and cit1∆ cells, and BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, or Cit1-mCherry were grown to mid-log phase in YPD and diluted to OD₆₀₀ = 0.01 in YPG. 10-fold serial dilutions were performed and 5 μl was placed on solid media (YPG) and grown at 30°C for three days. Images are representative of three independent trials.</p

Perceived brightness and stability of mTFP1 relative to GFP, mCitrine, and mCherry.

<p>BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, or Cit1-mCherry were imaged on a wide-field microscope. Single-plane, time-lapse imaging was performed at the center of the cell at 1 sec intervals for 120 sec using a metal halide lamp and appropriate filters at 216 gain and 200 ms exposure for each fluorophore. A) Integrated fluorescence density at time = 0 was measured using Image J as described in Materials and Methods. *** = p < 0.001. Error bars are SEM. B) Integrated fluorescence density was measured at each time point and normalized to the integrated fluorescence density at time = 0 and graphed as % fluorescence remaining as a function of time. Error bars are SEM. n = 28–44 cells per strain. Data is representative of 3 independent trials.</p

Utilization of mTFP1, mCitrine, mCherry and DAPI for 3- and 4-color, live-cell imaging.

<p>A) BY4741 cells expressing Cit1-mTFP1, Pho88-mCitrine, and Erg6-mCh were imaged either untreated (left panels) or after staining with 1 μg/mL DAPI for 10 min (right panel) at 30°C as described in Materials and Methods. B) Images of BY4741 cells expressing Erg6-mTagBFP2, Cit1-mTFP, Pho88-CmCitrine, and Vph1-mCherry. For A-B, Z-series were collected with wide-field microscopy at 0.5 μm intervals throughout the entire cell using a metal-halide lamp and appropriate filters using 216 gain and the following exposure times: 100 ms for Cit1-mTFP1, 200 ms for Pho88-mCitrine, 200 ms for Erg6-mCherry, 100 ms for DAPI, and 400 ms for mTagBFP2. Cell outlines were drawn over phase images. Scale bar = 1 μm. n = nucleus. mtDNA = mitochondrial DNA. nER = nuclear ER. cER = cortical ER.</p

Spectral compatibility of mitochondria-targeted FPs.

<p>BY4741 cells expressing Cit1-GFP, Cit1-mTFP1, Cit1-mCitrine, and Cit1-mCherry were imaged on a wide-field microscope. Z-series were collected at 0.5 μm intervals throughout the entire cell using a metal-halide lamp with standard GFP, CFP, YFP, and rhodamine filters using 216 gain and 200 ms exposure times. Full filter specifications are listed in Materials and Methods. Only the center wavelengths for our filters for values of excitation and emission filters are listed for ease of readability.</p