Single-cell visualization and quantification of trace metals in Chlamydomonas lysosome-related organelles

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)–based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

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Single-cell Visualization and Quantification of Trace Metals in Chlamydomonas Lysosome-Related Organelles

10.1101/2021.02.12.431000 ◽

2021 ◽

Author(s):

Stefan Schmollinger ◽

Si Chen ◽

Daniela Strenkert ◽

Colleen Hui ◽

Martina Ralle ◽

...

Keyword(s):

Trace Metal ◽

Chlamydomonas Reinhardtii ◽

Single Cell ◽

Imaging Techniques ◽

Single Cells ◽

Experimental System ◽

Absolute Quantification ◽

Global Scale ◽

Oxygenic Photosynthesis ◽

High Calcium

AbstractThe acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass-spectrometry (MS)-based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared to MS-based imaging techniques, XFM offers the advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii, to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal over-accumulation (Fe, Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions, and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.Significance statementTransition metals are of crucial importance for primary productivity; their scarcity limits crop yield in agriculture and carbon sequestration at global scale. Copper (Cu), iron (Fe) and manganese (Mn) are among the most important trace elements that enable the redox chemistry in oxygenic photosynthesis. The single-celled, eukaryotic green alga Chlamydomonas reinhardtii is a choice experimental system for studying trace metal homeostasis in the context of phototrophy, offering all the advantages of a classical microbial system with a well-characterized photosystem and trace metal metabolism machinery of relevance to plants. This project identifies and differentiates different trace metal storage sites in Chlamydomonas and uncovers the dynamics of trace metal storage and mobilization in situations of fluctuating resources.

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Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

Nucleic Acids Research ◽

10.1093/nar/gku661 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9880-9891 ◽

Cited By ~ 41

Author(s):

Arne H. Smits ◽

Rik G.H. Lindeboom ◽

Matteo Perino ◽

Simon J. van Heeringen ◽

Gert Jan C. Veenstra ◽

...

Keyword(s):

Single Cell ◽

Ribosomal Proteins ◽

Single Cells ◽

Transcript Level ◽

Absolute Quantification ◽

Protein Quantification ◽

Protein Levels ◽

Recent Developments ◽

Basal Transcription Factors ◽

Cell Proteome

Abstract While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

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Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

10.1101/2020.11.20.390971 ◽

2020 ◽

Author(s):

Noa Liscovitch-Brauer ◽

Antonino Montalbano ◽

Jiale Deng ◽

Alejandro Méndez-Mancilla ◽

Hans-Hermann Wessels ◽

...

Keyword(s):

Single Cell ◽

Large Scale ◽

Expression Profiles ◽

Low Cost ◽

Single Cells ◽

Gene Expression Profiles ◽

Chromatin Accessibility ◽

Myelogenous Leukemia ◽

Chromatin Remodelers ◽

Genetic Perturbations

AbstractPooled CRISPR screens have been used to identify genes responsible for specific phenotypes and diseases, and, more recently, to connect genetic perturbations with multi-dimensional gene expression profiles. Here, we describe a method to link genome-wide chromatin accessibility to genetic perturbations in single cells. This scalable, cost-effective method combines pooled CRISPR perturbations with a single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC). Using a human and mouse species-mixing experiment, we show that CRISPR-sciATAC separates single cells with a low doublet rate. Then, in human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 21 chromatin-related genes that are frequently mutated in cancer and 84 subunits and cofactors of chromatin remodeling complexes, generating chromatin accessibility data for ~30,000 single cells. Using this large-scale atlas, we correlate loss of specific chromatin remodelers with changes in accessibility — globally and at the binding sites of individual transcription factors. For example, we show that loss of the H3K27 methyltransferase EZH2 leads to increased accessibility at heterochromatic regions involved in embryonic development and triggers expression of multiple genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze dynamic changes in nucleosome spacing upon loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, low-cost single-cell method that can be applied broadly to study the role of genetic perturbations on chromatin in normal and disease states.

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In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

ISTFA 1999: Conference Proceedings from the 25th International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa1999p0311 ◽

1999 ◽

Author(s):

Gunnar Zimmermann ◽

Richard Chapman

Keyword(s):

Electron Beam ◽

Single Cell ◽

Single Cells ◽

Ion Beam ◽

Gate Oxide ◽

Ion Milling ◽

Dual Beam ◽

Sem Imaging ◽

Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara < 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

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Faculty Opinions recommendation of Single-Cell Multiomics: Multiple Measurements from Single Cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727213649.793550351 ◽

2018 ◽

Author(s):

Jing-Dong Jackie Han

Keyword(s):

Single Cell ◽

Single Cells ◽

Multiple Measurements

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Absolute Quantification of MicroRNAs in a Single Cell with Chemiluminescence Detection Based on Rolling Circle Amplification on a Microchip Platform

Analytical Chemistry ◽

10.1021/acs.analchem.1c01463 ◽

2021 ◽

Author(s):

Shengyu Chen ◽

Jingjin Zhao ◽

Chunhuan Xu ◽

Ivan Yu. Sakharov ◽

Shulin Zhao

Keyword(s):

Single Cell ◽

Rolling Circle Amplification ◽

Absolute Quantification ◽

Chemiluminescence Detection ◽

Rolling Circle

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Visually precise, low-damage, single-cell spatial manipulation with single-pixel resolution

Chemical Science ◽

10.1039/d0sc05534d ◽

2021 ◽

Vol 12 (11) ◽

pp. 4111-4118

Author(s):

Qi Zhang ◽

Yunlong Shao ◽

Boye Li ◽

Yuanyuan Wu ◽

Jingying Dong ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Pixel Resolution ◽

Single Pixel

We achieved the low-damage spatial puncture of single cells at specific visual points with an accuracy of <65 nm.

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Single cell transcriptome atlas of mouse mammary epithelial cells across development

Breast Cancer Research ◽

10.1186/s13058-021-01445-4 ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Bhupinder Pal ◽

Yunshun Chen ◽

Michael J. G. Milevskiy ◽

François Vaillant ◽

Lexie Prokopuk ◽

...

Keyword(s):

Epithelial Cells ◽

Single Cell ◽

Developmental Stages ◽

Mammary Epithelial Cells ◽

Expression Profiles ◽

Single Cells ◽

Chromatin Accessibility ◽

Mammary Epithelial ◽

Cell Transcriptome ◽

Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

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SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

Science Advances ◽

10.1126/sciadv.abe3610 ◽

2021 ◽

Vol 7 (8) ◽

pp. eabe3610

Author(s):

Conor J. Kearney ◽

Stephin J. Vervoort ◽

Kelly M. Ramsbottom ◽

Izabela Todorovski ◽

Emily J. Lelliott ◽

...

Keyword(s):

Single Cell ◽

Posttranslational Modification ◽

Cellular Differentiation ◽

Single Cells ◽

Simultaneous Detection ◽

Surface Protein ◽

Rna Seq ◽

Cell Level ◽

Simultaneous Integration ◽

Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

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