The Heterocyst-Specific NsiR1 Small RNA Is an Early Marker of Cell Differentiation in Cyanobacterial Filaments

ABSTRACT Differentiation of single cells along filaments of cyanobacteria constitutes one of the simplest developmental patterns in nature. In response to nitrogen deficiency, certain cells located in a semiregular pattern along filaments differentiate into specialized nitrogen-fixing cells called heterocysts. The process involves the sequential activation of many genes whose expression takes place, either exclusively or at least more strongly, in those cells undergoing differentiation. In the model cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120, increased transcription of hetR, considered the earliest detectable heterocyst-specific transcript, has been reported to occur in pairs or even in clusters of cells, thus making it difficult to identify prospective heterocysts during the early stages of differentiation, before any morphological change is detectable. The promoter of nsiR1 (nitrogen stress inducible RNA1), a heterocyst-specific small RNA, constitutes a minimal sequence promoting heterocyst-specific transcription. Using confocal fluorescence microscopy, I have analyzed expression of a gfp reporter transcriptionally fused to P nsiR1 . The combined analysis of green fluorescence (reporting transcriptional activity from P nsiR1 ) and red fluorescence (an indication of progress in the differentiation of individual cells) shows that expression of P nsiR1 takes place in single cells located in a semiregular pattern before any other morphological or fluorescence signature of differentiation can be observed, thus providing an early marker for cells undergoing differentiation. IMPORTANCE Cyanobacterial filaments containing heterocysts constitute an example of bacterial division of labor. When using atmospheric nitrogen, these filaments behave as multicellular organisms in which two different cell types (vegetative cells and nitrogen-fixing heterocysts) coexist and cooperate to achieve growth of the filament as a whole. The molecular basis governing the differentiation of individual vegetative cells, and thus the establishment of a one-dimensional pattern from cells that are apparently the same, remains one of the most intriguing aspects of this differentiation process. Recent evidence suggests that, at any given time, some cells in the filaments are more likely than others to become heterocysts when nitrogen limitation is encountered. The robust heterocyst-specific nsiR1 promoter, which is induced very early during differentiation, provides a valuable tool to analyze issues such as early candidacy or the possible role of transcriptional noise in determining the fate of specific cells in cyanobacterial filaments.

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Role of Transcriptional Read-Through in PRE Activity in Drosophila melanogaster

Acta Naturae ◽

10.32607/20758251-2016-8-2-79-86 ◽

2016 ◽

Vol 8 (2) ◽

pp. 79-86 ◽

Cited By ~ 3

Author(s):

P. V. Elizar’ev ◽

D. V. Lomaev ◽

D. A. Chetverina ◽

P. G. Georgiev ◽

M. M. Erokhin

Keyword(s):

Dna Sequences ◽

Cell Types ◽

Transcription Terminator ◽

Response Elements ◽

Polycomb Response Elements ◽

The Individual ◽

Multicellular Organisms ◽

Different Cell Types ◽

Main Factor

Maintenance of the individual patterns of gene expression in different cell types is required for the differentiation and development of multicellular organisms. Expression of many genes is controlled by Polycomb (PcG) and Trithorax (TrxG) group proteins that act through association with chromatin. PcG/TrxG are assembled on the DNA sequences termed PREs (Polycomb Response Elements), the activity of which can be modulated and switched from repression to activation. In this study, we analyzed the influence of transcriptional read-through on PRE activity switch mediated by the yeast activator GAL4. We show that a transcription terminator inserted between the promoter and PRE doesnt prevent switching of PRE activity from repression to activation. We demonstrate that, independently of PRE orientation, high levels of transcription fail to dislodge PcG/TrxG proteins from PRE in the absence of a terminator. Thus, transcription is not the main factor required for PRE activity switch.

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Thioredoxin targets are regulated in heterocysts of cyanobacterium Anabaena sp. PCC 7120 in a light-independent manner

Journal of Experimental Botany ◽

10.1093/jxb/erz561 ◽

2019 ◽

Vol 71 (6) ◽

pp. 2018-2027

Author(s):

Shoko Mihara ◽

Kazunori Sugiura ◽

Keisuke Yoshida ◽

Toru Hisabori

Keyword(s):

Redox Regulation ◽

Regulation System ◽

Regulation Mechanism ◽

Light Conditions ◽

Nitrogen Fixing ◽

Vegetative Cells ◽

Target Proteins ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

Abstract In the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, glucose 6-phosphate dehydrogenase (G6PDH) plays an important role in producing the power for reducing nitrogenase under light conditions. Our previous study showed that thioredoxin suppresses G6PDH by reducing its activator protein OpcA, implying that G6PDH is inactivated under light conditions because thioredoxins are reduced by the photosynthetic electron transport system in cyanobacteria. To address how Anabaena sp. PCC 7120 maintains G6PDH activity even under light conditions when nitrogen fixation occurs, we investigated the redox regulation system in vegetative cells and specific nitrogen-fixing cells named heterocysts, individually. We found that thioredoxin target proteins were more oxidized in heterocysts than in vegetative cells under light conditions. Alterations in the redox regulation mechanism of heterocysts may affect the redox states of thioredoxin target proteins, including OpcA, so that G6PDH is activated in heterocysts even under light conditions.

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A systems perspective of heterocellular signaling

Essays in Biochemistry ◽

10.1042/ebc20180015 ◽

2018 ◽

Vol 62 (4) ◽

pp. 607-617 ◽

Cited By ~ 3

Author(s):

Alan Wells ◽

H. Steven Wiley

Keyword(s):

Cell Types ◽

Regulatory Processes ◽

Technical Developments ◽

Building Models ◽

Realistic Description ◽

Multiple Cell ◽

Solid Foundation ◽

And Function ◽

Multicellular Organisms ◽

Different Cell Types

Signal exchange between different cell types is essential for development and function of multicellular organisms, and its dysregulation is causal in many diseases. Unfortunately, most cell-signaling work has employed single cell types grown under conditions unrelated to their native context. Recent technical developments have started to provide the tools needed to follow signaling between multiple cell types, but gaps in the information they provide have limited their usefulness in building realistic models of heterocellular signaling. Currently, only targeted assays have the necessary sensitivity, selectivity, and spatial resolution to usefully probe heterocellular signaling processes, but these are best used to test specific, mechanistic models. Decades of systems biology research with monocultures has provided a solid foundation for building models of heterocellular signaling, but current models lack a realistic description of regulated proteolysis and the feedback processes triggered within and between cells. Identification and understanding of key regulatory processes in the extracellular environment and of recursive signaling patterns between cells will be essential to building predictive models of heterocellular systems.

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Intercellular Diffusion of a Fluorescent Sucrose Analog via the Septal Junctions in a Filamentous Cyanobacterium

mBio ◽

10.1128/mbio.02109-14 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 57

Author(s):

Dennis J. Nürnberg ◽

Vicente Mariscal ◽

Jan Bornikoel ◽

Mercedes Nieves-Morión ◽

Norbert Krauß ◽

...

Keyword(s):

Gap Junctions ◽

Cell Types ◽

Cell Junctions ◽

Filamentous Cyanobacterium ◽

Nitrogen Fixing ◽

Filamentous Cyanobacteria ◽

Triple Mutant ◽

Vegetative Cells ◽

Metabolite Exchange ◽

Import System

ABSTRACTMany filamentous cyanobacteria produce specialized nitrogen-fixing cells called heterocysts, which are located at semiregular intervals along the filament with about 10 to 20 photosynthetic vegetative cells in between. Nitrogen fixation in these complex multicellular bacteria depends on metabolite exchange between the two cell types, with the heterocysts supplying combined-nitrogen compounds but dependent on the vegetative cells for photosynthetically produced carbon compounds. Here, we used a fluorescent tracer to probe intercellular metabolite exchange in the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. We show that esculin, a fluorescent sucrose analog, is incorporated by a sucrose import system into the cytoplasm of Anabaena cells. The cytoplasmic esculin is rapidly and reversibly exchanged across vegetative-vegetative and vegetative-heterocyst cell junctions. Our measurements reveal the kinetics of esculin exchange and also show that intercellular metabolic communication is lost in a significant fraction of older heterocysts. SepJ, FraC, and FraD are proteins located at the intercellular septa and are suggested to form structures analogous to gap junctions. We show that a ΔsepJΔfraCΔfraDtriple mutant shows an altered septum structure with thinner septa but a denser peptidoglycan layer. Intercellular diffusion of esculin and fluorescein derivatives is impaired in this mutant, which also shows a greatly reduced frequency of nanopores in the intercellular septal cross walls. These findings suggest that FraC, FraD, and SepJ are important for the formation of junctional structures that constitute the major pathway for feeding heterocysts with sucrose.IMPORTANCEAnabaena and its relatives are filamentous cyanobacteria that exhibit a sophisticated form of prokaryotic multicellularity, with the formation of differentiated cell types, including normal photosynthetic cells and specialized nitrogen-fixing cells called heterocysts. The question of how heterocysts communicate and exchange metabolites with other cells in the filament is key to understanding this form of bacterial multicellularity. Here we provide the first information on the intercellular exchange of a physiologically important molecule, sucrose. We show that a fluorescent sucrose analog can be imported into the Anabaena cytoplasm by a sucrose import system. Once in the cytoplasm, it is rapidly and reversibly exchanged among all of the cells in the filament by diffusion across the septal junctions. Photosynthetically produced sucrose likely follows the same route from cytoplasm to cytoplasm. We identify some of the septal proteins involved in sucrose exchange, and our results indicate that these proteins form structures functionally analogous to metazoan gap junctions.

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Computational approaches towards reducing contamination in single-cell RNA-seq data

10.1101/2020.07.15.205062 ◽

2020 ◽

Author(s):

Siamak Yousefi ◽

Hao Chen ◽

Jesse F. Ingels ◽

Melinda S. McCarty ◽

Arthur G. Centeno ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Real Life ◽

Cell Types ◽

Cell Capture ◽

Rna Seq ◽

Sequence Analyses ◽

Cell Functions ◽

Biological Interpretation ◽

Different Cell Types

SUMMARYSingle cell RNA sequencing has enabled quantification of single cells and identification of different cell types and subtypes as well as cell functions in different tissues. Single cell RNA sequence analyses assume acquired RNAs correspond to cells, however, RNAs from contamination within the input data are also captured by these assays. The sequencing of background contamination as well as unwanted cells making their way to the final assay Potentially confound the correct biological interpretation of single cell transcriptomic data. Here we demonstrate two approaches to deal with background contamination as well as profiling of unwanted cells in the assays. We use three real-life datasets of whole-cell capture and nucleotide single-cell captures generated by Fluidigm and 10x technologies and show that these methods reduce the effect of contamination, strengthen clustering of cells and improves biological interpretation.

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Mapping multicellular programs from single-cell profiles

10.1101/2020.08.11.245472 ◽

2020 ◽

Author(s):

Livnat Jerby-Arnon ◽

Aviv Regev

Keyword(s):

Single Cell ◽

Spatial Data ◽

Spatial Information ◽

Single Cells ◽

Cell Types ◽

Risk Genes ◽

Health And Disease ◽

Different Cell Types ◽

Cellular Components ◽

Acting In Concert

ABSTRACTTissue homeostasis relies on orchestrated multicellular circuits, where interactions between different cell types dynamically balance tissue function. While single-cell genomics identifies tissues’ cellular components, deciphering their coordinated action remains a major challenge. Here, we tackle this problem through a new framework of multicellular programs: combinations of distinct cellular programs in different cell types that are coordinated together in the tissue, thus forming a higher order functional unit at the tissue, rather than only cell, level. We develop the open-access DIALOGUE algorithm to systematically uncover such multi-cellular programs not only from spatial data, but even from tissue dissociated and profiled as single cells, e.g., by single-cell RNA-Seq. Tested on spatial transcriptomes from the mouse hypothalamus, DIALOGUE recovered spatial information, predicted the properties of a cell’s environment only based on its transcriptome, and identified multicellular programs that mark animal behavior. Applied to brain samples and colon biopsies profiled by scRNA-Seq, DIALOGUE identified multicellular configurations that mark Alzheimer’s disease and ulcerative colitis (UC), including a program spanning five cell types that is predictive of response to anti-TNF therapy in UC patients and enriched for UC risk genes from GWAS, each acting in different cell types, but all cells acting in concert. Taken together, our study provides a novel conceptual and methodological framework to unravel multicellular regulation in health and disease.

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Unfolding and identification of membrane proteins from native cell membranes

10.1101/732933 ◽

2019 ◽

Author(s):

Nicola Galvanetto ◽

Sourav Maity ◽

Nina Ilieva ◽

Zhongjie Ye ◽

Alessandro Laio ◽

...

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Single Cells ◽

Total Content ◽

Cell Types ◽

Single Cell Level ◽

Cell Level ◽

Single Molecule Force Spectroscopy ◽

Native Cell ◽

Different Cell Types

AbstractIs the mechanical unfolding of proteins just a technological feat applicable only to synthetic preparations or can it provide useful information even for real biological samples? Here, we describe a pipeline for analyzing native membranes based on high throughput single-molecule force spectroscopy. The protocol includes a technique for the isolation of the plasma membrane of single cells. Afterwards, one harvests hundreds of thousands SMSF traces from the sample. Finally, one characterizes and identifies the embedded membrane proteins. This latter step is the cornerstone of our approach and involves combining, within a Bayesian framework, the information of the shape of the SMFS Force-distance which are observed more frequently, with the information from Mass Spectrometry and from proteomic databases (Uniprot, PDB). We applied this method to four cell types where we classified the unfolding of 5-10% of their total content of membrane proteins. The ability to mechanically probe membrane proteins directly in their native membrane enables the phenotyping of different cell types with almost single-cell level of resolution.

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Massively multiplex single-cell Hi-C

10.1101/065052 ◽

2016 ◽

Cited By ~ 4

Author(s):

Vijay Ramani ◽

Xinxian Deng ◽

Kevin L Gunderson ◽

Frank J Steemers ◽

Christine M Disteche ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Cell Types ◽

Cell Heterogeneity ◽

Proof Of Concept ◽

Chromosome Conformation ◽

Large Numbers ◽

Conformational Properties ◽

Novel Method ◽

Different Cell Types

AbstractWe present combinatorial single cell Hi-C, a novel method that leverages combinatorial cellular indexing to measure chromosome conformation in large numbers of single cells. In this proof-of-concept, we generate and sequence combinatorial single cell Hi-C libraries for two mouse and four human cell types, comprising a total of 9,316 single cells across 5 experiments. We demonstrate the utility of single-cell Hi-C data in separating different cell types, identify previously uncharacterized cell-to-cell heterogeneity in the conformational properties of mammalian chromosomes, and demonstrate that combinatorial indexing is a generalizable molecular strategy for single-cell genomics.

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Massively parallel single cell lineage tracing using CRISPR/Cas9 induced genetic scars

10.1101/205971 ◽

2017 ◽

Cited By ~ 6

Author(s):

Bastiaan Spanjaard ◽

Bo Hu ◽

Nina Mitic ◽

Jan Philipp Junker

Keyword(s):

Single Cell ◽

Computational Analysis ◽

Systematic Approach ◽

Single Cells ◽

Cell Lineage ◽

Transcriptome Profiling ◽

Cell Types ◽

Lineage Tracing ◽

Lineage Trees ◽

Different Cell Types

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many different cell types. Single-cell RNA-sequencing (scRNA-seq) has become a widely-used method due to its ability to identify all cell types in a tissue or organ in a systematic manner 1–3. However, a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here, we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses, LINNAEUS (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences) can be used as a systematic approach for identifying the lineage origin of novel cell types, or of known cell types under different conditions.

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Requirement of Fra proteins for communication channels between cells in the filamentous nitrogen-fixing cyanobacteriumAnabaenasp. PCC 7120

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1512232112 ◽

2015 ◽

Vol 112 (32) ◽

pp. E4458-E4464 ◽

Cited By ~ 16

Author(s):

Amin Omairi-Nasser ◽

Vicente Mariscal ◽

Jotham R. Austin ◽

Robert Haselkorn

Keyword(s):

Electron Tomography ◽

Atmospheric Nitrogen ◽

Channel Formation ◽

Nitrogen Fixing ◽

Vegetative Cells ◽

Metabolite Exchange ◽

Minor Part ◽

A Minor ◽

Pcc 7120

The filamentous nitrogen-fixing cyanobacteriumAnabaenasp. PCC 7120 differentiates specialized cells, heterocysts, that fix atmospheric nitrogen and transfer the fixed nitrogen to adjacent vegetative cells. Reciprocally, vegetative cells transfer fixed carbon to heterocysts. Several routes have been described for metabolite exchange within the filament, one of which involves communicating channels that penetrate the septum between adjacent cells. Severalfragene mutants were isolated 25 y ago on the basis of their phenotypes: inability to fix nitrogen and fragmentation of filaments upon transfer from N+ to N− media. Cryopreservation combined with electron tomography were used to investigate the role of threefragene products in channel formation. FraC and FraG are clearly involved in channel formation, whereas FraD has a minor part. Additionally, FraG was located close to the cytoplasmic membrane and in the heterocyst neck, using immunogold labeling with antibody raised to the N-terminal domain of the FraG protein.

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