scholarly journals Experimental and computational framework for a dynamic protein atlas of human cell division

2017 ◽  
Author(s):  
Yin Cai ◽  
M. Julius Hossain ◽  
Jean-Karim Hériché ◽  
Antonio Z. Politi ◽  
Nike Walther ◽  
...  
Keyword(s):  
Cell Division ◽  
Human Cell ◽  
Protein Complexes ◽  
Morphological Changes ◽  
Protein Localization ◽  
Image Data ◽  
Cell Types ◽  
Correlation Spectroscopy ◽  
Concentration Data ◽  
Dynamic Interactions

SUMMARYEssential biological functions, such as mitosis, require tight coordination of hundreds of proteins in space and time. Localization, timing of interactions and changes in cellular structure are all crucial to ensure correct assembly, function and regulation of protein complexes1-4. Live cell imaging can reveal protein distributions and dynamics but experimental and theoretical challenges prevented its use to produce quantitative data and a model of mitosis that comprehensively integrates information and enables analysis of the dynamic interactions between the molecular parts of the mitotic machinery within changing cellular boundaries.To address this, we generated a 4D image data-driven, canonical model of the morphological changes during mitotic progression of human cells. We used this model to integrate dynamic 3D concentration data of many fluorescently knocked-in mitotic proteins, imaged by fluorescence correlation spectroscopy-calibrated microscopy5. The approach taken here in the context of the MitoSys consortium to generate a dynamic protein atlas of human cell division is generic. It can be applied to systematically map and mine dynamic protein localization networks that drive cell division in different cell types and can be conceptually transferred to other cellular functions.

Okadaic acid induces morphological changes, apoptosis and cell cycle alterations in different human cell types

2011 ◽  
Vol 13 (6) ◽  
pp. 1831 ◽  
Author(s):  
Vanessa Valdiglesias ◽  
Blanca Laffon ◽  
Eduardo Pásaro ◽  
Josefina Méndez
Keyword(s):  
Cell Cycle ◽  
Okadaic Acid ◽  
Human Cell ◽  
Morphological Changes ◽  
Cell Types ◽  
Cell Cycle Alterations

scholarly journals A scalable strategy for high-throughput GFP tagging of endogenous human proteins

2016 ◽  
Vol 113 (25) ◽  
pp. E3501-E3508 ◽  
Author(s):  
Manuel D. Leonetti ◽  
Sayaka Sekine ◽  
Daichi Kamiyama ◽  
Jonathan S. Weissman ◽  
Bo Huang
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Protein Function ◽  
Large Scale ◽  
Protein Complexes ◽  
Protein Localization ◽  
Human Cell Lines ◽  
Guide Rna ◽  
Human Genes ◽  
Endogenous Loci

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.

scholarly journals Quantitative behavior of protein complexes in human cells

2018 ◽  
Author(s):  
Morteza H Chalabi ◽  
Vasileios Tsiamis ◽  
Lukas Käll ◽  
Fabio Vandin ◽  
Veit Schwämmle
Keyword(s):  
Web Service ◽  
Human Cell ◽  
Protein Complexes ◽  
Complex Function ◽  
Cell Types ◽  
Human Cells ◽  
Control Mechanisms ◽  
Complex Behavior ◽  
Link Type

AbstractTranslational and post-translational control mechanisms in the cell result in widely observable differences between measured gene transcription and protein abundances. Herein, protein complexes are among the most tightly controlled entities by selective degradation of their individual proteins. They furthermore act as control hubs that regulate highly important processes in the cell and exhibit a high functional diversity due to their ability to change their composition and their structure. To better understand and predict these functional states, extensive characterization of complex composition, behavior, and abundance is necessary. Mass spectrometry provides an unbiased approach to directly determine protein abundances across cell populations and thus to profile a comprehensive abundance map of proteins. We investigated the behavior of protein subunits in known complexes by comparing their abundance profiles across up to 140 cell types available in ProteomicsDB. After thorough assessment of different randomization methods and statistical scoring algorithms, we developed a computational tool to quantify the significance of concurrent profiles within a complex, therefore providing insights into the conservation of their composition across human cell types. We identified the intrinsic structures in complex behavior that allow to determine which proteins orchestrate complex function. This analysis can be extended to investigate common profiles within arbitrary protein groups. With the CoExpresso web service, we offer a potent scoring scheme to assess proteins for their co-regulation and thereby offer insight into their potential for forming functional groups like protein complexes. CoExpresso can be accessed through http://computproteomics/Apps/CoExpresso. Source code and R scripts for database generation are available at https://bitbucket.org/veitveit/coexpresso.Author summaryMany proteins form multi-functional assemblies called protein complexes instead of working as singly units. These complexes control most processes in the cell making the full characterization of their behavior inevitable to understand cellular control mechanisms. Detailed knowledge about complex behavior will elucidate biomarkers and drug targets that exhibit and correct aberrant cell states, respectively. We investigated abundance changes of the protein complex components over more than 100 different human cell types. By using statistical scoring models, we estimated the evidence for the co-regulation of the proteins and revealed which proteins form subunits with impact on complex function and composition. By providing the interactive web service CoExpresso, any combination of proteins can be tested for their co-regulation in human cells.

Ultrastructural Study of a differentiating human colon carcinoma cell line

1990 ◽  
Vol 48 (3) ◽  
pp. 208-209
Author(s):  
Sylvie Polak-Charcon ◽  
Mehrdad Hekmati ◽  
Yehuda Ben Shaul
Keyword(s):  
Cell Line ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Human Colon ◽  
Cell Types ◽  
Culture Conditions ◽  
Morphological Evidence ◽  
Human Colon Carcinoma Cell ◽  
Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

scholarly journals Tumour Microenvironment: Roles of the Aryl Hydrocarbon Receptor, O-GlcNAcylation, Acetyl-CoA and Melatonergic Pathway in Regulating Dynamic Metabolic Interactions across Cell Types—Tumour Microenvironment and Metabolism

2020 ◽  
Vol 22 (1) ◽  
pp. 141
Author(s):  
George Anderson
Keyword(s):  
Aryl Hydrocarbon Receptor ◽  
Glycogen Synthase ◽  
Tumour Progression ◽  
Cell Types ◽  
Tumour Microenvironment ◽  
Future Research ◽  
Acetyl Coa ◽  
Dynamic Interactions ◽  
Aryl Hydrocarbon ◽  
Metabolic Interactions

This article reviews the dynamic interactions of the tumour microenvironment, highlighting the roles of acetyl-CoA and melatonergic pathway regulation in determining the interactions between oxidative phosphorylation (OXPHOS) and glycolysis across the array of cells forming the tumour microenvironment. Many of the factors associated with tumour progression and immune resistance, such as yin yang (YY)1 and glycogen synthase kinase (GSK)3β, regulate acetyl-CoA and the melatonergic pathway, thereby having significant impacts on the dynamic interactions of the different types of cells present in the tumour microenvironment. The association of the aryl hydrocarbon receptor (AhR) with immune suppression in the tumour microenvironment may be mediated by the AhR-induced cytochrome P450 (CYP)1b1-driven ‘backward’ conversion of melatonin to its immediate precursor N-acetylserotonin (NAS). NAS within tumours and released from tumour microenvironment cells activates the brain-derived neurotrophic factor (BDNF) receptor, TrkB, thereby increasing the survival and proliferation of cancer stem-like cells. Acetyl-CoA is a crucial co-substrate for initiation of the melatonergic pathway, as well as co-ordinating the interactions of OXPHOS and glycolysis in all cells of the tumour microenvironment. This provides a model of the tumour microenvironment that emphasises the roles of acetyl-CoA and the melatonergic pathway in shaping the dynamic intercellular metabolic interactions of the various cells within the tumour microenvironment. The potentiation of YY1 and GSK3β by O-GlcNAcylation will drive changes in metabolism in tumours and tumour microenvironment cells in association with their regulation of the melatonergic pathway. The emphasis on metabolic interactions across cell types in the tumour microenvironment provides novel future research and treatment directions.

scholarly journals Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wiruntita Chankeaw ◽  
Sandra Lignier ◽  
Christophe Richard ◽  
Theodoros Ntallaris ◽  
Mariam Raliou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Localization ◽  
Expression Profiles ◽  
Cell Types ◽  
Molecular Signature ◽  
Receptor Protein ◽  
Specific Gene ◽  
Specific Cell ◽  
Biological Processes ◽  
Endometrial Cells

Abstract Background A number of studies have examined mRNA expression profiles of bovine endometrium at estrus and around the peri-implantation period of pregnancy. However, to date, these studies have been performed on the whole endometrium which is a complex tissue. Consequently, the knowledge of cell-specific gene expression, when analysis performed with whole endometrium, is still weak and obviously limits the relevance of the results of gene expression studies. Thus, the aim of this study was to characterize specific transcriptome of the three main cell-types of the bovine endometrium at day-15 of the estrus cycle. Results In the RNA-Seq analysis, the number of expressed genes detected over 10 transcripts per million was 6622, 7814 and 8242 for LE, GE and ST respectively. ST expressed exclusively 1236 genes while only 551 transcripts were specific to the GE and 330 specific to LE. For ST, over-represented biological processes included many regulation processes and response to stimulus, cell communication and cell adhesion, extracellular matrix organization as well as developmental process. For GE, cilium organization, cilium movement, protein localization to cilium and microtubule-based process were the only four main biological processes enriched. For LE, over-represented biological processes were enzyme linked receptor protein signaling pathway, cell-substrate adhesion and circulatory system process. Conclusion The data show that each endometrial cell-type has a distinct molecular signature and provide a significantly improved overview on the biological process supported by specific cell-types. The most interesting result is that stromal cells express more genes than the two epithelial types and are associated with a greater number of pathways and ontology terms.

scholarly journals Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Radek Kaňa ◽  
Gábor Steinbach ◽  
Roman Sobotka ◽  
György Vámosi ◽  
Josef Komenda
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Protein Interactions ◽  
Thylakoid Membrane ◽  
Protein Complexes ◽  
Correlation Spectroscopy ◽  
Membrane Microdomains ◽  
Fast Diffusion ◽  
Light Harvesting Complexes ◽  
Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

scholarly journals Paired arrangement of kinetochores together with microtubule pivoting and dynamics drive kinetochore capture in meiosis I

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Gheorghe Cojoc ◽  
Ana-Maria Florescu ◽  
Alexander Krull ◽  
Anna H. Klemm ◽  
Nenad Pavin ◽  
...  
Keyword(s):  
Cell Division ◽  
Fission Yeast ◽  
General Feature ◽  
Protein Complexes ◽  
Spindle Pole ◽  
Single Object ◽  
Homologous Chromosomes ◽  
Angular Movement ◽  
Spindle Microtubules ◽  
Meiosis I

Abstract Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3–4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.

scholarly journals Regulation of Division and Differentiation of Plant Stem Cells

2018 ◽  
Vol 34 (1) ◽  
pp. 289-310 ◽  
Author(s):  
Edith Pierre-Jerome ◽  
Colleen Drapek ◽  
Philip N. Benfey
Keyword(s):  
Stem Cell ◽  
Cell Division ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Cell Types ◽  
Stem Cell Maintenance ◽  
Cell Position ◽  
Dependent Cell ◽  
Gene Regulatory ◽  
Plant Stem

A major challenge in developmental biology is unraveling the precise regulation of plant stem cell maintenance and the transition to a fully differentiated cell. In this review, we highlight major themes coordinating the acquisition of cell identity and subsequent differentiation in plants. Plant cells are immobile and establish position-dependent cell lineages that rely heavily on external cues. Central players are the hormones auxin and cytokinin, which balance cell division and differentiation during organogenesis. Transcription factors and miRNAs, many of which are mobile in plants, establish gene regulatory networks that communicate cell position and fate. Small peptide signaling also provides positional cues as new cell types emerge from stem cell division and progress through differentiation. These pathways recruit similar players for patterning different organs, emphasizing the modular nature of gene regulatory networks. Finally, we speculate on the outstanding questions in the field and discuss how they may be addressed by emerging technologies.

scholarly journals Profiling Bioactivity of the ToxCast Chemical Library Using BioMAP Primary Human Cell Systems

2009 ◽  
Vol 14 (9) ◽  
pp. 1054-1066 ◽  
Author(s):  
Keith A. Houck ◽  
David J. Dix ◽  
Richard S. Judson ◽  
Robert J. Kavlock ◽  
Jian Yang ◽  
...  
Keyword(s):  
Human Cell ◽  
Regulatory Networks ◽  
Cell Types ◽  
Environmental Chemicals ◽  
Data Sets ◽  
Chemical Library ◽  
Data Set ◽  
Cell Systems ◽  
Disease Biology ◽  
Nfκb Pathway

The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, leverage cellular regulatory networks to detect and distinguish chemicals with a broad range of target mechanisms and biological processes relevant to human toxicity. Here the authors use the BioMAP human cell systems to characterize effects relevant to human tissue and inflammatory disease biology following exposure to the 320 environmental chemicals in the Environmental Protection Agency’s (EPA’s) ToxCast phase I library. The ToxCast chemicals were assayed at 4 concentrations in 8 BioMAP cell systems, with a total of 87 assay endpoints resulting in more than 100,000 data points. Within the context of the BioMAP database, ToxCast compounds could be classified based on their ability to cause overt cytotoxicity in primary human cell types or according to toxicity mechanism class derived from comparisons to activity profiles of BioMAP reference compounds. ToxCast chemicals with similarity to inducers of mitochondrial dysfunction, cAMP elevators, inhibitors of tubulin function, inducers of endoplasmic reticulum stress, or NFκB pathway inhibitors were identified based on this BioMAP analysis. This data set is being combined with additional ToxCast data sets for development of predictive toxicity models at the EPA. ( Journal of Biomolecular Screening 2009:1054-1066)

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