Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

Mahen, Robert; Koch, Birgit; Wachsmuth, Malte; Politi, Antonio Z.; Perez‐Gonzalez, Alexis; Mergenthaler, Julia; Yin, Chen; Ellenberg, Jan

doi:10.1091/mbc.e14-06-1091

Cited by 55 publications

(80 citation statements)

References 22 publications

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“…These experiments reported that Eg5 was enriched at spindle poles relative to microtubules. The discrepancy in the results from the two experimental approaches supports the view that the method of protein tagging can affect protein distribution in cells as reported by others (see Section 4) (Dambournet et al, 2014;Mahen et al, 2014).…”

Section: Tpx2 Is Enriched Relative To Eg5supporting

confidence: 74%

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Mann

Wadsworth

2018

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The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

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Section: Tpx2 Is Enriched Relative To Eg5supporting

confidence: 74%

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Mann

Wadsworth

2018

Cytoskeleton

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“…HeLa Kyoto cells are hypotriploid with on average 64 chromosomes, thus during mitosis the cells have on average 64*2 = 128 kinetochores 12 . The cell lines have been generated for this project or previously 16,[26][27][28][29][30][31][32] are listed in Supplementary Table 1 with their providers indicated. Several cell lines were generated within this project as follows: The cell lines expressing PLK1-mEGFP, CEP192-mEGFP and mEGFP-NUP107 were generated using the Zinc finger nuclease (ZFN) pipeline as in 29 .…”

Section: Figure Legendsmentioning

confidence: 99%

“…For these cell lines, both gRNAs ( Supplementary Table 2) and the donor plasmid were designed based on ENSEMBL release 75 and transfected together into HeLa Kyoto cells with jetPrime (Polyplus) according to the manufacturer's instruction. A single clone was selected using our previously developed validation pipeline 6,29 . For 4 out of 20 genome-edited cell lines (BUB1B-EGFP, TPR-mEGFP, mEGFP-NUP107 and CEP192-mEGFP) we detected in the Western blots (anti-GFP, Roche cat#11814460001) a band of the size of free GFP.…”

Section: Figure Legendsmentioning

confidence: 99%

“…Image processing and calibration were performed according to 5 . Before each calibrated live cell confocal microscopy experiment, the focal volume was calibrated using a 10-50 nM solution of Alexa488 (Life Technologies), and single mEGFP brightness was calibrated by performing FCS measurements on HeLa Kyoto cells expressing mEGFP 29 . All FCS measurements were processed using Fluctuation where D was the diffusion coefficient of the Alexa488, which is 464 μm 2 /s at 37 °C, and the structural parameter.…”

Section: Figure Legendsmentioning

confidence: 99%

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

Yin

Hossain

Hèriché

et al. 2017

Preprint

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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.

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“…The use of genomic DNA instead of cDNA vectors in gene therapy has made a way for vital accurate gene expression, as these vectors contain all endogenous control elements in terms of achieving a close to physiologic expression level. The majority of studies have relied on transgenes using cDNA, which actually does not faithfully mimic physiologic gene expression, because it does not contain most of the endogenous cis ‐acting regulatory sequences (50). To date, little effort has gone into detailed analyses of the genomic environments of genetic element‐containing transgenes.…”

Section: Discussionmentioning

confidence: 99%

Nanoparticle‐mediated rhodopsin cDNA but not intron‐containing DNA delivery causes transgene silencing in a rhodopsin knockout model

et al. 2015

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Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NPmediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NPsgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/ matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications.-Zheng, M., Mitra, R. N., Filonov, N. A., Han, Z. Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model. FASEB J. 30, 1076-1086 (2016). www.fasebj.org

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Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

Cited by 55 publications

References 22 publications

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

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

Nanoparticle‐mediated rhodopsin cDNA but not intron‐containing DNA delivery causes transgene silencing in a rhodopsin knockout model

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