Gene Knockout Using Transcription Activator-like Effector Nucleases (TALENs) Reveals That Human NDUFA9 Protein Is Essential for Stabilizing the Junction between Membrane and Matrix Arms of Complex I

Stroud, David A.; Formosa, Luke E.; Wijeyeratne, Xiaonan W.; Nguyen, Thanh Ngoc; Ryan, Michael

doi:10.1074/jbc.c112.436766

Cited by 74 publications

(54 citation statements)

References 32 publications

(43 reference statements)

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“…21,30,32,46,67 Transcription activator-like effector nucleases (TALENs) are a second technology that is used for targeted mutagenesis applications. To date, TALENs have been successfully used to mutagenize mammalian cell lines, [68][69][70][71][72][73][74][75] plants, 69,[76][77][78] yeast, 79 Drosophila melanogaster, 72,80 nematodes, 81 silkworm, 82,83 Xenopus laevis, 72,84,85 mice, [86][87][88] rats, [88][89][90] swine 91 medaka, 92 and zebrafish. 72,[93][94][95][96][97][98][99] Similar to ZFNs, TALENs also induce doublestrand breaks that are repaired through homologous recombination, or NHEJ to generate insertions and deletions that alter gene function.…”

Section: Introductionmentioning

confidence: 99%

Evaluating the Mutagenic Activity of Targeted Endonucleases Containing aSharkeyFokI Cleavage Domain Variant in Zebrafish

et al. 2013

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Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.

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Section: Introductionmentioning

confidence: 99%

Evaluating the Mutagenic Activity of Targeted Endonucleases Containing aSharkeyFokI Cleavage Domain Variant in Zebrafish

et al. 2013

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“…HCT116 colorectal cancer cells (a gift from Richard Youle, NIH) using TALEN (transcription activator-like effector nuclease) binding pairs (PMID 22484455) designed to target the common exon of the three isoforms of human VDAC2 as previously described. 45 Clones were screened for VDAC2-deficiency using an antibody recognizing human VDAC2 (Boris Reljic).…”

Section: Methodsmentioning

confidence: 99%

Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

et al. 2014

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In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-x L . Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.

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“…TALEN and CRISPR/Cas9 cell lines were sorted through fluorescence (GFP and mCherry for TALEN pairs, GFP for CRISPR/Cas9) flow cytometry to give single cells to ensure clonality. Genomic verification of all cell lines was performed as reported previously (Stroud et al, 2013). Control MEFs were used as controls.…”

Section: Generation Of Gene-edited Mefsmentioning

confidence: 99%

Cooperative and independent roles of Drp1 adaptors Mff and MiD49/51 in mitochondrial fission

Osellame

Singh

Stroud

et al. 2016

Journal of Cell Science

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272

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Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used geneediting technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

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Gene Knockout Using Transcription Activator-like Effector Nucleases (TALENs) Reveals That Human NDUFA9 Protein Is Essential for Stabilizing the Junction between Membrane and Matrix Arms of Complex I

Cited by 74 publications

References 32 publications

Evaluating the Mutagenic Activity of Targeted Endonucleases Containing aSharkeyFokI Cleavage Domain Variant in Zebrafish

Evaluating the Mutagenic Activity of Targeted Endonucleases Containing aSharkeyFokI Cleavage Domain Variant in Zebrafish

Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

Cooperative and independent roles of Drp1 adaptors Mff and MiD49/51 in mitochondrial fission

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