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Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Spadafora D, Kozhukhar N, Alexeyev MF - PLoS ONE (2016)

Bottom Line: Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content.Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number.This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.

ABSTRACT
Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

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Respiration and growth rates.4B6 cells and cloned with LigA-supported mtDNA replication #1,#2, and #4 were transduced with retroviruses encoding either WT or catalytically inactive K510V mutant mLig3. A-C, OCR was determined in the parental (A) and transduced (B and C) cells. D, doubling time of the resulting clones. ***, P<0.001, two-way ANOVA with Dunnett’s post-hoc test.
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pone.0152705.g004: Respiration and growth rates.4B6 cells and cloned with LigA-supported mtDNA replication #1,#2, and #4 were transduced with retroviruses encoding either WT or catalytically inactive K510V mutant mLig3. A-C, OCR was determined in the parental (A) and transduced (B and C) cells. D, doubling time of the resulting clones. ***, P<0.001, two-way ANOVA with Dunnett’s post-hoc test.

Mentions: In patients with mtDNA depletion syndromes, mtDNA copy number is severely reduced, and mitochondrial respiratory function is compromised [10]. Yet population studies suggest that mtDNA content in tissues of healthy (and therefore, having normal respiratory function) individuals may vary by as much as 10-fold [23–26, 31]. Moreover, it has been observed that reduced mtDNA copy number has no major effect on mitochondrial transcript levels or enzyme activities in various tissues [32, 33]. Overall, these observations suggest that while, within limits, alterations in mtDNA copy number may have little physiological effect, a severe mtDNA depletion is detrimental. Therefore, we set out to investigate physiological consequences of mtDNA depletion in cell lines, in which mtDNA replication is supported by LigA. First, we measured the baseline mitochondrial respiration in WT 4B6 cells and in clones #1,#2 and #4 (Fig 4A). Despite elevated mtDNA copy number, the oxygen consumption rate (OCR) in clone #1 was the same as in WT 4B6 cells. However, in clones #2 and #4, which have reduced mtDNA copy number baseline respiration was suppressed as compared to WT 4B6 cells (Fig 4A). Introducing either WT or K510V mutant mLig3 into either 4B6 cells, or into clone #1 had little effect on the baseline OCR (Fig 4B). In contrast, transduction of clones #3 and #4 with WT, but not K510V mutant mLig3 led to a significant increase in baseline OCR (Fig 4C), suggesting that reduced mtDNA copy number is responsible for the reduced OCR. Also, empirical observations of variability in growth rates between parental and virus-transduced cell lines suggested precise measurements of proliferation rates. Doubling times in cell lines were inversely proportional to respiration. Transduction of the WT 4B6 cells and a high mtDNA copy number clone #1 with either WT or mutant Lig3 had no effect on growth rates, whereas transduction of low mtDNA copy number clones #2 and #4 with either WT or, surprisingly, with mutant mLig3 resulted in a significant acceleration of growth (Fig 4D).


Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Spadafora D, Kozhukhar N, Alexeyev MF - PLoS ONE (2016)

Respiration and growth rates.4B6 cells and cloned with LigA-supported mtDNA replication #1,#2, and #4 were transduced with retroviruses encoding either WT or catalytically inactive K510V mutant mLig3. A-C, OCR was determined in the parental (A) and transduced (B and C) cells. D, doubling time of the resulting clones. ***, P<0.001, two-way ANOVA with Dunnett’s post-hoc test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4816344&req=5

pone.0152705.g004: Respiration and growth rates.4B6 cells and cloned with LigA-supported mtDNA replication #1,#2, and #4 were transduced with retroviruses encoding either WT or catalytically inactive K510V mutant mLig3. A-C, OCR was determined in the parental (A) and transduced (B and C) cells. D, doubling time of the resulting clones. ***, P<0.001, two-way ANOVA with Dunnett’s post-hoc test.
Mentions: In patients with mtDNA depletion syndromes, mtDNA copy number is severely reduced, and mitochondrial respiratory function is compromised [10]. Yet population studies suggest that mtDNA content in tissues of healthy (and therefore, having normal respiratory function) individuals may vary by as much as 10-fold [23–26, 31]. Moreover, it has been observed that reduced mtDNA copy number has no major effect on mitochondrial transcript levels or enzyme activities in various tissues [32, 33]. Overall, these observations suggest that while, within limits, alterations in mtDNA copy number may have little physiological effect, a severe mtDNA depletion is detrimental. Therefore, we set out to investigate physiological consequences of mtDNA depletion in cell lines, in which mtDNA replication is supported by LigA. First, we measured the baseline mitochondrial respiration in WT 4B6 cells and in clones #1,#2 and #4 (Fig 4A). Despite elevated mtDNA copy number, the oxygen consumption rate (OCR) in clone #1 was the same as in WT 4B6 cells. However, in clones #2 and #4, which have reduced mtDNA copy number baseline respiration was suppressed as compared to WT 4B6 cells (Fig 4A). Introducing either WT or K510V mutant mLig3 into either 4B6 cells, or into clone #1 had little effect on the baseline OCR (Fig 4B). In contrast, transduction of clones #3 and #4 with WT, but not K510V mutant mLig3 led to a significant increase in baseline OCR (Fig 4C), suggesting that reduced mtDNA copy number is responsible for the reduced OCR. Also, empirical observations of variability in growth rates between parental and virus-transduced cell lines suggested precise measurements of proliferation rates. Doubling times in cell lines were inversely proportional to respiration. Transduction of the WT 4B6 cells and a high mtDNA copy number clone #1 with either WT or mutant Lig3 had no effect on growth rates, whereas transduction of low mtDNA copy number clones #2 and #4 with either WT or, surprisingly, with mutant mLig3 resulted in a significant acceleration of growth (Fig 4D).

Bottom Line: Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content.Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number.This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of South Alabama, 307 University Blvd, Mobile, Alabama, 36688, United States of America.

ABSTRACT
Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

Show MeSH
Related in: MedlinePlus