Isolation of functional pure mitochondria by superparamagnetic microbeads

Hornig-Do, Hue-Tran; Günther, Gritt; Bust, Maria; Lehnartz, Patricia; Bosio, Andreas; Wiesner, Rudolf J.

doi:10.1016/j.ab.2009.02.040

Cited by 129 publications

(100 citation statements)

References 22 publications

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“…Similarly, DC showed 4.47 fold increase in the activity of Complex IV compared to monocytes (6.74 ± 0.46 vs 1.42 ± 0.18 nmol/min/10 6 cells, respectively). In order to compare directly the expression of mitochondrial proteins between monocytes and DC, a new magnetic sorting method was used to isolate pure functional mitochondria from cultured cells, which turned out suitable also for a small number of human primary cells (Hornig-Do et al, 2009). WB analysis showed that the expression of the outer membrane proteins TOM22, VDAC, and NDUFS3 in mitochondria isolated from an equal nucleus number was significantly increased in DC compared to monocytes (Fig.…”

Section: Resultsmentioning

confidence: 99%

An active mitochondrial biogenesis occurs during dendritic cell differentiation

Zaccagnino

Saltarella

Maiorano

et al. 2012

The International Journal of Biochemistry & Cell Biology

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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. b s t r a c tDendritic cells (DC) are sentinels of the immune system deriving from circulating monocyte precursors recruited to sites of inflammation. In a previous report (Del Prete et al., 2008) we showed that, after differentiation, DC exhibited increased number of condensed mitochondria and dynamic changes in their energy metabolism. A study is presented here showing that the DC differentiation process is characterized by increased expression level and activity of mitochondrial respiratory complexes, as well as by an increased mitochondrial DNA (mtDNA) copy number. Moreover, DC are equipped with more efficient antioxidant protection systems, over expressed most likely to detoxify increased ROS production, as a consequence of the much higher mitochondrial activity. Kinetic analysis of the three main mitochondrial biogenesis-associated genes revealed that the peak in PPAR␥ coactivator-1alpha (PGC-1␣) gene expression was suddenly reached few hours after the onset of the differentiation. While PGC-1␣ expression rapidly declines, the mitochondrial transcription factor A (TFAM) and nuclear respiratory factor-1 (NRF-1) expression gradually increased. These findings demonstrate that an active mitochondrial biogenesis occurs during DC differentiation and further suggest that an early input by the master regulator of mitochondrial biogenesis PGC-1␣ is needed to trigger the subsequent activation of the downstream transcription factors, NRF-1 and TFAM in this process.

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Section: Resultsmentioning

confidence: 99%

An active mitochondrial biogenesis occurs during dendritic cell differentiation

Zaccagnino

Saltarella

Maiorano

et al. 2012

The International Journal of Biochemistry & Cell Biology

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“…Mitochondria were isolated from HeLa cells persistently infected with Coxiella using magnetic beads labeled with antibodies against the mitochondrial outer membrane protein Tom22, to ensure the specific enrichment of mitochondria. This approach has previously been reported to produce a high yield of pure and intact mitochondria (25,26). Immunoblotting for a mitochondrial protein, Bak, demonstrated that this protocol significantly enriched for mitochondria, and probing for the endoplasmic reticulum (ER) protein PDI showed that minimal ER was present in these mitochondrial preparations (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we observed that transfected 3ϫFLAG-tagged CBU0077 ( 3ϫFLAG CBU0077), expressed from pFLAG-0077, colocalized with the lysosomal marker LAMP-1 (8). Thus, it was not surprising to observe that ectopically expressed 3ϫFLAG CBU0077 specifically colocalized with LAMP-1 on the 25, and samples were collected at the same time points described above. Graphs represent the fold change in genome equivalents (Ϯ standard deviation) relative to those on day 0 for the Coxiella NM PhII wild type (black squares), Δcbu0077 clone 1 (white circles), and Δcbu0077 clone 2 (white triangles) from at least five independent experiments.…”

Section: Resultsmentioning

confidence: 99%

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection

et al. 2017

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Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella. Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiella effector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3ϫFLAG or MceA tagged with 2ϫhemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.KEYWORDS Coxiella burnetii, bacterial effector, mitochondria, prenylation, hostpathogen interactions C oxiella burnetii is the causative agent of human Q fever, a zoonotic infection with a worldwide distribution. The extremely low infectious dose, the ease of aerosol dissemination, and the environmental stability of this pathogen all contribute to Coxiella being classified as a category B select agent by the U.S. Centers for Disease Control and Prevention (1, 2).During human infection, inhaled Coxiella bacteria predominantly infect alveolar macrophages, where they develop a unique intracellular replicative niche termed the Coxiella-containing vacuole (CCV). Internalized bacteria are trafficked through the endocytic pathway until they reach the acidic and hydrolytic confines of the lysosome.

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“…In order to maximize mitochondria enrichment efficiency and minimize contaminations from other organelles, we employed a newly developed method for mitochondria isolation based on superparamagnetic microbeads conjugated to anti-TOM22 antibody (44). The protocol is fast, reproducible, and standardized, resulting in mitochondria of high purity, with minimal contamination from cytoskeleton, cytosol, Golgi apparatus, endosome, endoplasmic reticulum, and nucleus (44).…”

Section: Resultsmentioning

confidence: 99%

Quantitative Proteomics Discloses MET Expression in Mitochondria as a Direct Target of MET Kinase Inhibitor in Cancer Cells

Guo

Zhu

Gan

et al. 2010

Molecular & Cellular Proteomics

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Cancer cells with MET overexpression are paradoxically more sensitive to MET inhibition than cells with baseline MET expression. The underlying molecular mechanisms are incompletely understood. Here, we have traced early responses of SNU5, a MET-overexpressing gastric cancer cell line, exposed to sublethal concentration of PHA-665752, a selective MET inhibitor, using iTRAQ-based quantitative proteomics. More than 1900 proteins were quantified, of which >800 proteins were quantified with at least five peptides. Proteins whose expression was perturbed by PHA-665752 included oxidoreductases, transfer/carrier proteins, and signaling proteins. Strikingly, 38% of proteins whose expression was confidently assessed to be perturbed by MET inhibition were mitochondrial proteins. Upon MET inhibition by a sublethal concentration of PHA-665752, mitochondrial membrane potential increased and mitochondrial permeability transition pore was inhibited concomitant with widespread changes in mitochondrial protein expression. We also showed the presence of highly activated MET in mitochondria, and striking suppression of MET activation by 50 nM PHA-665752. Taken together, our data indicate that mitochondria are a direct target of MET kinase inhibition, in addition to plasma membrane MET. Effects on activated MET in the mitochondria of cancer cells that are sensitive to MET inhibition might constitute a novel and critical noncanonical mechanism for the efficacy of MET-targeted therapeutics. Molecular & Cellular Proteomics 9: 2629 -2641, 2010.

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Isolation of functional pure mitochondria by superparamagnetic microbeads

Cited by 129 publications

References 22 publications

An active mitochondrial biogenesis occurs during dendritic cell differentiation

An active mitochondrial biogenesis occurs during dendritic cell differentiation

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection

Quantitative Proteomics Discloses MET Expression in Mitochondria as a Direct Target of MET Kinase Inhibitor in Cancer Cells

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