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1.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199774

RESUMO

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.


Assuntos
Mitocôndrias/enzimologia , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Ribonuclease P/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Mitocôndrias/patologia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
2.
J Vis Exp ; (170)2021 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-33900289

RESUMO

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.


Assuntos
Drosophila melanogaster , Ovário/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Feminino , Peróxido de Hidrogênio/farmacologia , Microscopia , Mitocôndrias/efeitos dos fármacos , Ovário/citologia , Oxidantes/farmacologia
3.
Nucleic Acids Res ; 44(13): 6409-22, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27131785

RESUMO

Proteins encoded by mitochondrial DNA are translated using mitochondrially encoded tRNAs and rRNAs. As with nuclear encoded tRNAs, mitochondrial tRNAs must be processed to become fully functional. The mitochondrial form of ribonuclease P (mt:RNase P) is responsible for 5'-end maturation and is comprised of three proteins; mitochondrial RNase P protein (MRPP) 1 and 2 together with proteinaceous RNase P (PRORP). However, its mechanism and impact on development is not yet known. Using homology searches, we have identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). Here, we show that each protein is essential and localizes with mitochondria. Furthermore, reducing levels of each causes mitochondrial deficits, which appear to be due at least in part to defective mitochondrial tRNA processing. Overexpressing two members of the complex, Mulder and Roswell, is also lethal, and in the case of Mulder, causes abnormal mitochondrial morphology. These data are the first evidence that defective mt:RNase P causes mitochondrial dysfunction, lethality and aberrant mitochondrial tRNA processing in vivo, underscoring its physiological importance. This in vivo mt:RNase P model will advance our understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , DNA Mitocondrial/genética , Proteínas de Drosophila/genética , Proteínas Mitocondriais/genética , Ribonuclease P/genética , Animais , Drosophila/genética , Regulação da Expressão Gênica , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , RNA de Transferência/genética , Mutações Sintéticas Letais/genética
4.
Biol Open ; 5(2): 195-203, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26834020

RESUMO

Mitochondrial function is tied to the nucleus, in that hundreds of proteins encoded by nuclear genes must be imported into mitochondria. While post-translational import is fairly well understood, emerging evidence supports that mitochondrial site-specific import, or co-translational import, also occurs. However, the mechanism and the extent to which it is used are not fully understood. We have previously shown Clueless (Clu), a conserved multi-domain protein, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, and genetically and physically interacts with the PINK1-Parkin pathway. The human ortholog of Clu, Cluh, was shown to bind nuclear-encoded mitochondrially destined mRNAs. Here we identify the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, we show Clu interacts with the ribosome at the mitochondrial outer membrane. Taken together, these data support a model whereby Clu binds to and mitochondrially targets mRNAs to facilitate mRNA localization to the outer mitochondrial membrane, potentially for site-specific or co-translational import. This role may link the presence of efficient mitochondrial protein import to mitochondrial quality control through the PINK1-Parkin pathway.

5.
Front Pharmacol ; 6: 168, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26321954

RESUMO

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic ß-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.

6.
Dis Model Mech ; 8(6): 577-89, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26035866

RESUMO

Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD). However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Feminino , Humanos , Proteínas Mitocondriais/metabolismo , Mutação/genética , Fenótipo , Ligação Proteica
7.
BMC Physiol ; 14: 12, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25488103

RESUMO

BACKGROUND: There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing Kv channel activity and decreasing the action potential duration. Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes. RESULTS: Acute exposure of myocytes to 10 mM sodium acetate caused a marked, but transient, decrease in systolic sarcomere shortening (1.49 ± 0.20% vs. 5.58 ± 0.49% in control), accompanied by a significant increase in diastolic sarcomere length (1.81 ± 0.01 µm vs. 1.77 ± 0.01 µm in control), with a near linear dose response in the 1-10 mM range. Unlike palmitate, acetate caused no change in action potential duration; however, acetate markedly increased mitochondrial Ca(2+) uptake. Moreover, pretreatment of cells with the mitochondrial Ca(2+) uptake blocker, Ru-360 (10 µM), markedly suppressed the effect of acetate on contraction. CONCLUSIONS: Lehninger and others have previously demonstrated that the anions of weak aliphatic acids such as acetate stimulate Ca(2+) uptake in isolated mitochondria. Here we show that this effect of acetate appears to extend to isolated cardiac myocytes where it transiently modulates cell contraction.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Contração Miocárdica , Acetato de Sódio/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Acetato de Sódio/farmacologia
8.
Exp Parasitol ; 135(1): 87-95, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23792131

RESUMO

Antigens obtained from the intestinal tract of filarial nematodes have been proposed as potential safe and effective vaccine candidates. Because they may be 'hidden' from the immune response during natural infection, yet accessible by antibodies induced by vaccination, intestinal antigens may have a low potential for eliciting allergic responses when vaccinating previously infected individuals. Despite prior promising data, vaccination with intestinal antigens has yet to be tested in a permissive model of filariasis. In this study we investigated the efficacy of vaccination with filarial intestinal antigens in the permissive Litomosoides sigmodontis BALB/c model of filariasis, and we evaluated the extent to which these antigens are recognized by the immune system during and after infection. Infected BALB/c mice developed lower IgG antibody responses to soluble intestinal antigens (GutAg) than to soluble antigens of whole worms (LsAg). Similarly, GutAg induced less proliferation and less production of IL-4 and IFNγ from splenocytes of infected mice than LsAg. In contrast to these differences, active infection resulted in equivalent levels of circulating GutAg-specific IgE and LsAg-specific IgE levels. Consistent with this, basophil activation, as assessed by flow cytometric staining of intracellular basophil IL-4 expression, was equivalent in response to GutAg and LsAg. Vaccination with GutAg adsorbed to CpG/alum induced GutAg specific IgG1 and IgG2A production, with GutAg specific IgG titers greater than 5-fold higher than those measured in previously infected animals. Despite this response to GutAg vaccination, vaccinated mice harbored similar parasite burdens 8 weeks post infection when compared to non-vaccinated controls. These studies demonstrate that soluble antigens obtained from the intestinal tracts of L. sigmodontis have some qualities of 'hidden' antigens, but they still sensitize mice to allergic reactions and fail to protect against future infection when given as a vaccine adsorbed to alum/CPG.


Assuntos
Antígenos de Helmintos/administração & dosagem , Filariose/prevenção & controle , Filarioidea/imunologia , Vacinação/normas , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Basófilos/imunologia , Citocinas/análise , Modelos Animais de Doenças , Feminino , Filariose/imunologia , Gerbillinae , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Intestinos/imunologia , Larva/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia
9.
PLoS One ; 8(1): e54283, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23342118

RESUMO

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.


Assuntos
Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/genética , Larva , Proteínas Nucleares/genética , Fosforilação Oxidativa
10.
Dis Model Mech ; 2(9-10): 490-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19638420

RESUMO

Parkinson's disease has been linked to altered mitochondrial function. Mutations in parkin (park), the Drosophila ortholog of a human gene that is responsible for many familial cases of Parkinson's disease, shorten life span, abolish fertility and disrupt mitochondrial structure. However, the role played by Park in mitochondrial function remains unclear. Here, we describe a novel Drosophila gene, clueless (clu), which encodes a highly conserved tetratricopeptide repeat protein that is related closely to the CluA protein of Dictyostelium, Clu1 of Saccharomyces cerevisiae and to similar proteins in diverse metazoan eukaryotes from Arabidopsis to humans. Like its orthologs, loss of Drosophila clu causes mitochondria to cluster within cells. We find that strong clu mutations resemble park mutations in their effects on mitochondrial function and that the two genes interact genetically. Conversely, mitochondria in park homozygotes become highly clustered. We propose that Clu functions in a novel pathway that positions mitochondria within the cell based on their physiological state. Disruption of the Clu pathway may enhance oxidative damage, alter gene expression, cause mitochondria to cluster at microtubule plus ends, and lead eventually to mitochondrial failure.


Assuntos
Sequência Conservada , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genes de Insetos , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Alelos , Animais , Proteínas de Drosophila/química , Drosophila melanogaster/ultraestrutura , Feminino , Regulação da Expressão Gênica , Microtúbulos/metabolismo , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Músculos/patologia , Músculos/ultraestrutura , Mutação/genética , Proteínas Nucleares/química , Folículo Ovariano/metabolismo , Transporte Proteico , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Ubiquitina-Proteína Ligases
11.
Development ; 133(17): 3371-7, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16887820

RESUMO

Mitochondria in many species enter the young oocyte en mass from interconnected germ cells to generate the large aggregate known as the Balbiani body. Organelles and germ plasm components frequently associate with this structure. Balbiani body mitochondria are thought to populate the germ line, ensuring that their genomes will be inherited preferentially. We find that milton, a gene whose product was previously shown to associate with Kinesin and to mediate axonal transport of mitochondria, is needed to form a normal Balbiani body. In addition, germ cells mutant for some milton or Kinesin heavy chain (Khc) alleles transport mitochondria to the oocyte prematurely and excessively, without disturbing Balbiani body-associated components. Our observations show that the oocyte acquires the majority of its mitochondria by competitive bidirectional transport along microtubules mediated by the Milton adaptor. These experiments provide a molecular explanation for Balbiani body formation and, surprisingly, show that viable fertile offspring can be obtained from eggs in which the normal program of mitochondrial acquisition has been severely perturbed.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Mitocôndrias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Oócitos/fisiologia , Oogênese/fisiologia , Animais , Western Blotting , Padronização Corporal , Divisão Celular/fisiologia , Clonagem de Organismos , Dineínas/fisiologia , Cinesina/fisiologia , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Development ; 130(8): 1579-90, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12620983

RESUMO

Maternally inherited mitochondria and other cytoplasmic organelles play essential roles supporting the development of early embryos and their germ cells. Using methods that resolve individual organelles, we studied the origin of oocyte and germ plasm-associated mitochondria during Drosophila oogenesis. Mitochondria partition equally on the spindle during germline stem cell and cystocyte divisions. Subsequently, a fraction of cyst mitochondria and Golgi vesicles associates with the fusome, moves through the ring canals, and enters the oocyte in a large mass that resembles the Balbiani bodies of Xenopus, humans and diverse other species. Some mRNAs, including oskar RNA, specifically associate with the oocyte fusome and a region of the Balbiani body prior to becoming localized. Balbiani body development requires an intact fusome and microtubule cytoskeleton as it is blocked by mutations in hu-li tai shao, while egalitarian mutant follicles accumulate a large mitochondrial aggregate in all 16 cyst cells. Initially, the Balbiani body supplies virtually all the mitochondria of the oocyte, including those used to form germ plasm, because the oocyte ring canals specifically block inward mitochondrial transport until the time of nurse cell dumping. Our findings reveal new similarities between oogenesis in Drosophila and vertebrates, and support our hypothesis that developing oocytes contain specific mechanisms to ensure that germ plasm is endowed with highly functional organelles.


Assuntos
Drosophila melanogaster/fisiologia , Mitocôndrias/genética , Oócitos/fisiologia , Oogênese/fisiologia , Animais , Padronização Corporal , Divisão Celular/fisiologia , Feminino , Complexo de Golgi/metabolismo , Humanos , Microscopia Eletrônica , Oócitos/citologia , Ovário/anatomia & histologia , RNA/metabolismo , Transgenes
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