Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum‐rich domain of the ascidian egg myoplasm

Ishii, Hirokazu; Kunihiro, Shiori; Tanaka, Manabu; Hatano, Kyoko; Nishikata, Takahito

doi:10.1111/dgd.12003

Cited by 11 publications

(19 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6 F–H) distributed throughout most of the cytoplasm. NN18 labelled exclusively mitochondria in these preparations, however the use of sub-cellular fractionation followed by immunnoblotting indicates that p58 is also present in non-mitochondrial cytoplasm [61].…”

Section: Resultsmentioning

confidence: 77%

Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity

et al. 2013

Self Cite

View full text Add to dashboard Cite

Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.

show abstract

Section: Resultsmentioning

confidence: 77%

Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity

et al. 2013

Self Cite

View full text Add to dashboard Cite

show abstract

“…These subunits were unexpectedly found to exist in the cytosol and are suggested to play unknown roles other than mitochondrial respiration (Ishii et al . ). However, mitochondria are very few in the cortical region of the myoplasm (Speksnijder et al .…”

Section: Introductionmentioning

confidence: 97%

“…; Ishii et al . ). Most of the mitochondria are localized to the internal region of the myoplasm (mitochondria‐rich myoplasm).…”

Section: Introductionmentioning

confidence: 97%

See 1 more Smart Citation

Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria‐rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo

et al. 2014

Self Cite

View full text Add to dashboard Cite

In ascidian eggs, cytoplasmic and cortical reorganization, previously called ooplasmic segregation, occurs in two phases during the first cell cycle. In the second phase of reorganization, the mitochondria-rich cytoplasm (myoplasm) moves to the future posterior side, concurrent with sperm aster migration along the egg cortex. Although this reorganization is the critical step for establishing the anteroposterior axis, its molecular mechanism is not fully understood. In this study, we showed that low concentrations of the mitochondrial inhibitor sodium azide (NaN 3 ), which showed the low toxicity in sperm, inhibited the second phase of reorganization without the microtubule depolymerization. In the NaN 3 -treated embryo, the sperm aster was not attracted to the cortex and altered its migration pathway; therefore, the myoplasm remained at the vegetal pole. Consequently, the anteroposterior axis was not established. Another mitochondrial inhibitor, oligomycin, did not affect these processes. These results suggest that NaN 3 inhibits unknown molecules that are important for the second phase of reorganization. Identifying the target molecule of NaN 3 will lead to a molecular understanding of cytoplasmic and cortical reorganization.

show abstract

“…Egg and sperm handling, dechorionation, and fertilization followed previous publications (Ishii et al . , ). Embryos were reared in filtered seawater at 18°C.…”

Section: Methodsmentioning

confidence: 99%

Microtubule array observed in the posterior‐vegetal cortex during cytoplasmic and cortical reorganization of the ascidian egg

2017

Self Cite

View full text Add to dashboard Cite

Body axis formation during embryogenesis results from asymmetric localization of maternal factors in the egg. Shortly before the first cleavage in ascidian eggs, cell polarity along the anteroposterior (A-P) axis is established and the cytoplasmic domain (myoplasm) relocates from the vegetal to the posterior region in a microtubule-dependent manner. Through immunostaining, tubulin accumulation during this reorganization is observable on the myoplasm cortex. However, more detailed morphological features of microtubules remain relatively unknown. In this study, we invented a new reagent that improves the immunostaining of cortical microtubules and successfully visualized a parallel array of thick microtubules. During reorganization, they covered nearly the entire myoplasm cortical region, beneath the posterior-vegetal cortex. We designated this microtubule array as CAMP (cortical array of microtubules in posterior vegetal region). During the late phase of reorganization, CAMP shrank and the myoplasm formed a crescent-like cytoplasmic domain. When the CAMP formation was inhibited by sodium azide, myoplasmic reorganization and A-P axis formation were both abolished, suggesting that CAMP is important for these two processes.

show abstract

Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum‐rich domain of the ascidian egg myoplasm

Cited by 11 publications

References 53 publications

Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity

Purification of Mitochondrial Proteins HSP60 and ATP Synthase from Ascidian Eggs: Implications for Antibody Specificity

Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria‐rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo

Microtubule array observed in the posterior‐vegetal cortex during cytoplasmic and cortical reorganization of the ascidian egg

Contact Info

Product

Resources

About