scholarly journals Mechanisms, pathophysiological roles and methods for analyzing mitophagy – recent insights

2018 ◽  
Vol 399 (2) ◽  
pp. 147-178 ◽  
Author(s):  
Jessica A. Williams ◽  
Wen-Xing Ding
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Molecular Tools ◽  
Mouse Tissues

Abstract In 2012, we briefly summarized the mechanisms, pathophysiological roles and methods for analyzing mitophagy. As then, the mitophagy field has continued to grow rapidly, and many new molecular mechanisms regulating mitophagy and molecular tools for monitoring mitophagy have been discovered and developed. Therefore, the purpose of this review is to update information regarding these advances in mitophagy while focusing on basic molecular mechanisms of mitophagy in different organisms and its pathophysiological roles. We also discuss the advantage and limitations of current methods to monitor and quantify mitophagy in cultured cells and in vivo mouse tissues.

scholarly journals ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility

2021 ◽  
Vol 118 (6) ◽  
pp. e2018355118
Author(s):  
Keisuke Shimada ◽  
Soojin Park ◽  
Haruhiko Miyata ◽  
Zhifeng Yu ◽  
Akane Morohoshi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Mitochondrial Dynamics ◽  
Helical Structure ◽  
Double Helical Structure ◽  
Mammalian Sperm ◽  
Peripheral Membrane Protein ◽  
Sheath Formation ◽  
Sperm Midpiece

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered Armc12-null mice, FLAG-tagged Armc12 knock-in mice, and TBC1 domain family member 21 (Tbc1d21)-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in Armc12-null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.

scholarly journals Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent

2000 ◽  
Vol 352 (3) ◽  
pp. 795-800 ◽  
Author(s):  
Tadao HASEGAWA ◽  
Hengyi XIAO ◽  
Fumiyasu HAMAJIMA ◽  
Ken-ichi ISOBE
Keyword(s):  
Dna Damage ◽  
Heat Shock ◽  
Cultured Cells ◽  
Similar Region ◽  
C Terminus ◽  
Mouse Tissues ◽  
Time Courses ◽  
Shock Proteins ◽  
Two Hybrid

GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and γ134.5 regions. A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned. The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo. For binding of the two proteins, the γ134.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important. GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis. Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different. In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 815-815
Author(s):  
Marilyn Parra ◽  
Xiuli An ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Start Codon ◽  
Exon 2 ◽  
Mouse Tissues

Abstract Abstract 815 Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3' splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3' splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs. To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

2008 ◽  
Vol 182 (6) ◽  
pp. 1127-1139 ◽  
Author(s):  
Ying-Chen Claire Hou ◽  
Suganthi Chittaranjan ◽  
Sharon González Barbosa ◽  
Kimberly McCall ◽  
Sharon M. Gorski
Keyword(s):  
Cell Death ◽  
Drosophila Melanogaster ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Mitogen Activated Protein Kinase ◽  
Nuclear Condensation ◽  
Effector Caspase ◽  
Apoptosis Protein ◽  
Egg Chambers

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells

2004 ◽  
Vol 32 (6) ◽  
pp. 1057-1059 ◽  
Author(s):  
A.D. Postle ◽  
H. Dombrowsky ◽  
H. Clarke ◽  
C.J. Pynn ◽  
G. Koster ◽  
...  
Keyword(s):  
Molecular Species ◽  
Cultured Cells ◽  
Ionization Mass ◽  
Membrane Composition ◽  
Mouse Tissues ◽  
Wide Range ◽  
Kinetics Of ◽  
Hydrolysis Of

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d6-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241− and m/z 247− respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

scholarly journals Senescent Cell-Secreted Netrin-1 Modulates Aging-Related Disorders by Recruiting Sympathetic Fibers

2020 ◽  
Vol 12 ◽  
Author(s):  
Ai Qing Yu ◽  
Jie Wang ◽  
Xiao Jia Zhou ◽  
Ke Yu Chen ◽  
You De Cao ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Molecular Mechanisms ◽  
Human Colon ◽  
Senescent Cell ◽  
Aged Mouse ◽  
Age Related ◽  
Mouse Tissues ◽  
6 Hydroxydopamine

Cellular senescence is implicated in several lines of aging-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibers (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SFs densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cells induce recruitment of SFs in vitro. Additionally, subcutaneous transplantation of 2BS fibroblasts in nude mice shows that transplanted senescent 2BS fibroblasts promote SFs infiltration. Intra-articular senolytic molecular injection can reduce SFs density and inhibit SFs infiltration caused by senescent cells in osteoarthritis (OA), suggesting senescent cells promote the infiltration of SFs in vivo in aged tissues. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective β receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective β receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE−/− mice) by reducing hepatic SNA. Taken together, this study concludes that senescent cell-secreted netrin-1 mediated SFs outgrowth and infiltration, which contributes to aging-related disorders, suggesting that clearing senescent cells or inhibiting SNA is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced aging-related pathologies.

scholarly journals Quantitative intravital imaging reveals in vivo dynamics of physiological-stress induced mitophagy

2020 ◽  
Author(s):  
Paul J. Wrighton ◽  
Arkadi Shwartz ◽  
Jin-Mi Heo ◽  
Eleanor D. Quenzer ◽  
Kyle A. LaBella ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Physiological Stress ◽  
Hypoxia Inducible Factor ◽  
Imaging Study ◽  
Time Lapse ◽  
Intravital Imaging ◽  
Response Dynamics ◽  
Pathway Activation

ABSTRACTMitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia, and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive, fluorescent probes mito-Keima and mito-EGFP-mCherry and used quantitative intravital imaging to illuminate mitophagy during physiological stresses—embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress-response dynamics, and time-lapse imaging revealed mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor bnip3 prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes bnip3la (nix), fundc1, pink1 or prkn (Parkin) had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.

scholarly journals Degradation intermediates as an indicator of mRNA and cell metabolism

2021 ◽  
Author(s):  
Yusheng Liu ◽  
Yiwei Zhang ◽  
Hu Nie ◽  
Falong Lu ◽  
Jiaqiang Wang
Keyword(s):  
Degradation Rate ◽  
Cultured Cells ◽  
Cell Metabolism ◽  
Single Cells ◽  
Mrna Degradation ◽  
Rate Analysis ◽  
Mouse Tissues ◽  
Degradation Intermediates ◽  
Mrna Degradation Rate

Traditional mRNA degradation rate measurements involves complex experimental design with RNA labeling or transcription blocking together with sampling at multiple timepoints. These experimental requirements limit the application of transcriptome-wide mRNA degradation rate analysis mainly in cultured cells, but rarely in in vivo samples. Therefore, a direct and simple strategy needs to be developed to study mRNA degradation rate. Here, we defined mRNA degradation intermediates as transcripts where decay is about to occur or has partially occurred in the 3′-untranslated regions after poly(A) tail deadenylation, and found that the proportion of mRNA degradation intermediates is a very simple and convenient indicator for evaluating the degradation rate of mRNA in mouse and human cell lines. In addition, we showed that a higher proportion of mRNA degradation intermediates is correlated with faster cell cycle and higher turnover rate of mouse tissues. Further, we validated that in mouse maturing oocytes where transcription is silent, the proportion of mRNA degradation intermediates is positively correlated with the mRNA degradation rate. Together, these results demonstrate that degradation intermediates can function as a good indicator of mRNA, cell, and tissue metabolism, and can be easily assayed by total RNA 3′-end sequencing from a single bulk cell sample without the need for drug treatment or multi-timepoint sampling. This finding is of great potential for studies on mRNA degradation rate at the molecular, cellular, or organic level, including samples or systems that cannot be assayed with previous methods. In addition, further application of the findings into single cells will likely greatly aid the identification and study of rare cells with unique cellular metabolism dynamics such as tissue stem cells and tumor cells.

Oligodendrocyte precursor (O-2A progenitor cell) migration; a model system for the study of cell migration in the developing central nervous system

Development ◽  
1993 ◽  
Vol 119 (Supplement) ◽  
pp. 219-225 ◽  
Author(s):  
B. W. Kiernan ◽  
Charles ffrench-Constant
Keyword(s):  
Nervous System ◽  
Cell Migration ◽  
Progenitor Cell ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Large Numbers ◽  
Multicellular Organisms ◽  
Developing Nervous System

Cell migration plays an important role in the development of complex multicellular organisms. The molecular mechanisms that regulate this migration arc therefore of great interest. Unfortunately, however, analysis of cell migration in vertebrates is hampered by the inaccessability of the cells and the difficulty of manipulating their environment within the embryo. This review focusses on one particular migratory cell population, the oligodendrocyte p1ecursor cell or O-2A progenitor cell, that gives rise to the myelin-forming oligodendrocytes within the CNS. These cells mi grate extensively during normal development. They can be purified and grown in large numbers in cell culture, so allowing the use of reductionist approaches using cell and molecular biology techniques. Moreover, cultured cells will migrate within the CNS following transplantation. As a result, the migration of these cells in vivo can be analysed following manipulation in vitro. Taken together, we believe that the different properties of these cells makes them excellent candidates for studies addressing the control of cell migration in the developing nervous system.

scholarly journals P0407THE INVESTIGATION OF SYNAPTOPODIN EXPRESSION OF PODOCYTES IN GLOMERONEPHRITIS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Zhigang Zhang
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Glomerular Diseases ◽  
Podocyte Injury ◽  
Adriamycin Nephropathy ◽  
Assembly Result ◽  
And Function ◽  
Rhoa Signaling ◽  
New Strategies

Abstract Background and Aims Synaptopodin, a proline-rich actin-associated protein, plays an important role in the regulation of podocytes processes structures and dynamics. The mutation or lack of synaptopodin may lead to the changes of podocytes structures and functions and cause the occurrence of proteinuria. But the underlying molecular mechanisms remain primarily elusive. Method we used cellular and pathological experiments to observe the expression changes synaptopodin in vivo and vitrio. Results The results showed that the reduction expression of synaptopodin and RhoA were found in the podocytes in different nephriris of human renal biopsy as well as in rat adriamycin nephropathy. The cultured cells treated with inflammatory cytokins such as TNF, IL-1 also showed decreased synaptopodin level in podocyte, which led to low RhoA level and disarrange the actin cytoskeleton assembly, result in the abnormal changes of podocyte morphology. Conclusion These data preliminarily proved that synaptopodin loss in podocyte injury plays an important role in the regulation of podocyte morphology and function through RhoA signaling pathway, and further researches are required to clarify the more mechanism, which may provide new strategies and methods for the prevention and treatment of glomerular diseases.

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