Studying Mitochondrial Network Formation by In Vivo and In Vitro Reconstitution Assay

2021 ◽  
pp. 333-341
Author(s):  
Wanqing Du ◽  
Xiangjun Di ◽  
Qian Peter Su
Keyword(s):  
Network Formation ◽  
Mitochondrial Network ◽  
In Vitro Reconstitution

scholarly journals All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1202
Author(s):  
Bojjibabu Chidipi ◽  
Syed Islamuddin Shah ◽  
Michelle Reiser ◽  
Manasa Kanithi ◽  
Amanda Garces ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Mitochondrial Fission ◽  
Normal Function ◽  
Mitochondrial Network ◽  
Protein Levels ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

scholarly journals Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Charlotte F Kelley ◽  
Thomas Litschel ◽  
Stephanie Schumacher ◽  
Dirk Dedden ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Network Formation ◽  
Focal Adhesions ◽  
Membrane Composition ◽  
Macromolecular Assemblies ◽  
In Vitro Reconstitution ◽  
Membrane Bound ◽  
Underlying Mechanisms ◽  
Higher Order Networks ◽  
Made In

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

scholarly journals TGF-β promotes microtube formation in glioblastoma through Thrombospondin 1

2021 ◽  
Author(s):  
Justin V. Joseph ◽  
Capucine R. Magaut ◽  
Simon Storevik ◽  
Luiz H. Geraldo ◽  
Thomas Mathivet ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Network Formation ◽  
Cell Communication ◽  
Treatment Resistance ◽  
Communication Structures ◽  
Oligodendroglial Tumors ◽  
Thrombospondin 1 ◽  
Stem Cell Lines

AbstractMicrotubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. To identify potential signaling pathways involved in MT formation we performed a bioinformatics analysis of TCGA data showing that the TGF-β pathway is highly activated in GBMs compared to oligodendroglial tumors. In particular we observed that signaling pathways involved in extracellular matrix organization are differentially expressed between these tumor entities. Using patient-derived GBM stem cell lines, we demonstrated that TGF-β1 stimulation promotes enhanced MT formation and communication via Calcium signaling. Inhibition of the TGF-β pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-β, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-β stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. In conclusion, TGF-β and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT driven invasion/ resistance network.

scholarly journals Intermediate step of cohesin’s ATPase cycle allows cohesin to entrap DNA

2018 ◽  
Vol 115 (39) ◽  
pp. 9732-9737 ◽  
Author(s):  
Gamze Ö. Çamdere ◽  
Kristian K. Carlborg ◽  
Douglas Koshland
Keyword(s):  
Atp Hydrolysis ◽  
Intermediate Step ◽  
Mutant Form ◽  
In Vitro Reconstitution ◽  
Chromatid Cohesion ◽  
The Family ◽  
Structural Maintenance Of Chromosomes ◽  
Dna Substrate

Cohesin is a four-subunit ATPase in the family of structural maintenance of chromosomes (SMC). Cohesin promotes sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. Cohesin performs these functions as a DNA tether and potentially a DNA-based motor. At least one of its DNA binding activities involves entrapment of DNA within a lumen formed by its subunits. This activity can be reconstituted in vitro by incubating cohesin with DNA, ATP, and cohesin loader. Previously we showed that a mutant form of cohesin (DE-cohesin) possesses the ability to bind and tether DNA in vivo. Using in vitro reconstitution assays, we show that DE-cohesin can form stable complexes with DNA without ATP hydrolysis. We show that wild-type cohesin with ADP aluminum fluoride (cohesinADP/AlFx) can also form stable cohesin–DNA complexes. These results suggest that an intermediate nucleotide state of cohesin, likely cohesinADP-Pi, is capable of initially dissociating one interface between cohesin subunits to allow DNA entry into a cohesin lumen and subsequently interacting with the bound DNA to stabilize DNA entrapment. We also show that cohesinADP/AlFx binding to DNA is enhanced by cohesin loader, suggesting a function for loader other than stimulating the ATPase. Finally, we show that loader remains stably bound to cohesinADP/AlFx after DNA entrapment, potentially revealing a function for loader in tethering the second DNA substrate. These results provide important clues on how SMC complexes like cohesin can function as both DNA tethers and motors.

Abstract 5: Neovascularization By Substance-p- Mobilized Epc And Msc In Vitro And In Vivo

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Hyun Sook Hong ◽  
Suna Kim ◽  
Youngsook Son
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Substance P ◽  
Network Formation ◽  
Skin Wound ◽  
Hematopoietic Stem ◽  
Vessel Structure

Bone marrow stem cells, especially, endothelial precursor cells (EPC), mesenchymal stem cells (MSC) or hematopoietic stem cell (HSC) are expected as reparative cells for the repair of a variety of tissue damages such as stroke and myocardial infarction, even though their role in the repair is not demonstrated. This report was investigated to find a role of Substance-p (SP) as a reparative agent in the tissue repair requiring EPC and MSC. In order to examine EPC (EPC SP ) and MSC (MSC SP ) mobilized by SP, we injected SP intravenously for consecutive 2 days and saline was injected as a vehicle. At 3 post injection, peripheral blood (PB) was collected.To get mesenchymal stem cells or endothelial progenitor cells, MNCs were incubated in MSCGM or EGM-2 respectively for 10 days. Functional characteristics of the EPC SP were proven by the capacity to form endothelial tubule network in the matrigel in vitro and in the matrigel plug assay in vivo. In contrast, MSC SP did not form a tube-like structure but formed a pellet-structure on matrigel. However, when both cells were premixed before the matrigel assay, much longer and branched tubular network was formed, in which a-SMA expressing MSC SP were decorating outside of the endothelial tube, especially enriched at the bifurcating point. MSC SP may contribute and reinforce elaborate vascular network formation in vivo by working as pericyte-like cells. Thus, the EPC SP and MSC SP were labeled with PKH green and PKH red respectively and their tubular network was examined. Well organized tubular network was formed, which was covered by PKH green labeled cells and was decorated in a punctate pattern by PKH red labeled cells. In order to investigate the role of EPC SP and MSC SP specifically in vivo, rabbit EPC SP and MSC SP were transplanted to full thickness skin wound. The vessel of EPC SP -transplanted groups was UEA-lectin+, which was not covered with a-SMA+ pericytes but EPC SP + MSC SP -transplanted groups showed, in part, a-SMA+ pericyte-encircled UEA-lectin+ vessels. This proved the specific role of MSC SP as pericytes. From these data, we have postulated that the collaboration of MSC and EPC is essential for normal vessel structure and furthermore, accelerated wound healing as ischemia diseases, which can be stimulated through by SP injection.

scholarly journals A stromal region of cytochromeb6fsubunit IV is involved in the activation of the Stt7 kinase inChlamydomonas

2017 ◽  
Vol 114 (45) ◽  
pp. 12063-12068 ◽  
Author(s):  
Louis Dumas ◽  
Francesca Zito ◽  
Stéphanie Blangy ◽  
Pascaline Auroy ◽  
Xenie Johnson ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Direct Interaction ◽  
Chloroplast Transformation ◽  
Kinase Domain ◽  
State Transitions ◽  
Light Harvesting Complexes ◽  
Gene Coding ◽  
In Vitro Reconstitution

The cytochrome (cyt)b6fcomplex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cytb6factivates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplastpetDgene, coding for subunit IV of the cytb6fcomplex, and complementation of a ΔpetDhost strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cytb6fsubunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cytb6fand recombinant Stt7 kinase domain show that cytb6fenhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cytb6fcomplex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane.

scholarly journals Drosophila development requires spectrin network formation.

1995 ◽  
Vol 128 (1) ◽  
pp. 71-79 ◽  
Author(s):  
H Deng ◽  
J K Lee ◽  
L S Goldstein ◽  
D Branton
Keyword(s):  
Network Formation ◽  
Recombinant Dna ◽  
Point Mutations ◽  
Drosophila Genome ◽  
Dependent Manner ◽  
Null Mutant ◽  
Alpha Spectrin ◽  
Spectrin Network

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta-spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head-end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.

scholarly journals Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Taro Ozaki ◽  
Kona Yamashita ◽  
Yuki Goto ◽  
Morito Shimomura ◽  
Shohei Hayashi ◽  
...  
Keyword(s):  
In Vitro Reconstitution
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