A viscosity sensitive fluorescent dye for real-time monitoring of mitochondria transport in neurons

Baek, Yeonju,Park, Sang Jun,Zhou, Xin,Kim, Gyungmi,Kim, Hwan Myung,Yoon, Juyoung Elsevier 2016 Biosensors & Bioelectronics Vol.86 No.-

<P><B>Abstract</B></P> <P>We present here a viscosity sensitive fluorescent dye, namely thiophene dihemicyanine (TDHC), that enables the specific staining of mitochondria. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this dye demonstrated its unique ability for robust staining of mitochondria with high photostability and ultrahigh signal-to-noise ratio (SNR). Moreover, TDHC also showed high sensitivity towards mitochondria membrane potential (ΔΨ<SUB>m</SUB>) and intramitochondria viscosity change. Consequently, this dye was utilized in real-time monitoring of mitochondria transport in primary cortical neurons. Finally, the Two-Photon Microscopy (TPM) imaging ability of TDHC was also demonstrated.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We presented here a viscosity sensitive dye that can remarkably improve signal-to-noise ratio for mitochondria specific staining. </LI> <LI> This dye showed high photostability, and high sensitivity to mitochondria membrane potential alteration. </LI> <LI> The dye was utilized in real-time monitoring mitochondria transport in primary cortical neurons. </LI> <LI> TPM imaging ability of the dye was also demonstrated. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>The viscosity sensitive fluorescent dye, which enable to specifically stain mitochondria with ultrahigh signal-to-noise ratio, was utilized in real-time monitoring mitochondria transport in primary cortical neurons.</P> <P>[DISPLAY OMISSION]</P>

Functional nanosome for enhanced mitochondria-targeted gene delivery and expression

Bae, Yoonhee,Jung, Min Kyo,Song, Su Jeong,Green, Eric S.,Lee, Seulgi,Park, Hyun-Sook,Jeong, Seung Hun,Han, Jin,Mun, Ji Young,Ko, Kyung Soo,Choi, Joon Sig Elsevier 2017 Mitochondrion Vol.37 No.-

<P><B>Abstract</B></P> <P>Mitochondria dysfunction plays a role in many human diseases. Therapeutic techniques for these disorders require novel delivery systems that can specifically target and penetrate mitochondria. In this study, we report a novel nanosome composed of dequalinium-DOTAP-DOPE (1,2 dioleoyl-3-trimethylammonium-propane-1,2-dioleoyl-<I>sn</I>-glycero-3-phosphoethanolamine) (DQA80s) as a potential mitochondria-targeting delivery vector. The functional DQAsome, DQA80s, showed enhanced transfection efficiency compared to a vector DQAsomes in HeLa cells and dermal fibroblasts. In addition, DQA80s/pDNA complexes exhibited rapid escape from the endosome into the cytosol. We observed the delivery of pDNA to mitochondria in living cells using flow cytometry, confocal microscopy, and TME imaging. More specifically, we confirmed our results by co-localization of hmtZsGreen constructs to mitochondria when delivered via DQAsomes and DQA80s in living cells. The mitochondria-targeting DQAsomes and DQA80s induced mitochondrial dysfunction through depolarization of mitochondrial membrane potential. Our data demonstrate that DQA80s show promise for use as a mitochondria-targeted carrier system for treatment of mitochondria diseases in vivo.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Formulation and characterization of novel nanosome as a potential mitochondria-targeting delivery vector </LI> <LI> DQA80s/pDNA complexes exhibited a rapid endolysosomal escape and enhanced mitochondrial targeting in living cells. </LI> <LI> DQA80s/hmtGFP construct complexes showed expression at mitochondrial region in living cells. </LI> <LI> DQA80s induced mitochondria dysfunction via depolarization of mitochondrial membrane potential in cancer cells. </LI> </UL> </P>

Asymmetric distribution of mitochondria in the vegetal marginal zone of the ascidian embryo

Ji Won Lee,Gil Jung Kim 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8

The mechanisms by which embryo exactly distributes mitochondria into the blastomeres during embryogenesis are one of the important issues in developmental biology. Although the mechanisms has been thought to be important for the proper embryonic development, our understanding has remained limited. In the present study, the distribution of mitochondria was examined in embryos of the ascidian, Halocynthia roretzi, by immunohistochemical staining with three-types of the mitochondria-specific antibodies and vital staining of mitochondria with a fluorescent probe, DiOC2(3). Results of the immunohistochemical staining coincided with that of vital staining, which is able to detect the distribution of mitochondria in cytoplasm of the embryo. Mitochondria was mainly segregated into the B4.1 posterior-vegetal blastomeres at the 8-cell stage. During the next stages, mitochondria was preferentially partitioned into cells of the B-line muscle and the A-line nerve cord precursor compared with each sister cell, endoderm in the 5th cleavage stage, and mesenchyme and notochord in the 6th cleavage stage. However, the mitochondria-rich cytoplasm is divided equally among the blastomeres of the animal hemisphere between the 8-cell and the 64-cell stages. When B6.2 blastomeres were isolated at the early 32-cell stage embryo and cultured in seawater, until control embryos reached the 64-cell stage, pattern of mitochondria distribution was similar to results of the coisolated B7.3 and B7.4 blastomeres from the 64-cell stage embryos. Therefore, it is likely that mitochondria are asymmetrically segregated into the marginal cells in the vegetal hemisphere of the ascidian embryo without cell-cell interaction.

Involvement of S6K1 in mitochondria function and structure in HeLa cells

Park, J.,Tran, Q.,Mun, K.,Masuda, K.,Kwon, S.H.,Kim, S.H.,Kim, D.H.,Thomas, G.,Park, J. Pergamon Press ; Elsevier Science Ltd 2016 Cellular signalling Vol.28 No.12

The major biological function of mitochondria is to generate cellular energy through oxidative phosphorylation. Apart from cellular respiration, mitochondria also play a key role in signaling processes, including aging and cancer metabolism. It has been shown that S6K1-knockout mice are resistant to obesity due to enhanced beta-oxidation, with an increased number of large mitochondria. Therefore, in this report, the possible involvement of S6K1 in regulating mitochondria dynamics and function has been investigated in stable lenti-shS6K1-HeLa cells. Interestingly, S6K1-stably depleted HeLa cells showed phenotypical changes in mitochondria morphology. This observation was further confirmed by detailed image analysis of mitochondria shape. Corresponding molecular changes were also observed in these cells, such as the induction of mitochondrial fission proteins (Drp1 and Fis1). Oxygen consumption is elevated in S6K1-depeleted HeLa cells and FL5.12 cells. In addition, S6K1 depletion leads to enhancement of ATP production in cytoplasm and mitochondria. However, the relative ratio of mitochondrial ATP to cytoplasmic ATP is actually decreased in lenti-shS6K1-HeLa cells compared to control cells. Lastly, induction of mitophagy was found in lenti-shS6K1-HeLa cells with corresponding changes of mitochondria shape on electron microscope analysis. Taken together, our results indicate that S6K1 is involved in the regulation of mitochondria morphology and function in HeLa cells. This study will provide novel insights into S6K1 function in mitochondria-mediated cellular signaling.

Liposomes containing cholesterol and mitochondria-penetrating peptide (MPP) for targeted delivery of antimycin A to A549 cells

Mallick, Sudipta,Thuy, Le Thi,Lee, Seulgi,Park, Jong-II,Choi, Joon Sig Elsevier 2018 Colloids and surfaces. B, Biointerfaces Vol.161 No.-

<P><B>Abstract</B></P> <P> <UL> <LI> Mitochondria are exclusively employed to produce energy required for the vital metabolic functions of the cell. However, mitochondria also play a key role in mammalian cell death. Dissipation in the mitochondria membrane potential causes cell death. Therefore, in cancer therapy, mitochondria are a novel target. Herein, we developed a nano-formulation of Antimycin A specifically targeted towards mitochondria and lung cancer; A549 cell. The liposomes were prepared using cholesterol and a mitochondria-penetrating peptide (MPP) having a phenylalanine-arginine-phenylalanine-lysine (FRFK) peptide sequence. The FRFK peptide was synthesized using solid phase peptide synthesis (SPPS) and contained cholesterol in the N-terminal end of the phenylalanine (Chol-FRFK). The synthesized material was confirmed using <SUP>1</SUP>H NMR, Fourier transform infrared spectroscopy (FT-IR) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/MS). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and Chol-FRFK, at a molar ratio of 1:1, were used for liposomal (Chol-FRFK/D) formulations; the sizes of the liposomes were confirmed using dynamic light scattering (DLS). Cytotoxicity was evaluated in A549 cells. Cellular uptake and mitochondria targeting were confirmed by flow cytometry and confocal microscopy, respectively. Antimycin A, a hydrophobic and mitochondrial electron transporter inhibitor was encapsulated into the Chol-FRFK/D liposomes. Our results indicate that Chol-FRFK/D liposomes may potentially be used for the nano-formulation of cytotoxic drugs and enhancing their bioavailability in cancer therapy. </LI> </UL> </P> <P><B>Highlights</B></P> <P> <UL> <LI> Idea of mitochondria targeting carrier to deliver mitochondria-specific drug, is proposed. </LI> <LI> Chol-FRFK was synthesized as a liposomal component. </LI> <LI> Chol-FRFK/D liposomes were prepared for delivery of Antimycin A to the mitochondria. </LI> <LI> Nano-formulation of Antimycin A showed higher toxicity in A549 cells. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Mitochondria-specific monoclonal antibodies in embryos of the ascidian, Halocynthia roretzi

Wang Jong Lee,Gil Jung Kim 한국발생생물학회 2017 한국발생생물학회 학술발표대회 Vol.2017 No.8

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. It is, however, still unclear what signaling and molecular event control polarized distribution of mitochondria in the early ascidian embryonic development. To obtain molecular markers for studying mechanisms involved in polarized distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondria-rich cytoplasm in all cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like a mesh structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. These antibodies showed that mitochondria were distributed evenly in the animal hemisphere blastomeres at cleavage stages, whereas did not in the vegetal hemisphere blastomeres. Mitochondria were transferred more into cells of the marginal zone, such as muscle and nerve cord lineage cells, than into cells of the central zone, such as mesenchyme, notochord and endoderm lineage, in the vegetal hemisphere. Therefore, it is suggested that these antibodies may be useful as markers for analysing mechanisms involved in polarized distribution of mitochondria during ascidian embryogenesis.

Dynamic Profiles of Ubiquitination and Autophagy Associated with Paternal Mitochondria Degradation during Mouse Postfertilization Development

Xing-Wei Liang,Yong-Xun Jin,Ga-Young An,Seul-Ki Lee,Jung-Woo Kwon,Xiang-Shun Cui,Nam-Hyung Kim 한국동물번식학회 2012 Reproductive & Developmental Biology(Supplement) Vol.36 No.2s

It is well established that mitochondrial genome is strictly maternally inherited in mammalian, despite the fact that paternal mitochondria enter into oocyte during fertilization. To date, although some mechanisms have been extrapolated to interpret the elimination of paternal mitochondria, the exact mechanism still is unclear. Recent studies suggest that autophagy process and the ubiquitin-mediated degradation pathway may be involved in elimination of paternal mitochondria. However, the dynamic profiles of autophagy and ubiquitination associated with paternal mitochondria degradation have not been determined in mouse model. Through immunostaining with specific antibody LC3 and Ubiquitin and confocal microscopy, we investigated the dynamic profiles of LC3 and Ubiquitin signals in mouse embryos during preimplantation development. In addition, embryos were stained with MitoTracker Red for tracking the degradation process of paternal mitochondria. Our results showed that paternal mitochondria gradually degraded during postfertilization development, and sporadic paternal mitochondria were found at least in 16 cell embryos. LC3 and Ubiquitin signals appeared in the midpiece of sperm at 3 h postfertilization, and they were strictly colocalizated with paternal mitochondria from zygote to 2 cell embryo. Nevertheless, the colocalization became loose at 4 cell embryos, and gradually disappeared beyond 4 cell embryos. Our results confirmed that autophagy process and the ubiquitin-mediated degradation pathway may take part in the postfertilization remove of paternal mitochondria.

A new Near Infrared mitochondria targeting bioprobe via control molecular hydrophobicity

성준,김은하 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

Mitochondria is one of the cell organelle playing a critical role as ATP synthesis, cell signaling, cell apoptosis, redox homeostasis. Recently, mitochondria are noticed with new drug target, and biomarker as a disease diagnosis. Therefore, fluorescent bioprobes monitoring mitochondria will be useful for high throughput screening to discover new drugs or disease diagnosis tool. In several studies, mitochondria targeting molecules are recommended to have a cationic charge and proper hydrophobicity. But there are not presentation about quantitative hydrophobicity to target mitochondria. So we controlled the hydrophobicity of NIR chromophore, silicon rhodamine to make a mitochondria targeting bioprobe. NIR region have advantage of live cell imaging, because of low phototoxicity, high signal to background ratio. In this poster, we just control molecular hydrophobicity to targeting mitochondria.

Cationic Oligopeptide-Conjugated Mitochondria Targeting Sequence as a Novel Carrier System for Mitochondria

유광식,최준식,한진,고경수 한국고분자학회 2014 Macromolecular Research Vol.22 No.1

Mitochondria are crucial subcellular organelles for cell death and energy production. Mitochondrial dysfunctioncontributes to many human diseases. Gaining access to this organelle would create a powerful means fortreating various diseases, including cancer. However, delivering bioactive materials to the mitochondria is hamperedby the presence of the plasma membrane and mitochondrial membrane. To overcome this problem, we introduceda short cationic oligopeptide composed of histidines and arginines (H3R9) to the mitochondria-targeting sequence(MTS). Synthesized MTS-H3R9 was efficiently trans-localized to the mitochondria in HeLa cells according to confocalmicroscopy and flow cytometry studies. By combining these two distinct characteristics, cell-penetrating andmitochondria-targeting, MTS-H3R9 displayed much facilitated intracellular uptake and localization to the mitochondria. Such mitochondria-targeting peptides conjugated with functional peptides will enable effective translocalizationof bioactive materials to the mitochondria.

Mitochondria-Specific Monoclonal Antibodies in Eggs and Embryos of the Ascidian Halocynthia roretzi

Baek, Yong Han,Lee, Wang Jong,Kim, Gil Jung The Korean Society of Developmental Biology 2017 발생과 생식 Vol.21 No.4

Ascidian embryos have become an important model for embryological studies, offering a simple example for mechanisms of cytoplasmic components segregation. It is a well-known example that the asymmetric segregation of mitochondria into muscle lineage cells occurs during ascidian embryogenesis. However, it is still unclear which signaling pathway is involved in this process. To obtain molecular markers for studying mechanisms involved in the asymmetric distribution of mitochondria, we have produced monoclonal antibodies, Mito-1, Mito-2 and Mito-3, that specifically recognize mitochondria-rich cytoplasm in cells of the ascidian Halocynthia roretzi embryos. These antibodies stained cytoplasm like reticular structure in epidermis cells, except for nuclei, at the early tailbud stage. Similar pattern was observed in vital staining of mitochondria with DiOC2, a fluorescent probe of mitochondria. Immunostaining with these antibodies showed that mitochondria are evenly distributed in the animal hemisphere blastomeres at cleavage stages, whereas not in the vegetal hemisphere blastomeres. Mitochondria were transferred to the presumptive muscle and nerve cord lineage cells of the marginal zone in the vegetal hemisphere more than to the presumptive mesenchyme, notochord and endoderm lineage of the central zone. Therefore, it is suggested that these antibodies will be useful markers for studying mechanisms involved in the polarized distribution of mitochondria during ascidian embryogenesis.