Team:Kyoto/CiC/Method

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Methods

Method for Subgoal A --- Liposomes with HIV-1 TAT peptide

Subgoal A is the construction of a liposome that penetrate through cell membranes.

Construction of Liposomes with HIV-1 TAT peptide

We designed a liposome with HIV-1 TAT peptide.

HIV-1 TAT is a Protein Transduction Domain (PTD)[2]. Liposomes made of peptidolipid with HIVｰ1 TAT is known to penetrate through cell membrane[1]. It is suggested that HIV-1 TAT promotes endocytosis,though the precise mechanism remains unclear.

Then we fused HIV-1 TAT with potential anchor proteins (LALAAAALALAAAALALAAAA)[3]. The LALAAAA side of this fusion protein is expected to be anchored in the lipid barrier of liposome. And the TAT side of the protein will be exposed to water so that TAT domain can touches the cell membrane. When exposed to cells, liposomes with this fusion protein will promote endocytosis and be taken into the cell. If such liposomes are fluorescent stained, they will show fluorescence in the cells.

We constructed plasmids encoding this fusion protein, referred to as (HIV-TAT)-(LALAAAA)3-generator in Parts section.

When this plasmid is mixed with mixture of liposomes and cell-free protein synthesis system, the fusion proteins will be synthesized and anchored on Liposomes[4].

We also performed similar experiments with chemically synthesized TAT-LALAAAA protein.

Confirmation of function of TAT-liposome

We chose HeLa cells for this experiment. HeLa cells were cultured at 37°C in D-MEM/F-12. The media was supplemented with 10% FBS and an antibitic/antimycotic solution for maintaining HeLa cells. To confirm the function of translocation into cells of TAT-anchored liposomes, HeLa cells were cultured with prepared TAT-liposome with NBD-PE, which is a fluorescent molecule bind to lipid. HeLa cells were plated in 3.5 cm glass bottom dish (6x10^5 cells/well), pre-incubated for 20 hours at 37°C, and the medium was replaced with Opti-MEM that containing the NBD-PE-liposome with or without TAT. The cells were incubated at 37°C for 4 hours and washed by fresh D-MEM/F-12 supplemented with 10% FBS twice, and then, obserbed. The fluorescent images were captured by IX81 microscope system using a 470- to 495-nm excitation filter and a 510- to 550-nm emission filter. The conforcal images were captured by Nikon A1Rsi conforcal microscopy system with 0.6 um step using a 488 nm semiconductor lazer for excitation and 525/50 nm emission filter. The result is heresig-GFP expression in HeLa.

Method for Subgoal B --- Liposomes with Mitochondrial Translocases

Subgoal B is construction of liposome with mitochondrial translocases and confirming whether mitochondrial proteins are imported into them.

Construction of Liposomes with Mitochondrial Translocases

Mitochondria have several types of translocases: TOM40 complex on outer membrane, TIM22 complex, TIM23 complex and OXA complex on Inner membrane. They catalyze protein transport across mitochondrial membrane. Precursor proteins with signal sequence for mitochondrial protein import are selectively sent to mitochondria.

We aimed to reconstitute all types of mitochondrial translocases on liposomes but later found it too difficult. Mitochondria have double-membraned sturucture, which we can hardly construct well. So, we decided to construct liposomes which correspond to mitochondrial Inner membrane.

It is known that mitochondria without their outer membrane, which is called mitoplasts, can efficiently import precursor proteins into the matrix space. It means that TIM23 can recognize signal sequence and catalyze protein tranport if exposed on their surface.

So if we successfully reconstitute liposomes with TIM23, these liposomes will take in proteins with mitochondrial signal sequence too. We named such liposomes "Prepared Liposome".

Prepared liposome is constructed in the ways described below.

1. Isolate mitochondrial fraction.
2. Disrupt outer membrane.
3. Mix mitoplasts with detergent.
4. Mix liposomes with mixture of the 3 steps
5. Get rid of detergent.

GFP with Mitochondrial Signal Sequence

We designed GFP with mitochondrial signal sequence, which we named this sig-GFP, and constructed plasmids encoding it, referred to as sig-GFP generator in Parts section. And we isolated mitochondrial fraction from yeast to confirm whether sig-GFP is imported into mitochondria in vitro. Mitochondrial fraction is mixed with cell-free protein synthesis system expressing sig-GFP. If sig-GFP works well, mitochondria will be filled with GFP. And it means sig-GFP can be imported into prepared liposome too.

Method for Subgoal C --- Liposomes with HIV-1 TAT and Mitochondrial Translocases

Subgoal C is construction of Liposomes with HIVｰ1 TAT and mitochondrial translocases, and confirming whether they go into host cells and import mitochondrial proteins there.

sig-GFP expression in HeLa

We constructed plasmids for HeLa cells to make them express sig-GFP. When sig-GFP is expressed in HeLa, it will be imported into mitochondria as a pseudo mitochondrial protein and fluorescence willl localize to mitochondria. If prepared liposomes successfully get into those transformed HeLa, in the same way, localization of fluorescence to prepared liposome will be shown. If we observed the localization, it is suggested that real mitochondrial proteins could also be imported into those prepared liposomes.

At first, we confirmed the function of signal sequence using the plasmid coding sig-GFP. Although the signal sequence is derived from yeast, we determined to use HeLa cell for the confirmation since the sequence was expected to work in other eukaryotic cells and yeast cell might be too small to observe its mitochondria. HeLa cells were cultured at 37°C in D-MEM/F-12 for this experiment. The media was supplemented with 10% FBS and an antibitic/antimycotic solution for maintaining HeLa cells. The incubator contained air that was enriched with 5% CO2. HeLa cells were plated in a 6 well glass bottom plate (6x105 cells/well), pre-incubated for 9 hours at 37°C, and the medium was replaced with fresh D-MEM/F12 supplemented with 10% FBS. The cells were transfected with 1.5 ug of pGFP or psigGFP using 3.75 ul of lipofectamine 2000 according to the standard protocols provided by invtrogen. After 12 hours or more of the transfection, the medium was replaced with fresh medium with or without 100 nM of mitotracker orange in order to visualize the location of mitochondria, and incubated at 37°C for 15 min. The stained cells were washed by fresh medium twice, and then microscopic images were captured. The microscopic images were captured by a IX81 microscope system using 470-495 nm or 530-550 nm filters were used for excitation of GFP or mitotracker orange, and 510-550 nm or 575-625 nm filters were used for emission, respectively. The conforcal images were captured by an A1Rsi conforcal microscopy system with 0.15 um step using 488 nm or 567 nm semiconductor laser for excitation of GFP or mitotracker orange, and 525/50 nm or 595/50 nm filters were used for emission, respectively.

Reference

Subgoal A

[1] Nobuhiro Yagi: Furukute atarashi miwakuno nanoryuusi Liposome(Classic yet new and attractive nanoparticle:liposome). yakuzaigaku, 68 (5), (2008)

[2] Hideki MATSUI, Kazuhito TOMIZAWA and Masayuki MATSUSHITA: Protein transduction by poly-arginine. Jpn.121，(2003)

[3] Yoshiaki Yano, Tomokazu Takemoto, Satoe Kobayashi, Hiroyuki Yasui, Hiromu Sakurai,Wakana Ohashi, Miki Niwa, Shiroh Futaki, Yukio Sugiura, and Katsumi Matsuzaki: Topological Stability and Self-Association of a Completely Hydrophobic Model Transmembrane Helix in Lipid Bilayers.Biochemistry, 2002, 41 (9)

[4] Shin-ichiro M. Nomura, Satoshi Kondoha, Wakiko Asayamaa, Akikazu Asada, Shigemichi. Nishikawa and Kazunari Akiyoshia: Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis.Journal of Biotechnology Volume 133, Issue 2, 20 January 2008

Subgoal B

[5] Agnieszka Chacinska, Carla M. Koehler, Dusanka Milenkovic, Trevor Lithgow and Nikolaus Pfanner: Importing Mitochondrial Proteins: Machineries and Mechanisms. Cell, Volume 138, Issue 4, 21 August 2009

[6] Martin van der Laan, Michael Meinecke, Jan Dudek, Dana P. Hutu, Maria Lind, Inge Perschil, Bernard Guiard, Richard Wagner, Nikolaus Pfanner, and Peter Rehling: Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nature Cell Biology volume 9, number 10, OCTOBER 2007

[7] Toshiya Endo, Hayashi Yamamoto and Masatoshi Esaki: Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles.Journal of Cell Science 116, 3259-3267 2003

[8] Toshiya Endo: mitochondria makuwo butaitosuru tanpakusituno koutuu. seibutubuturi, 48 (1), 004-010 (2008) (Toshiya Endo: protein traffic on mitochondrial membranes)

[9] DANIEL S. ALLISON AND GOTTFRIED SCHATZ: Artificial mitochondrial presequences, Cell Biology、Vol. 83, pp. 9011-9015, December 1986

[10] Masato Yano, Masaki Kanazawa, Kazutoyo Terada, Chewawiwat Namchai, Masaru Yamaizumi, Brendon Hansoni, Nicholas Hoogenraadi, and Masataka Mori: Visualization of Mitochondrial Protein Import in Cultured Mammalian Cells with Green Fluorescent Protein and Effects of Overexpression of the Human Import Receptor Tom20. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 13, Issue of March 28

[11] Benedikt Westermann and Walter Neupert: Mitochondria-targeted green fuorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae. Yeast 2000; 16

[12] Federico Katzen, Todd C. Peterson and Wieslaw Kudlicki: Membrane protein expression: no cells required. Trends in Biotechnology, Volume 27, Issue 8, 20 July 2009

Subgoal C