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Fluorescent investigation on process of tBid inducing membrane permeabilization

Ma Li He Xiao-Long Li Ming Hu Shu-Xin

Fluorescent investigation on process of tBid inducing membrane permeabilization

Ma Li, He Xiao-Long, Li Ming, Hu Shu-Xin
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  • The proapoptotic protein tBid is a member of Bcl-2 family, and it plays an important role in apoptosis by inducing mitochondrial outer membrane permeabilization (MOMP) and lysosomal membrane permeabilization (LMP). Previous studies have shown that the mechanism of tBid-dependent MOMP and LMP depends on tBid interacting with membranes. Researchers hold different opinions about whether tBid itself could induce MOMP and LMP. Some of the researchers insist that tBid must trigger other proteins like Bax or Bak inserting into the membrane, and assembly of tBid itself could not form pores large enough to release cytochrome c. Some others think that tBid just like Bax, can permeabilize mitochondrial outer membrane releasing cytochrome c and lysosomal membrane with the leakage of lysosomal cathepsin B. Here, we want to know whether the tBid itself can induce membrane permeabilization in our model system at low concentration. We use 3 ways to observe tBid and membranes interactions. They are confocal imaging of GUVs (giant unilamellar vesicles), traditional single molecular fluorescence assay, and a recently developed approach, single molecular surface-induced fluorescence attenuation (sm-SIFA). So we can obtain information from single vesicle level and single molecule level. At single vesicle level, we can directly find out whether the GUVs are permeabilized and at the same time the shape of the GUVs is changed. At a single molecule level, we can know the properties of one protein. Especially by using the sm-SIFA, we can obtain the insertion depth of exact residue. Combining the results obtained from different ways under the same conditions, we find that tBid itself can induce the model membrane to permeate, releasing the fluorescent molecules, by oligomerization. What is more, we suggest that the mechanism is that in oligomers some tBids can be inserted deep into the membrane although in oligomers not all the proteins have the same insertion depth. It is indicated that the conformations of tBids in oligomers are diversified. We also prove that the ways we use here are efficient. The GUVs and supported lipid bilayers are indeed tenable model systems. Sm-SIFA has a grand future in the study of protein and membrane interactions.
      Corresponding author: Hu Shu-Xin, hushuxin@iphy.ac.cn
    • Funds: Project supported by the Major Research Plan of the National Natural Science Foundation of China (Grant No. 91753104).
    [1]

    Golstein P 1998 Science 281 1283

    [2]

    Danial N N, Korsmeyer S J 2004 Cell 116 205

    [3]

    Lovell J F, Billen L P, Bindner S, Shamas-Din A, Fradin C, Leber B, Andrews D W 2008 Cell 135 1074

    [4]

    Shamas-Din A, Bindner S, Zhu W, Zaltsman Y, Campbell C, Gross A, Leber B, Andrews D W, Fradin C 2013 J. Biol. Chem. 288 22111

    [5]

    Subburaj Y, Cosentino K, Axmann M, Pedrueza-Villalmanzo E, Hermann E, Bleicken S, Spatz J, Garcia-Saez A J 2015 Nat. Commun. 6 8042

    [6]

    Roy M J, Vom A, Czabotar P E, Lessene G 2014 Br. J Pharmacol. 171 1973

    [7]

    Youle R J, Strasser A 2008 Nat. Rev. Mol. Cell Biol. 9 47

    [8]

    Czabotar P E, Lessene G, Strasser A, Adams J M 2014 Nat. Rev. Mol. Cell Biol. 15 49

    [9]

    Kaufmann T, Jost P J, Pellegrini M, Puthalakath H, Gugasyan R, Gerondakis S, Cretney E, Smyth M J, Silke J, Hakem R, Bouillet P, Mak T W, Dixit V M, Strasser A 2009 Immunity 30 56

    [10]

    Hutt K J 2015 Reproduction 149 R81

    [11]

    Billen L P, Shamas-Din A, Andrews D W 2009 Oncogene 27 S93

    [12]

    Kim H, Rafiuddin-Shah M, Tu H C, Jeffers J R, Zambetti G P, Hsieh J J, Cheng E H 2006 Nat. Cell Biol. 8 1348

    [13]

    Gross A, Yin X M, Wang K, Wei M C, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeyer S J 1999 J. Biol. Chem. 274 1156

    [14]

    Tait S W, Green D R 2010 Nat. Rev. Mol. Cell Biol. 11 621

    [15]

    Li H L, Zhu H, Xu C J, Yuan J Y 1998 Cell 94 491

    [16]

    Wei M C, Lindsten T, Mootha V K, Weiler S, Gross A, Ashiya M, Thompson C B, Korsmeyer S J 2000 Gene. Dev. 14 2060

    [17]

    Eskes R, Desagher S, Antonsson B, Martinou J C 2000 Mol. Cell. Biol. 20 929

    [18]

    Happo L, Strasser A, Cory S 2012 J. Cell Sci. 125 1081

    [19]

    Billen L P, Kokoski C L, Lovell J F, Leber B, Andrews D W 2008 Plos Biol. 6 e147

    [20]

    Guicciardi M E, Bronk S F, Werneburg N W, Yin X M, Gores G J 2005 Gastroenterology 129 269

    [21]

    Schendel S L, Azimov R, Pawlwski K, Godzik A, Kagan B L, Reed J C 1999 J. Biol. Chem. 274 21932

    [22]

    Grinberg M, Sarig R, Zaltsman Y, Frumkin D, Grammatikakis N, Reuveny E, Gross A 2002 J. Biol. Chem. 277 12237

    [23]

    Zhao K, Zhou H J, Zhao X Y, Wolff D W, Tu Y P, Liu H L, Wei T T, Yang F Y 2012 J. Lipid Res. 53 2102

    [24]

    Shivakumar S, Kurylowicz M, Hirmiz N, Manan Y, Friaa O, Shamas-Din A, Masoudian P, Leber B, Andrews D W, Fradin C 2014 Biophys. J. 106 2085

    [25]

    Bleicken S, Hofhaus G, Ugarte-Uribe B, Schroder R, Garcia-Saez A J 2016 Cell Death Dis. 7 e2121

    [26]

    Li Y, Qian Z Y, Ma L, Hu S X, Nong D G, Xu C H, Ye F F, Lu Y, Wei G H, Li M 2016 Nat. Commun. 7 12906

    [27]

    Swathi R S, Sebastian K L 2008 J. Chem. Phys. 129 054703

    [28]

    Swathi R S, Sebastian K L 2009 J. Chem. Phys. 130 086101

    [29]

    Zhao J P, Pei S F, Ren W C, Gao L B, Cheng H M 2010 ACS Nano 4 5245

    [30]

    Hummers Jr W S, Offeman R E 1958 J. Am. Chem. Soc. 80 1339

    [31]

    Wang Y, Tjandra N 2013 J. Biol. Chem. 288 35840

    [32]

    Oh K J, Barbuto S, Meyer N, Kim R S, Collier R J, Korsmeyer S J 2005 J. Biol. Chem. 280 753

  • [1]

    Golstein P 1998 Science 281 1283

    [2]

    Danial N N, Korsmeyer S J 2004 Cell 116 205

    [3]

    Lovell J F, Billen L P, Bindner S, Shamas-Din A, Fradin C, Leber B, Andrews D W 2008 Cell 135 1074

    [4]

    Shamas-Din A, Bindner S, Zhu W, Zaltsman Y, Campbell C, Gross A, Leber B, Andrews D W, Fradin C 2013 J. Biol. Chem. 288 22111

    [5]

    Subburaj Y, Cosentino K, Axmann M, Pedrueza-Villalmanzo E, Hermann E, Bleicken S, Spatz J, Garcia-Saez A J 2015 Nat. Commun. 6 8042

    [6]

    Roy M J, Vom A, Czabotar P E, Lessene G 2014 Br. J Pharmacol. 171 1973

    [7]

    Youle R J, Strasser A 2008 Nat. Rev. Mol. Cell Biol. 9 47

    [8]

    Czabotar P E, Lessene G, Strasser A, Adams J M 2014 Nat. Rev. Mol. Cell Biol. 15 49

    [9]

    Kaufmann T, Jost P J, Pellegrini M, Puthalakath H, Gugasyan R, Gerondakis S, Cretney E, Smyth M J, Silke J, Hakem R, Bouillet P, Mak T W, Dixit V M, Strasser A 2009 Immunity 30 56

    [10]

    Hutt K J 2015 Reproduction 149 R81

    [11]

    Billen L P, Shamas-Din A, Andrews D W 2009 Oncogene 27 S93

    [12]

    Kim H, Rafiuddin-Shah M, Tu H C, Jeffers J R, Zambetti G P, Hsieh J J, Cheng E H 2006 Nat. Cell Biol. 8 1348

    [13]

    Gross A, Yin X M, Wang K, Wei M C, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeyer S J 1999 J. Biol. Chem. 274 1156

    [14]

    Tait S W, Green D R 2010 Nat. Rev. Mol. Cell Biol. 11 621

    [15]

    Li H L, Zhu H, Xu C J, Yuan J Y 1998 Cell 94 491

    [16]

    Wei M C, Lindsten T, Mootha V K, Weiler S, Gross A, Ashiya M, Thompson C B, Korsmeyer S J 2000 Gene. Dev. 14 2060

    [17]

    Eskes R, Desagher S, Antonsson B, Martinou J C 2000 Mol. Cell. Biol. 20 929

    [18]

    Happo L, Strasser A, Cory S 2012 J. Cell Sci. 125 1081

    [19]

    Billen L P, Kokoski C L, Lovell J F, Leber B, Andrews D W 2008 Plos Biol. 6 e147

    [20]

    Guicciardi M E, Bronk S F, Werneburg N W, Yin X M, Gores G J 2005 Gastroenterology 129 269

    [21]

    Schendel S L, Azimov R, Pawlwski K, Godzik A, Kagan B L, Reed J C 1999 J. Biol. Chem. 274 21932

    [22]

    Grinberg M, Sarig R, Zaltsman Y, Frumkin D, Grammatikakis N, Reuveny E, Gross A 2002 J. Biol. Chem. 277 12237

    [23]

    Zhao K, Zhou H J, Zhao X Y, Wolff D W, Tu Y P, Liu H L, Wei T T, Yang F Y 2012 J. Lipid Res. 53 2102

    [24]

    Shivakumar S, Kurylowicz M, Hirmiz N, Manan Y, Friaa O, Shamas-Din A, Masoudian P, Leber B, Andrews D W, Fradin C 2014 Biophys. J. 106 2085

    [25]

    Bleicken S, Hofhaus G, Ugarte-Uribe B, Schroder R, Garcia-Saez A J 2016 Cell Death Dis. 7 e2121

    [26]

    Li Y, Qian Z Y, Ma L, Hu S X, Nong D G, Xu C H, Ye F F, Lu Y, Wei G H, Li M 2016 Nat. Commun. 7 12906

    [27]

    Swathi R S, Sebastian K L 2008 J. Chem. Phys. 129 054703

    [28]

    Swathi R S, Sebastian K L 2009 J. Chem. Phys. 130 086101

    [29]

    Zhao J P, Pei S F, Ren W C, Gao L B, Cheng H M 2010 ACS Nano 4 5245

    [30]

    Hummers Jr W S, Offeman R E 1958 J. Am. Chem. Soc. 80 1339

    [31]

    Wang Y, Tjandra N 2013 J. Biol. Chem. 288 35840

    [32]

    Oh K J, Barbuto S, Meyer N, Kim R S, Collier R J, Korsmeyer S J 2005 J. Biol. Chem. 280 753

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  • Received Date:  15 January 2018
  • Accepted Date:  09 April 2018
  • Published Online:  20 July 2019

Fluorescent investigation on process of tBid inducing membrane permeabilization

    Corresponding author: Hu Shu-Xin, hushuxin@iphy.ac.cn
  • 1. Key Laboratory of Soft Matter Physics, Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China;
  • 2. School of Physical Sciences, University of Chinese Academy of Sciences, Beijing 100049, China;
  • 3. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Fund Project:  Project supported by the Major Research Plan of the National Natural Science Foundation of China (Grant No. 91753104).

Abstract: The proapoptotic protein tBid is a member of Bcl-2 family, and it plays an important role in apoptosis by inducing mitochondrial outer membrane permeabilization (MOMP) and lysosomal membrane permeabilization (LMP). Previous studies have shown that the mechanism of tBid-dependent MOMP and LMP depends on tBid interacting with membranes. Researchers hold different opinions about whether tBid itself could induce MOMP and LMP. Some of the researchers insist that tBid must trigger other proteins like Bax or Bak inserting into the membrane, and assembly of tBid itself could not form pores large enough to release cytochrome c. Some others think that tBid just like Bax, can permeabilize mitochondrial outer membrane releasing cytochrome c and lysosomal membrane with the leakage of lysosomal cathepsin B. Here, we want to know whether the tBid itself can induce membrane permeabilization in our model system at low concentration. We use 3 ways to observe tBid and membranes interactions. They are confocal imaging of GUVs (giant unilamellar vesicles), traditional single molecular fluorescence assay, and a recently developed approach, single molecular surface-induced fluorescence attenuation (sm-SIFA). So we can obtain information from single vesicle level and single molecule level. At single vesicle level, we can directly find out whether the GUVs are permeabilized and at the same time the shape of the GUVs is changed. At a single molecule level, we can know the properties of one protein. Especially by using the sm-SIFA, we can obtain the insertion depth of exact residue. Combining the results obtained from different ways under the same conditions, we find that tBid itself can induce the model membrane to permeate, releasing the fluorescent molecules, by oligomerization. What is more, we suggest that the mechanism is that in oligomers some tBids can be inserted deep into the membrane although in oligomers not all the proteins have the same insertion depth. It is indicated that the conformations of tBids in oligomers are diversified. We also prove that the ways we use here are efficient. The GUVs and supported lipid bilayers are indeed tenable model systems. Sm-SIFA has a grand future in the study of protein and membrane interactions.

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