Novel in vitro reconstitution assay for Autophagy and Mitochondrial Metabolism
I have longstanding interest in understanding autolysosome composition and dynamics, the fundamental autophagy (literally, “self-eating” in Greek) mechanism implicated in cancer, neurodegenerative diseases, infectious diseases, and metabolic diseases and awarded Nobel Prize 2017.
From 2011 to 2016, by leading 3 PhDs and 2 RAs from Peking, Tsinghua University and Chinese Academy of Science, I have established an easy-to-use single-molecule in vitro reconstitution assay to study motor protein induced membrane deformation, reformation and dynamics. This in vitro pure system provides an unprecedented flexibility in manipulating motor protein conformation, lipid composition, vesicle size, Ca2+ ion concentration and ATP/ADP binding condition, which cannot be genetically or physiologically controlled in intracellular environment. Notably, Development Cell Editor Dr Ilil Carmi commented our work as “an extremely convenient in vitro knock-down treatment could be achieved through the reconstitution assay” [1].
Using this powerful in vitro experimental platform, our team have demonstrated that the kinesin-1 KIF5B, through interactions with PtdIns(4,5)P2, drives tubulation by pulling on the autolysosome membrane, revealing a motor-based membrane deformation process that helps maintain lysosomal homeostasis [1,2]. This work was recommended and highlighted by F1000 Prime by Prof. Wei GUO and Prof. Bin WU: “This paper provides a working model to explain how microtubule motor proteins are involved in lysosome membrane dynamics.”
A following work using this in vitro reconstitution system for mitochondrial metabolism, uncovered an essential mechanism for mitochondrial network formation driven by KIF5B and mediated by MFN1/2 and revealed that different parts of the mitochondrial network are formed by different mechanisms [3], cover story in Cell Research.
The well-established in vitro reconstitution assay with single-molecule sensitivity has further been applied to understand CapZ and actin in autophagy [4], ATP/ADP in kinesin motility [5], Ca2+ in myosin motion [6], Golgi derived vesicles [7], Mitochondria DNA dynamics [8] and I was listed as contributing authors in these papers. Several more invited book chapter and review papers have been published in the following years [9-11].
[1] Du, W.*, Qian Peter Su*(equal contribution), Y. Chen*, Y. Zhu, D. Jiang, Y. Sun#, and L. Yu#. 2016. Kinesin-1 Drives Autolysosome Tubulation. Developmental Cell (F1000 Prime highlight) 37: p. 326-336.
[2] Su Qian Peter, W. Du, Q. Ji, B. Xue, Y. Zhu, D. Jiang, J, Lou#, L. Yu#, and Y. Sun#. 2016. Vesicle Size Regulates Nanotube Formation in the Cell. Scientific Reports 6:24002.
[3] Wang, C.*, W. Du*, Qian Peter Su*(equal contribution), M. Zhu, P. Feng, Y. Li, Y. Zhou, N. Mi, Y. Zhu, D. Jiang, S. Zhang, Z. Zhang, Y. Sun#, and L. Yu#. 2015. Dynamic tubulation of mitochondria drives mitochondrial network formation. Cell Research (cover story) 25(10): p. 1108-1120.
[4] Mi, N.*, Y. Chen*, S. Wang*, M. Chen, M. Zhao, G. Yang, M. Ma, Qian Peter Su, et al, Y. Sun, Z. Chen#, and L. Yu#. 2015. CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane. Nature Cell Biology 17(9): p. 1112-23.
[5] Guan, R.*, L. Zhang*, Qian Peter Su, K.J. Mickolajczyk, et al, Y.F. Zhao#, and Z. Chen#. 2017. Crystal structure of Zen4 in the APO state reveals a missing conformation of kinesin. Nature Comms 8:14951.
[6] Shen, M.*, N. Zhang*, S. Zheng*, et al, Qian Peter Su, Y. Sun, K. Ye and X.D. Li. 2016. Calmodulin in Complex with the First IQ Motif of Myosin-5a Functions as an Intact Ca Sensor. PNAS 133.40: E5812-20.
[7] Hao H., J. Niu, B. Xue, Qian Peter Su, M. Liu, J. Yang, J. Qin, S. Zhao, C. Wu and Y. Sun. 2020. Golgi-associated microtubules are fast cargo tracks and required for persistent migration. EMBO Report e48385
[8] Qin J., Y. Guo, B. Xue, P. Shi, Y. Chen, Qian Peter Su ,H. Hao, S. Zhao, C. Wu, L. Yu, D. Li and Y. Sun. 2020. ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation. Nature Communications 11:4471.
[9] Du W*., Qian Peter Su*#(equal contribution, corresponding author). 2019. Single-molecule in vitro reconstitution assay for Kinesin-1 driven membrane dynamics. Biophysical Reviews 1-7
[10] Chen Y.*, Qian Peter Su*(equal contribution), Y. Sun#, L. Yu#. 2018. Study the autophagic lysosome reformation in cells and by in vitro reconstitution system. Methods in Molecular Biology 1880 163-172.
[11] Chen Y.*, Qian Peter Su*(equal contribution), Y. Sun#, L. Yu#. 2018. Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems. Current Prot. in Cell Biology 11 1-11.24.
From 2011 to 2016, by leading 3 PhDs and 2 RAs from Peking, Tsinghua University and Chinese Academy of Science, I have established an easy-to-use single-molecule in vitro reconstitution assay to study motor protein induced membrane deformation, reformation and dynamics. This in vitro pure system provides an unprecedented flexibility in manipulating motor protein conformation, lipid composition, vesicle size, Ca2+ ion concentration and ATP/ADP binding condition, which cannot be genetically or physiologically controlled in intracellular environment. Notably, Development Cell Editor Dr Ilil Carmi commented our work as “an extremely convenient in vitro knock-down treatment could be achieved through the reconstitution assay” [1].
Using this powerful in vitro experimental platform, our team have demonstrated that the kinesin-1 KIF5B, through interactions with PtdIns(4,5)P2, drives tubulation by pulling on the autolysosome membrane, revealing a motor-based membrane deformation process that helps maintain lysosomal homeostasis [1,2]. This work was recommended and highlighted by F1000 Prime by Prof. Wei GUO and Prof. Bin WU: “This paper provides a working model to explain how microtubule motor proteins are involved in lysosome membrane dynamics.”
A following work using this in vitro reconstitution system for mitochondrial metabolism, uncovered an essential mechanism for mitochondrial network formation driven by KIF5B and mediated by MFN1/2 and revealed that different parts of the mitochondrial network are formed by different mechanisms [3], cover story in Cell Research.
The well-established in vitro reconstitution assay with single-molecule sensitivity has further been applied to understand CapZ and actin in autophagy [4], ATP/ADP in kinesin motility [5], Ca2+ in myosin motion [6], Golgi derived vesicles [7], Mitochondria DNA dynamics [8] and I was listed as contributing authors in these papers. Several more invited book chapter and review papers have been published in the following years [9-11].
[1] Du, W.*, Qian Peter Su*(equal contribution), Y. Chen*, Y. Zhu, D. Jiang, Y. Sun#, and L. Yu#. 2016. Kinesin-1 Drives Autolysosome Tubulation. Developmental Cell (F1000 Prime highlight) 37: p. 326-336.
[2] Su Qian Peter, W. Du, Q. Ji, B. Xue, Y. Zhu, D. Jiang, J, Lou#, L. Yu#, and Y. Sun#. 2016. Vesicle Size Regulates Nanotube Formation in the Cell. Scientific Reports 6:24002.
[3] Wang, C.*, W. Du*, Qian Peter Su*(equal contribution), M. Zhu, P. Feng, Y. Li, Y. Zhou, N. Mi, Y. Zhu, D. Jiang, S. Zhang, Z. Zhang, Y. Sun#, and L. Yu#. 2015. Dynamic tubulation of mitochondria drives mitochondrial network formation. Cell Research (cover story) 25(10): p. 1108-1120.
[4] Mi, N.*, Y. Chen*, S. Wang*, M. Chen, M. Zhao, G. Yang, M. Ma, Qian Peter Su, et al, Y. Sun, Z. Chen#, and L. Yu#. 2015. CapZ regulates autophagosomal membrane shaping by promoting actin assembly inside the isolation membrane. Nature Cell Biology 17(9): p. 1112-23.
[5] Guan, R.*, L. Zhang*, Qian Peter Su, K.J. Mickolajczyk, et al, Y.F. Zhao#, and Z. Chen#. 2017. Crystal structure of Zen4 in the APO state reveals a missing conformation of kinesin. Nature Comms 8:14951.
[6] Shen, M.*, N. Zhang*, S. Zheng*, et al, Qian Peter Su, Y. Sun, K. Ye and X.D. Li. 2016. Calmodulin in Complex with the First IQ Motif of Myosin-5a Functions as an Intact Ca Sensor. PNAS 133.40: E5812-20.
[7] Hao H., J. Niu, B. Xue, Qian Peter Su, M. Liu, J. Yang, J. Qin, S. Zhao, C. Wu and Y. Sun. 2020. Golgi-associated microtubules are fast cargo tracks and required for persistent migration. EMBO Report e48385
[8] Qin J., Y. Guo, B. Xue, P. Shi, Y. Chen, Qian Peter Su ,H. Hao, S. Zhao, C. Wu, L. Yu, D. Li and Y. Sun. 2020. ER-mitochondria contacts promote mtDNA nucleoids active transportation via mitochondrial dynamic tubulation. Nature Communications 11:4471.
[9] Du W*., Qian Peter Su*#(equal contribution, corresponding author). 2019. Single-molecule in vitro reconstitution assay for Kinesin-1 driven membrane dynamics. Biophysical Reviews 1-7
[10] Chen Y.*, Qian Peter Su*(equal contribution), Y. Sun#, L. Yu#. 2018. Study the autophagic lysosome reformation in cells and by in vitro reconstitution system. Methods in Molecular Biology 1880 163-172.
[11] Chen Y.*, Qian Peter Su*(equal contribution), Y. Sun#, L. Yu#. 2018. Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems. Current Prot. in Cell Biology 11 1-11.24.