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Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria

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Abstract

The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases.

Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 μm polycarbonate membrane filter to generate fusogenic plasma membrane vesicles (fPMVs), which contain bioactive molecules and organelles but not the nucleus. fPMVs can be endocytosed by target cells, while the cargo is released due to low-pH induced membrane fusion. These nucleus-free fPMVs are efficient at delivery of cytoplasmic proteins and mitochondria, leading to recovery of mitochondrial biogenesis and proliferative ability in mitochondria-deficient cells.

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Acknowledgments

This research was supported by the Li Ka Shing Foundation; the Natural Science Foundation of China (http://www.nsfc.gov.cn/ Grant No. 30971665, 81172894, 81370925); and the Education Department of Guangdong (http://www.gdhed.edu.cn/ Grant No. cxzd1123).

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Authors and Affiliations

  1. Multidisciplinary Research Center, Shantou University, Shantou, Guangdong, 515063, China

    Hao-peng Lin, De-jin Zheng, Yun-pan Li, Na Wang, Shao-jun Chen & Chi-ju Wei

  2. Laboratory of Cell Senescence, Shantou University Medical College, Shantou, Guangdong, 515041, China

    Yu-cai Fu

  3. Department of Endocrinology, First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, 515041, China

    Wen-can Xu

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  1. Hao-peng Lin
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Correspondence to Chi-ju Wei.

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Hao-peng Lin and De-jin Zheng contributed equally to this research.

Electronic supplementary material

Supplementary Figure 1

Functional verification of mitochondria between Ad293 cells and fPMVs. (A) Western blot analysis of MTCO1 in Ad293 cells and fPMVs. TOM20 was used as a loading control. (B) Estimation of the expression levels of MTCO1 in Ad293 cells and fPMVs. The ratio of MTCO1 to TOM20 in cell lysates was arbitrarily assigned as 1. Data are represented by mean ± sem. N = 5. (*p < 0.05) (GIF 9 kb)

High Resolution Image (TIFF 287 kb)

Supplementary Figure 2

Restoration of mitochondrial membrane potential in Rho0 HeLa cells. (A) TMRE staining in Rho0 cells 5 days after PMVs or fPMVs incubation. Scale bar =100 μm. (B) Estimation of mitochondrial membrane potential in rescued Rho0 cells was shown. The △Ψ of PMVs added group was arbitrarily assigned as 1. Data are represented by mean ± sem. N = 5. (*p < 0.05) (GIF 56 kb)

High Resolution Image (TIFF 5023 kb)

Supplementary Figure 3

Recovery of Rho0 mitochondrial function depended on mito-transfer. (A) Morphology of Ad293 cells and R6G-treated Ad293 cells. Scale bar =100 μm. (B) Mitochondrial dysfunction of R6G-treated cells was confirmed by western blot analysis of MTCO1 expression. TOM20 was used as a loading control. (C) Culture of Rho0 cells in the presence of fPMVs with/without R6G pretreatment. Scale bar =100 μm. (D) Cell number of rescued Rho0 cells after 5 day cultivation. Data are represented by mean ± sem. N = 5. (*p < 0.05) (GIF 29 kb)

High Resolution Image (TIFF 1550 kb)

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Lin, Hp., Zheng, Dj., Li, Yp. et al. Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria. Biomed Microdevices 18, 41 (2016). https://doi.org/10.1007/s10544-016-0066-y

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