通过细胞中细胞质纳米管桥接评估线粒体的转运

Shravani Mukherjee; Tanveer Ahmad; Anurag Agrawal

doi:10.21769/BioProtoc.1542

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Peer-reviewed

Assessing Mitochondrial Transport via Cytoplasmic Nanotubular Bridges in Cells

通过细胞中细胞质纳米管桥接评估线粒体的转运

Shravani Mukherjee

TA Tanveer Ahmad

Anurag Agrawal email

发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1542 浏览次数: 8648

评审: Arsalan DaudiAnonymous reviewer(s)

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Cover of The EMBO Journal, featuring study using the protocol.

May 2014

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Abstract

This protocol aims to study intercellular transport of mitochondria, dynamic cellular organelles via tunnelling nanotubes (TNT), a cell membrane extension of cytoskeletal elements. The nanotubular bridges or the tunnelling nanotube highways are one of the emerging new cell-to-cell communication systems which mediates exchange of cellular materials, most importantly as in our observation, mitochondria. Mesenchymal stem cells (MSC) have been well studied to be endowed with a highly efficient intercellular mitochondrial donation ability and this property is now proven crucial to its functional role of rescue in cellular therapy.

Keywords: Mitochondrial transfer (线粒体移植)

Nanotube (纳米管)

Intercellular transport (胞间运输)

Miro1 (miro1)

Materials and Reagents

Cells
Mesenchymal stem cells (Human or Mouse derived in passage 3-7), any fibroblasts cell lines (HSMC, 3T3) or primary cells. Even epithelial [BEAS-2b (ATCC, catalog number: CRL-9609 ), NHBE (Lonza, catalog number: CC-2540 ), A549, LA-4, MLE- 12] and endothelial cells (HUVEC) also show such capacity but at comparatively minimal efficiency.
Reagents
1. CellLight^® Mitochondria-GFP, BacMam 2.0 (Life Technologies, catalog number: C10600 )
2. CellLight^® Mitochondria-RFP, BacMam 2.0 (Life Technologies, catalog number: C10505 )
3. MitoTracker^® Green FM (Life Technologies, catalog number: M7514 )
4. MitoTracker^® Deep Red FM (Life Technologies, catalog number: M22426 )
5. Alexa Fluor^® 594 phalloidin (Life Technologies, catalog number: A12381 ) or fluorescein phalloidin (Life Technologies, catalog number: F432 )
6. Alexa Fluor^® 488 phalloidin (Life Technologies, catalog number: A12379 )
7. 2% paraformaldehyde (SigmaAldrich, catalog number: P6148 ) diluted in 1x PBS
8. ProLong^® Gold Antifade Mountant with DAPI (Life Technologies, catalog number: P36931 )
9. Respective cell culture media
  1. DMEM (Sigma-Aldrich, catalog number: D5523 ) + 10% FBS for Human MSC (HuMSC), Fibroblasts 3T3, A549
  2. Mouse MesenCult™ (STEMCELL Technologies, catalog number: 05512 ) for mouse MSC
  3. Ham’s-F 12 (Sigma-Aldrich, catalog number: N6760 ) + 15% FBS for LA-4, MLE- 12
  4. BEGM (Lonza, catalog number: CC3170 ) for NHBE and BEAS-2b
  5. M-199 (Sigma-Aldrich, catalog number: M2520 ) + 15% FBS (Life Technologies, Invitrogen^TM, catalog number: 10270 ) for HUVECs (primary cultured)
10. TritonX-100 (Sigma-Aldrich, catalog number: T9284 )
11. 1x phosphate buffer saline (PBS)

Equipment

Lab-tek II Chambered Coverglass w/cover #1.5 Borosilicate Sterile (Sigma-Aldrich)
75 x 25 mm microscope slides (J. Melvin Freed Brand)
22 x 22 Esco microscope square cover glass with no. 1 thickness (Erie Scientific Company)
Live cell fluorescent microscope (Leica Microsystems, model: DMI6000 )
Confocal microscope (Leica Microsystems, model: TCSSP5 )
BD FACS Aria (BD Biosciences)
BD FACS Calibur (BD Biosciences)

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Mukherjee, S., Ahmad, T. and Agrawal, A. (2015). Assessing Mitochondrial Transport via Cytoplasmic Nanotubular Bridges in Cells. Bio-protocol 5(15): e1542. DOI: 10.21769/BioProtoc.1542.
Ahmad, T., Mukherjee, S., Pattnaik, B., Kumar, M., Singh, S., Kumar, M., Rehman, R., Tiwari, B. K., Jha, K. A., Barhanpurkar, A. P., Wani, M. R., Roy, S. S., Mabalirajan, U., Ghosh, B. and Agrawal, A. (2014). Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy. EMBO J 33(9): 994-1010.