Nanopore-mediated protein delivery enabling three-color single-molecule tracking in living cells

Zhongwen Chen, Yuhong Cao, Chun Wei Lin, Steven Alvarez, Dongmyung Oh, Peidong Yang, Jay T. Groves

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Original languageEnglish (US)
Article numbere2012229118
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number5
StatePublished - Feb 2 2021
Externally publishedYes


  • EGFR signaling
  • Electroporation
  • Ex vivo protein delivery
  • Nanopore
  • Single molecule

ASJC Scopus subject areas

  • General


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