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Studying Cellular Dynamics Using Proximity-Dependent Biotinylation: Somatic Cell Reprogramming.

Reuben Samson1,2, Francesco Zangari1,2, Anne-Claude Gingras3,4

  • 1Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, ON, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|September 4, 2023
PubMed
Summary
This summary is machine-generated.

This study introduces time-resolved proximity-dependent biotinylation (PDB) protocols for studying dynamic cellular processes like reprogramming. Optimized miniTurbo PDB methods enable rapid proteomic analysis in primary cells, advancing cell fate research.

Keywords:
BioIDBiotinFibroblastLentivirusMass spectrometryProtein-protein interactionProteomicsProximity labelingProximity-dependent biotinylationReprogrammingStreptavidin

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Area of Science:

  • Cell Biology
  • Proteomics
  • Biochemistry

Background:

  • Understanding cellular reorganization during reprogramming and differentiation is key to elucidating morphological and fate changes.
  • Proximity-dependent biotinylation (PDB) methods offer advantages over traditional biochemical purification for proteomic characterization of subcellular compartments.
  • First-generation PDB enzymes like BirA* (BioID) typically require long labeling times (8-24 hours).

Purpose of the Study:

  • To define and optimize protocols for time-resolved PDB in primary cells.
  • To apply optimized PDB techniques to study somatic cell reprogramming in a mouse fibroblast model.
  • To provide a reference protocol for adapting PDB to other dynamic cellular processes.

Main Methods:

  • Utilized highly active PDB enzymes, such as miniTurbo, for rapid biotinylation signals (minutes).
  • Developed and employed an inducible lentiviral toolkit for BioID applications.
  • Optimized and applied lentivirally delivered miniTurbo constructs in a mouse fibroblast model of somatic cell reprogramming.

Main Results:

  • Successfully optimized time-resolved PDB protocols using miniTurbo constructs.
  • Demonstrated the application of these protocols to study the dynamic process of somatic cell reprogramming.
  • Established a baseline reference for researchers using PDB in dynamic cellular studies.

Conclusions:

  • Time-resolved PDB, particularly with miniTurbo, enables efficient proteomic analysis of dynamic cellular events.
  • The developed protocols facilitate the study of somatic cell reprogramming and can be adapted for other cellular processes.
  • This work advances the understanding of mechanistic underpinnings in cell fate changes.