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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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Fluorescence activation imaging of localization, distribution, and level of miRNA in various organelles inside cells

Chuanli Wu1, Xiaoyan Liu1, Yiyi Zheng1

  • 1Jiangsu Key Laboratory of New Power Batteries, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, PR China.

Talanta
|May 23, 2018
PubMed

Insights

This study introduces a new method to visualize microRNA (miRNA) in cellular organelles using a special fluorescent probe. The technique successfully mapped miRNA-150 levels within mitochondria and lysosomes in cancer cells.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • MicroRNAs (miRNAs) play crucial roles in gene regulation and cellular processes.
  • Understanding miRNA localization and levels within organelles is vital for comprehending their functions.
  • Current methods for imaging intracellular miRNA distribution and concentration are limited.

Purpose of the Study:

  • To develop and validate a novel activatable fluorescence probe for imaging miRNA localization, distribution, and levels in subcellular organelles.
  • To demonstrate the probe's capability using miR-150 as an example in human cervical cancer cells (HeLa).
  • To assess the probe's performance in terms of specificity, signal-to-background ratio, and nuclease resistance.

Main Methods:

  • Design and synthesis of an activatable fluorescence probe sensitive to miRNA binding-induced conformational changes.
  • Application of the probe in HeLa and MCF-7 cancer cells to visualize miR-150.
  • Confocal microscopy to determine miRNA localization, distribution, and relative levels in organelles like mitochondria and lysosomes.
  • Evaluation of probe specificity, signal-to-background ratio, and resistance to cellular nucleases.

Main Results:

  • The developed probe successfully imaged the localization and distribution of miR-150 in various organelles, primarily mitochondria and lysosomes.
  • Lysosomal miR-150 levels were found to be higher than mitochondrial levels in both HeLa and MCF-7 cells.
  • MiRNA levels exhibited a height-dependent distribution (z-direction) within the cells.
  • The approach demonstrated simultaneous imaging of multiple miRNAs (e.g., miR-150, miR-214) in different organelles.

Conclusions:

  • The novel activatable fluorescence probe enables precise imaging of miRNA localization, distribution, and levels within subcellular organelles.
  • This approach provides a valuable tool for studying the functional roles of specific miRNAs in cancer cells.
  • The probe's high specificity, signal-to-background ratio, and nuclease resistance make it suitable for complex biological environments.
Keywords:
Activatable fluorescence probesCancer cellsCancer diagnosisMiRNA imaging

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