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Visualizing Lysosomal Membrane Permeabilization by Fluorescent Dextran Release.

Anne-Marie Ellegaard1, Marja Jäättelä1, Jesper Nylandsted1

  • 1Unit for Cell Death and Metabolism, Center for Autophagy, Recycling and Disease, Danish Cancer Society Research Center, DK-2100, Copenhagen, Denmark.

Cold Spring Harbor Protocols
|October 3, 2015
PubMed
Summary
This summary is machine-generated.

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This study presents a new method to monitor lysosomal membrane permeabilization (LMP), a key cell death pathway. The technique uses fluorescent dextran to visualize lysosomal integrity in real-time, aiding cell death research.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Lysosomal membrane permeabilization (LMP) is a crucial programmed cell death pathway activated by various cellular stresses.
  • Monitoring LMP is essential for understanding cell death mechanisms and responses to cytotoxic stimuli.

Purpose of the Study:

  • To develop and validate a novel, real-time method for observing LMP.
  • To characterize the pore sizes formed during LMP using size exclusion.

Main Methods:

  • Utilizing the endocytic capacity of cells to load lysosomes with fluorescent dextran.
  • Observing the cytosolic translocation of fluorescent dextran post-LMP induction.
  • Employing time-lapse imaging for real-time monitoring of LMP.
  • Using differently sized fluorescent dextrans to determine pore size exclusion limits.

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Main Results:

  • Healthy cells exhibit punctate fluorescent dextran staining within intact lysosomes.
  • Upon LMP, fluorescent dextran diffuses into the cytoplasm, showing a diffuse staining pattern.
  • The method allows for real-time tracking of LMP progression.
  • Different dextran sizes enable the estimation of lysosomal pore dimensions.

Conclusions:

  • The fluorescent dextran method provides a robust and accessible way to monitor LMP in real-time.
  • This technique facilitates the study of LMP dynamics and the characterization of lysosomal pores.
  • The findings contribute to a deeper understanding of programmed cell death pathways.