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Related Experiment Videos

Imaging of proteolytic activity using a conditional cell surface receptor.

Mahaveer Swaroop Bhojani1, Daniel A Hamstra, Daniel C Chang

  • 1University of Michigan Medical Center, Ann Arbor, MI 48109, USA.

Molecular Imaging
|September 7, 2006
PubMed
Summary
This summary is machine-generated.

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Researchers developed a novel imaging platform to detect cellular protease activation during apoptosis. This method visualizes caspase-3 activity by detecting a released single-chain antibody on the cell surface, enabling real-time apoptosis imaging.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Imaging

Background:

  • Programmed cell death (apoptosis) is vital for multicellular organisms, and its dysregulation is linked to diseases like cancer and neurodegeneration.
  • Caspases, particularly caspase-3, are key proteases executing apoptosis by cleaving specific substrates like the DEVD sequence.

Purpose of the Study:

  • To develop a novel imaging platform for sensing cellular protease activation, specifically caspase-3.
  • To enable real-time, noninvasive imaging of apoptosis and high-throughput screening of therapeutic agents.

Main Methods:

  • A recombinant chimeric protein was engineered, comprising a cell-surface-targeted single-chain antibody (sFv) fused to a Golgi retention signal, with a DEVD sequence as a caspase-3 cleavage site.
  • Upon apoptosis induction, caspase-3 cleaves the fusion protein, releasing the sFv to the cell surface.

Related Experiment Videos

  • Immunofluorescence microscopy was used to visualize the translocated sFv on the cell surface.

    • Main Results:

    • The fusion protein was successfully localized to Golgi bodies and remained intracellular in non-apoptotic cells.
    • Apoptosis induction led to caspase-3-dependent cleavage, releasing the sFv to the cell surface.
    • The appearance of sFv on the cell surface served as a reliable indicator of apoptosis.

    Conclusions:

    • The developed imaging platform enables protease-dependent visualization of apoptosis.
    • This methodology offers a unique approach for real-time apoptosis imaging and high-throughput screening of apoptosis-modulating drugs.