Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe
S Mizukami1, K Kikuchi, T Higuchi
1Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan.
Abstract:
Microscopic visualization of intracellular enzyme activity can provide information about the physiological role of the enzyme. Caspases are cysteine proteases that have critical roles in the execution of apoptosis. General fluorometric substrates of caspase-3, such as DEVD-MCA, are unsuitable for imaging because they are excited at short wavelength, so we designed and synthesized novel fluorescent probes that are excited at suitable wavelengths for detecting caspase-3 activity in living cells. Using one of these probes, we succeeded in microscopic visualization of caspase-3-like activity within HeLa cells treated with etoposide. The caspase-3-like activity was increased in the cytosol at first, then expanded to the whole cell.
Insights
Researchers developed novel fluorescent probes for visualizing caspase-3 activity in living cells. This advancement allows for microscopic imaging of apoptosis execution, revealing enzyme activity dynamics within cells.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Imaging
Background:
- Intracellular enzyme activity visualization offers insights into enzyme physiological roles.
- Caspases, critical cysteine proteases in apoptosis execution, require effective visualization methods.
- Existing fluorometric substrates for caspase-3 lack suitable excitation wavelengths for live-cell imaging.
Purpose of the Study:
- To design and synthesize novel fluorescent probes for detecting caspase-3 activity.
- To enable microscopic visualization of caspase-3 activity in living cells.
- To overcome limitations of existing short-wavelength-excited substrates.
Main Methods:
- Design and synthesis of novel fluorescent probes with suitable excitation wavelengths.
- Application of a synthesized probe for detecting caspase-3-like activity.
- Microscopic imaging of HeLa cells treated with etoposide.
Main Results:
- Successful microscopic visualization of caspase-3-like activity in living HeLa cells.
- Demonstrated increased caspase-3-like activity in the cytosol following etoposide treatment.
- Observed expansion of caspase-3-like activity from the cytosol to the entire cell.
Conclusions:
- Novel fluorescent probes are effective for visualizing caspase-3 activity in live cells.
- The developed probes overcome limitations of traditional substrates for cellular imaging.
- This method provides a dynamic view of caspase-3 activation during apoptosis.
