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Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles
Aurélien Briens1, Maxime Gauberti1, Jérôme Parcq1
11. Institut National de la Santé et de la Recherche Médicale (INSERM), INSERM UMR-S U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, GIP Cyceron, Université Caen-Normandie, Caen, France.
Abstract:
Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.
Insights
Researchers developed a new microscopy method to measure cell-derived microparticles (MPs). This technique characterizes MP size, antigens, and activity, aiding disease diagnosis and prognosis.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Nanotechnology
Background:
- Cell-derived microparticles (MPs) are vesicles carrying cellular components, potentially useful for disease diagnosis.
- Current methods struggle to reliably measure MP size, number, and biological activity.
- Functional characterization of MPs is crucial for understanding their role in human diseases.
Purpose of the Study:
- To develop a novel protocol for direct imaging and functional characterization of cell-derived MPs.
- To establish reliable assays for measuring MP size, membrane antigens, and proteolytic activity.
- To demonstrate the method's applicability in disease contexts, including ischemic stroke.
Main Methods:
- Developed a protocol using high-resolution laser-scanning confocal microscopy.
- Utilized annexin-V coated micro-wells for MP trapping.
- Created fluorescent reporter assays for size measurement, antigen detection, and nano-zymography (proteolytic activity).
Main Results:
- Successfully imaged and functionally characterized engineered MPs.
- Demonstrated detection of tissue-type plasminogen activator (tPA), urokinase, and plasmin on MPs.
- Identified tPA-bearing fibrinolytic MPs in ischemic stroke patient plasma and mouse models.
Conclusions:
- The developed microscopy method enables direct imaging and functional characterization of cell-derived MPs.
- This technique offers high specificity for detecting MP-associated antigens and proteolytic activities.
- The method shows promise for the diagnostic and prognostic evaluation of diseases involving MPs, such as ischemic stroke.
