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Label-fracture of plasma membranes isolated from measles virus-infected cells
1Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Germany.
Abstract:
We present a method that permits correlation of the intramembrane architecture of plasma membrane fracture faces with the distribution of specific molecules at the corresponding cytoplasmic or exoplasmic membrane surfaces. HeLa cells infected with measles virus were used as a model system. Large fragments of the dorsal membrane were isolated after the virus glycoproteins were tagged at the outer cell surface with immune serum and protein A-gold markers. In a second step, different virus polypeptides at the inner cell surface were also identified by a smaller gold label. Thereafter, the isolated plasma membranes were frozen and freeze-fractured. The complementary fracture faces were shadowed with heavy metals and carbon and examined in the transmission electron microscope without cleaning of remaining biological material. Thus, the micromorphology of the replicated fracture faces and the topochemistry of virus components localized at the corresponding leaflets of the plasmalemma could be seen on the same image at high resolution. Of note is that the freeze-fracture morphology of the protoplasmic face is related to the molecular composition of the cytoplasmic surface, as revealed by antibody tagging.
Insights
This study introduces a novel method to link plasma membrane structure to molecule distribution using measles virus-infected HeLa cells. The technique allows high-resolution visualization of intramembrane architecture and surface molecule localization simultaneously.
Area of Science:
- Cell Biology
- Virology
- Biophysics
Background:
- Understanding plasma membrane organization is crucial for cellular processes.
- Current methods often lack the resolution to correlate intramembrane structure with surface molecule distribution.
Purpose of the Study:
- To develop a method correlating intramembrane architecture with molecular distribution on plasma membrane surfaces.
- To visualize virus component localization within infected cell membranes.
Main Methods:
- Utilized measles virus-infected HeLa cells as a model system.
- Employed immunogold labeling to tag viral glycoproteins on both outer and inner cell surfaces.
- Performed freeze-fracture electron microscopy on isolated plasma membranes.
- Examined complementary fracture faces via transmission electron microscopy without cleaning.
Main Results:
- Successfully correlated freeze-fracture morphology of plasma membrane fracture faces with the distribution of specific viral molecules.
- Achieved high-resolution visualization of both intramembrane structure and surface-localized viral components on the same image.
- Demonstrated that freeze-fracture morphology of the protoplasmic face relates to the cytoplasmic surface's molecular composition.
Conclusions:
- The developed method enables simultaneous high-resolution analysis of plasma membrane intramembrane structure and surface molecular topochemistry.
- This technique provides new insights into the relationship between membrane architecture and the localization of viral components.
- The findings highlight the utility of correlative freeze-fracture electron microscopy for studying cell surface-associated molecular events.