Related Experiment Video
Updated: Jun 4, 2025
Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography
Published on: August 5, 2021
Ultrastructural expansion microscopy (U-ExM) visualization of malaria parasite dense granules using RESA as a
Junpei Fukumoto1, Takafumi Tsuboi2, Eizo Takashima1
1Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Ehime 790-8577, Japan.
Abstract:
Dense granules (DG) are understudied apical organelles in merozoites, the malaria parasite stage that invades erythrocytes. Only six proteins have been identified which localize to DGs, despite that DG proteins play crucial roles in multiple steps of intraerythrocytic parasite development. To develop a tool for investigating DG structure and function, this study applied ultrastructural expansion microscopy (U-ExM) to visualize the ring-infected erythrocyte surface antigen (RESA) in Plasmodium falciparum merozoites. Merozoites were expanded to approximately four times their original size, allowing the identification of DGs without the need for electron microscopy. RESA localization in merozoite DGs was confirmed by staining with a combination of anti-RESA mAb and protein staining by NHS-ester. The translocation of RESA to the infected erythrocyte membrane was also observed in early ring-stage parasites. These results are in good agreement with the RESA localization reported using immunoelectron microscopy (IEM). By using U-ExM, the identification of novel DG proteins will be facilitated without time-consuming IEM, thereby enhancing our understanding of erythrocyte parasitism by P. falciparum.
Insights
Ultrastructural expansion microscopy (U-ExM) visualizes dense granules (DGs) in malaria parasites, aiding the study of erythrocyte invasion. This method facilitates identifying novel DG proteins, enhancing understanding of Plasmodium falciparum development.
Area of Science:
- Cell Biology
- Parasitology
- Microscopy
Background:
- Dense granules (DGs) are apical organelles in malaria merozoites crucial for erythrocyte invasion.
- Limited knowledge exists regarding DG protein composition and function.
- Investigating DGs is vital for understanding intraerythrocytic parasite development.
Purpose of the Study:
- To develop and apply ultrastructural expansion microscopy (U-ExM) as a tool for studying malaria parasite dense granules.
- To visualize the localization of the ring-infected erythrocyte surface antigen (RESA) within Plasmodium falciparum merozoite DGs.
- To establish a method for identifying novel DG proteins without relying on electron microscopy.
Main Methods:
- Application of ultrastructural expansion microscopy (U-ExM) to Plasmodium falciparum merozoites.
- Expansion of merozoite samples approximately fourfold for enhanced visualization.
- Immunofluorescence staining using anti-RESA monoclonal antibody (mAb) and NHS-ester for protein detection.
Main Results:
- U-ExM successfully visualized dense granules (DGs) in Plasmodium falciparum merozoites.
- Localization of RESA within merozoite DGs was confirmed.
- RESA translocation to the infected erythrocyte membrane in early ring-stage parasites was observed.
- Results align with previous findings from immunoelectron microscopy (IEM).
Conclusions:
- U-ExM is an effective tool for visualizing malaria parasite dense granules and their protein components.
- This technique facilitates the identification of novel DG proteins, advancing the study of Plasmodium falciparum.
- U-ExM offers a less time-consuming alternative to IEM for DG research, improving understanding of erythrocyte parasitism.
