Oral Presentation 26th ACMM “2020 Visions in Microscopy”

Investigating Malaria Cell Biology using Correlative Super-Resolution & Electron Microscopy (#130)

Oliver Looker 1 2 , Adam Blanch 1 , Boyin Liu 1 3 , Juan Nunez-Iglesias 1 4 , Paul McMillan 1 5 , Matthew Dixon 1 , Leann Tilley 1
  1. Department of Biochemistry & Molecular Biology, University of Melbourne, Parkville
  2. Current Affiliation, Biomedical Imaging Facility, University of New South Wales, Sydney
  3. Current Affiliation:, Thermo Fisher Scientific, Melbourne
  4. Current Affiliation, Monash Micro Imaging, Monash University, Clayton
  5. Biological Optical Microscopy Platform, University of Melbourne, Parkville

Malaria is caused by an infection of the human host with the intracellular parasite, Plasmodium falciparum. During the disease-causing stages of infection, the parasite invades host erythrocytes (Red Blood Cells) and undergoes cycles of asexual reproduction.

In order to evade detection in the host, the parasite mediates adhesion of infected erythrocytes to blood vessel walls. This adhesion is caused by the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1), which is produced by the parasite and is trafficked out to the surface of the erythrocyte. Trafficking of PfEMP1 involves parasite derived structures called Maurer’s clefts and knobs, which are approx. 500nm and 90nm in diameter respectively.

The nano-scale size of these structures prohibits the use of many conventional fluorescence microscopy techniques. Therefore, we have combined Super-Resolution Microscopy and Electron Microscopy techniques, including 3D-Structured Illumination Microscopy (3D-SIM), direct STochastic Optical Reconstruction Microscopy (dSTORM) and Scanning Electron Microscopy (SEM) to investigate the cell biology of these structures.

Using 3D-SIM, we have investigated the organisation of Maurer’s clefts, showing that there are different protein domains within these structures, which are vital for correct trafficking of PfEMP1 to the surface of the erythrocyte. Using dSTORM we have shown that knobs are generated by the association of the Knob Associated Histidine Rich Protein (KAHRP) with the erythrocyte cytoskeleton. During the life cycle the KAHRP aggregates come together to form a donut shaped pattern located at the edge of the knob. We also show that PfEMP1 is trafficked to the erythrocyte membrane before becoming associated with pre-formed knobs at the surface.

We have also developed a method for performing correlative dSTORM and Scanning Electron Microscopy (SEM) and utilised this to investigate knob production in relation to cytoskeletal rearrangements during the life cycle. We show that cytoskeletal rearrangement is required for correct knob production and PfEMP1 trafficking.

  1. Looker et al PLoS Pathog. 2019 May 9;15(5):e1007761. doi: 10.1371/journal.ppat.1007761