Budding pouches and associated bubbles: 3D visualization of exo-membrane structures in plasmodium falciparum gametocytes


The complex life cycle of Plasmodium falciparum (P. falciparum) includes sexual development and asexual replication in the human host’s red blood cells and sexual reproduction in the mosquito vector. While the asexual stages cause the pathogenesis of malaria and the ensuing morbidity and mortality, the sexual stages of the parasite, known as gametocytes, are necessary for effective parasite transmission from humans to mosquitoes. In their asexual stages of development, P. falciparum gametocytes exhibit distinctive morphology, metabolism, and protein expression profiles. In the gametocyte stage, a wide range of “exomembrane structures” are newly formed in addition to the striking changes in their appearance. Rearrangements of the localization of F-actin and tubulin as well as changes to the spectrin network of the parasite cell membrane in stages III to V are linked to the structural modification in infected erythrocytes. The Maurer’s clefts (MCs) membrane is home to the well-studied P. falciparum skeleton-binding protein 1 (PfSBP1), an exported integral membrane protein. Early after the parasite’s invasion, membrane-limited vacuoles or sack-like structures called MCs form in the erythrocyte’s cytosol. The function, localization, and three-dimensional structural information of P. falciparum gametocytes are poorly understood, and only a small amount of structural information, typically two-dimensional, has been reported using traditional electron microscopy or fluorescence microscopy.

In a new study published in the journal Frontiers in Cellular and Infection Microbiology, researchers  Dr. Eri Saki Hayakawa and Professor Nobuhiko Ohno from Jichi Medical University together with Dr. Marina Wayama from Hitachi High-Tech Corporation, Professor Fuyuki Tokumasu from Nagasaki University, Dr. Mami Matsumoto from Nagoya City University Graduate School of Medical Sciences and Professor Jiro Usukura from Nagoya University, National Institute for Physiological Sciences, Japan merged unroofing and stereoscopic tilt imaging methods to allow direct measurements of the internal organization of gametocyte-parasitic cells. Their multi-imaging methods were successful in locating distinct but intricate membrane structures inside gametocytes that are absent from the parasites’ asexual life stages.

Drs. Eri Saki Hayakawa, Jiro Usukura, and Fuyuki Tokumasu previously developed an unroofing strategy to directly observe MCs in asexual parasitized erythrocytes by removing the upper part of the cell’s membrane followed by transmission electron microscopy in order to better visualize intracellular organelle and exomembrane structures. Through their direct intracellular findings using unroofing- Transmission Electron Microscopy (TEM) and Serial Block-Face Scanning Electron Microscopy (SBF-SEM), they gained new knowledge about the stereoscopic exomembranes in gametocyte-infected erythrocytes. Researchers discovered that MCs are attached to the host erythrocyte membrane skeletons by a variety of tethers. The typical MCs, which can be distinguished from other membranous structures solely by the appearance in asexual stages, were not found in their data. In addition, the erythrocyte membrane skeleton was frequently connected to tiny bubbles. In order to reveal the exo-membrane structures in gametocytes, the authors used unroofing-TEM, SBF-SEM, and fluorescence microscopy to examine the intracellular structures of gametocytes. In gametocytes, their research revealed “balloon/pouch”-like structures budding from the parasitophorous vacuole membrane (PVM), some of which contained several layers of other balloons. In addition, numerous bubbles that resembled MC-like membranes but were smaller than asexual MCs appeared on the inner surface of the erythrocyte membrane, or PVM. Exo-membranes in erythrocytes may be reformated by P.  falciparum to accommodate stage-specific biological activities during their sexual development, according to a recent study. Although not significant, some differences in the appearance of membranes were observed among the number of cells seen in the experiments. These differences are likely the result of the different physical effects that various unroofing conditions have on gametocyte stages.

In summary, Dr. Eri Saki Hayakawa and her colleagues used the unroofing technique to obtain stereoscopic structures of exo-membranes in gametocyte-erythrocytes, providing unique structural data and knowledge for upcoming cell biological studies. Future investigations utilizing a combination of genetically altering well-known marker proteins and cutting-edge imaging modalities will be necessary to identify each gametocyte-specific exo-membrane system and its function precisely.

Budding pouches and associated bubbles: 3D visualization of exo-membrane structures in plasmodium falciparum gametocytes - Medicine Innovates

About the author

Eri Saki H. Hayakawa received her Ph.D. in 1998 at Hokkaido University, School of Engineering (in Biophysics). She received the Research fellowship for young scientists by JSPS (Japanese Society for the Promotion of science) as a pre- and postdoctoral fellow. Then, she spent 3 years in National Institute on Alcohol Abuse and Alcoholism (NIAAA)-NIH as a visiting fellow in Dr. Burton J. Littman’s lab and studied rhodopsin-lipid interactions. After she won PRESTO Research Fellow position organized by JST (Japan science and technology agency), she studied biophysically using atomic force microscopy and other physical measurements, the effect of tuberculosis-glycolipid on cholesterol domain formation as a model for survival skills of tuberculosis in Dr. James A. Dvorak’s lab at National Institute of Allergy and Infectious Diseases (NIAID)-NIH.

Currently, her work mainly focuses on characterizing the protein and lipid transport system in the erythrocytic stages of Plasmodium falciparum using cutting edge imaging technologies including holographic microscopy and electron microscopy. She recently reported transportations of cholesterol-rich membranes inside parasitized erythrocytes for the first time and demonstrate a possible “membrane recycling” occurred in asexual stages of P. falciparum-erythrocytes (Hayakawa et al., 2020, Scientific report). She also reported three-dimensional structures of Maurer’s clefts, associating fine structures, and their interactions with cytoskeleton (Hayakawa et al., 2015, Experimental Parasitology). This intracellular structure is created by parasites and known as a key protein transport carrier, but their associations with the cytoskeleton has not be directly visualized. She recently extended her study to gametocytes, the sexual stage of P. falciparum, and found a variety of lipid membranes, i.e. pouches and bubbles, appeared in the parasitized erythrocytes using unroofing technique that is an indication of parasite-induced host erythrocyte modifications for specific needs in each parasite stage.


Hayakawa E, Wayama M, Tokumasu F, Ohno N, Matsumoto M, Usukura J. Budding pouches and associated bubbles: 3D visualization of exo-membrane structures in plasmodium falciparum gametocytes. Frontiers in cellular and infection microbiology. 2022 Jan 1;12.

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