In this FY, we accomplished and advanced several important projects. They are (1) Identifications of unique properties of parasitophorous vacuole membrane (PVM), (2) study of time-dependent trafficking of tetracysteine (TC)-tagged parasite KAHRP protein, (3) study of protein trafficking modulations in parasitized hemoglobin S (HbS) erythrocytes. (4) Direct visualizations of whole MC by electron microscopy, (5) Microscopy study of gametocyte metabolism and drug resistance relationship, and (6) Initiation of new project studying hemoglobin distributions using hyperspectral imaging. (1) We continued studying parasite-derived membranes with unique biophysical properties using fluorescence lifetime microscopy (FLIM). During invasion process, PVM surrounds invading parasite. PVM continuously increase in its size as parasites mature. In addition, new membranes emerge from PVM and form Mauere's cleft (MC). This structure seems to play crucial roles in trafficking and delivery processes for parasite-derived proteins. However, the source of lipids enough to support the size increase in the new membrane structure is not known. A key to solve this question is cholesterol content as parasite does not have a cholesterol synthesis mechanism. In addition, cholesterol is one of the most important molecules for membrane biophysical properties. The potentially unique properties of PVM and MC could provide important insights for their functions responsible for efficient trafficking of parasite proteins to erythrocyte membrane. In previous FYs, we identified the lipid exchange/transfer between parasite and host erythrocyte membranes during intraerythrocytic stages monitored by the translocations of membrane dye, Di4-ANEPPDHQ, and cholesterol analogue, bodipy-cholesterol. These results suggest that lipids are transferred between erythrocyte and parasites, and transfer mechanism may not be the same between cholesterol and nonsterol lipids such as phospholipids. In this FY, we repeat several experiments to confirm results we obtained. The manuscript for these data is now under preparation. (2) In previous FYs, successfully engineered two TC constracts, KAHRP-GFP-TC and KAHRP-TC, and established protocol and methods for studying parasite protein trafficking using TC tag. Previous results suggest that (a) FlAsH and ReAsH-TC labeling can provide alternative fluorescence labeling technique, (b) the use of these tags result in high background level, but BAL (2, 3 dimercaptopropanol) can suppress this background for image analyses, (c) 5 hours was required for newly synthesized proteins appear in MC and 9 hours for erythrocyte membrane appearance, (d) not all proteins were delivered to erythrocyte cytoplasm, but remained inside parasite. These results show that the TC technology provides a dynamic aspects of protein delivery mechanism which is not possible with the common GFP-labeling otherwise. In this FY, we studied BAL effects on background suppression and normal protein delivery. We found that BAL concentration in pre-treatment prior to FlAsH or ReAsH labeling could be lowered to 100 umol/L from 650 umol/L we originally designed. However, post BAL treatment has to maintain high concentration. These data suggest that TC technology needs to be used with close attentions on background level which depends on cell types and amino acid profiles. The results of this project have been published in an Aug issue of PLoS ONE. (3) In this FY, we initiated studying protein trafficking modulations in erythrocytes with HbS. Children with abnormal hemoglobins such as HbS and HbC were previously shown to be protected against severe form of malaria. We have indentified multiple irregularities in protein expressions in parasitized HbC erythrocytes including accelerated band 3 clustering, and denser raft fractions. We also found that HbC erythrocytes showed lower membrane potential and increased hemichrome formations. Our current model indicates that increased hemichrome binds to erythrocyte membrane and reduces space where proteins can be transported. These changes in cell matrix could also exist in HbS erythrocytes. We used KAHRP-GFP-TC expressing parasites and found that parasitized HbS erythrocytes resulted in significantly larger GFP signal suggesting that KAHARP clustered during its trafficking. We are currently studying the cause of the larger clusters in HbS by focusing on oxidations and cell aging. (4) We expanded studying 3D morphology of MC membrane. MCs are formed during intraerythrocytic stage of P. falciparum and previously suggested to serve as a mid-station of certain parasite proteins for their maturations. The morphology of MCs has been shown using 3D reconstructions of transmission electron microscope images. Although these data provide a picture of MC morphology and orientations, fine structures of MCs and their relationships with erythrocyte membrane are still not revealed. In this FY, we directly visualized cytoplasmic side of parasitized erythrocytes to study MC morphology by applying unroofing technique which physically removes a part of cell membrane by gentle ultrasonic waves. Combination with fast freezing replica technique, we successfully revealed native MC structures. Our electron microscope data show for the first time that MCs interconnect with erythrocyte membrane skeletons and other MCs via fine, thread-like extensions. We also found these threads also extends larger areas on erythrocyte membrane skeletons and modify the membrane skeleton mesh size. These data suggest that proteins stationed in MC could be transferred to erythrocyte membrane along with the MC extensions. The manuscript describing these data is now under preparations. (5) We continued studying gametocyte metabolism using microscopy approach. In previous FY, we identified that compared to intraerythrocytic stages of parasites, gametocytes have (a) a high lipid content, but similar cholesterol level, (b) reduced reactive oxygen species, and (d) similar mitochondrial membrane potential. In this FY, we studied requirement of mitochondrial activity for gametocyte developments. We treated gametocytes with CCCP (carbonyl cyanide m-chlorophenyl hydrazone) to disturb mitochondrial membrane potential and monitored gametocyte viability. Gametocyte activity was changed when CCCP concentration is more than 100 uM, but mitochondrial membrane potential visualized with Mitotracker did not show significant change at 100 uM. However, we found a large inter-gametocyte difference in membrane potential in any concentrations of CCCP including control sample. These data suggested that complex physiological variability exist in gametocyte developments. We are currently testing other metabolism indicators to study specific physiological targets. (6) In this FY, we initiated a project studying hemoglobin distributions within a erythrocytes using hyperspectral imaging. Hemoglobin molecules have specific absorption spectra with several characteristic peaks. As hemoglobin degrades, these peaks shift to different wavelengths. Hyperspectral imaging uses a specially designed optics and changes wavelength of incident light, and scans from UV to near IR regions. Intensities of captured images at each wavelengths were compared to original light intensities and absorption spectra were produced. This technique has multiple advantages as absorption spectra of any area of cell can be analyzed that potentially help estimating hemoglobin amount and the level of degradations. Initial stage of this project established analysis protocol and correctly estimated oxyhemoglobins and hemozoins. This result has been accepted to J. of Selected Topics in Quantum Electronics. We are currently developing technique to estimate hemoglobin concentration with in specific area of cell.
