Major
Neuroscience
Anticipated Graduation Year
2022
Access Type
Restricted Access
Abstract
The cytoskeleton of eukaryotic cells is integral for the maintenance of cell structure, intracellular transport, polarization, and locomotion. It is composed of actin, actin microfilaments, intermediate filaments, and microtubules. In higher eukaryotes, microtubules (MTs) are largely dependent on microtubule-associated proteins (MAPs) that prevent them from disassembling at low temperatures or in the presence of nocodazole, a synthetic tubulin-binding agent that prevents mitosis and induces apoptosis. Here, our lab veers further into the role of microtubule-associated proteins in the malaria parasite Plasmodium. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. Plasmodium transforms into specific stages, such as the ookinete stage. The ookinete displays a highly polarized banana shape that allows the parasite to become motile and escape the blood meal to continue its development in the mosquito. During the life cycle, the parasites undergo several cycles of extreme population growth and mitosis in order to continue transmission and its role as pathogenesis in the host. In our lab, we cloned and expressed Plasmodium proteins in a human cell line and identified two novel microtubule-binding proteins (MAPs) PbSAXO and PbTLAP2 in the host cell. We also demonstrated that PbSAXO can stabilize microtubules at cold temperatures (4∞C). This investigation of Plasmodium proteins in a human cell line was possible because human and parasite tubulins share 80% protein sequence identity.
To build off these findings, we plan to demonstrate the cellular functions of TALP2 and SAXO in the malaria parasite. In order to achieve this, we will remove each gene from the genome of the parasite and investigate the consequences by using the genome-editing tool CRISPR. Our lab has recently established CRISPR of the mouse malaria parasite Plasmodium berghei and successfully deleted SAXO from its genome. This investigation did not demonstrate any significant phenotype. After demonstrating that SAXO and TLAP2 bind to microtubules, we hypothesize that one MAP may be able to compensate for the other. We also hypothesize that TLAP2 may be a more prominent MAP and therefore cause no phenotype to emerge in the deletion of SAXO. To test our hypothesis, we plan to use CRISPR to delete the TLAP2 gene from the parasite genome and analyze the phenotype, if one emerges. Afterward, we plan to generate a SAXO-TLAP2 double deletion parasite line to determine whether or not TLAP2 is an essential gene.
Faculty Mentors & Instructors
Dr. Stefan Kanzok, Principal Investigator, Biology; Grifin Berge, Graduate Student, Biology; Manny Widuch, Graduate Student Mentor
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Investigating Microtubules in the Human U2-OS Cell Line
The cytoskeleton of eukaryotic cells is integral for the maintenance of cell structure, intracellular transport, polarization, and locomotion. It is composed of actin, actin microfilaments, intermediate filaments, and microtubules. In higher eukaryotes, microtubules (MTs) are largely dependent on microtubule-associated proteins (MAPs) that prevent them from disassembling at low temperatures or in the presence of nocodazole, a synthetic tubulin-binding agent that prevents mitosis and induces apoptosis. Here, our lab veers further into the role of microtubule-associated proteins in the malaria parasite Plasmodium. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. Plasmodium transforms into specific stages, such as the ookinete stage. The ookinete displays a highly polarized banana shape that allows the parasite to become motile and escape the blood meal to continue its development in the mosquito. During the life cycle, the parasites undergo several cycles of extreme population growth and mitosis in order to continue transmission and its role as pathogenesis in the host. In our lab, we cloned and expressed Plasmodium proteins in a human cell line and identified two novel microtubule-binding proteins (MAPs) PbSAXO and PbTLAP2 in the host cell. We also demonstrated that PbSAXO can stabilize microtubules at cold temperatures (4∞C). This investigation of Plasmodium proteins in a human cell line was possible because human and parasite tubulins share 80% protein sequence identity.
To build off these findings, we plan to demonstrate the cellular functions of TALP2 and SAXO in the malaria parasite. In order to achieve this, we will remove each gene from the genome of the parasite and investigate the consequences by using the genome-editing tool CRISPR. Our lab has recently established CRISPR of the mouse malaria parasite Plasmodium berghei and successfully deleted SAXO from its genome. This investigation did not demonstrate any significant phenotype. After demonstrating that SAXO and TLAP2 bind to microtubules, we hypothesize that one MAP may be able to compensate for the other. We also hypothesize that TLAP2 may be a more prominent MAP and therefore cause no phenotype to emerge in the deletion of SAXO. To test our hypothesis, we plan to use CRISPR to delete the TLAP2 gene from the parasite genome and analyze the phenotype, if one emerges. Afterward, we plan to generate a SAXO-TLAP2 double deletion parasite line to determine whether or not TLAP2 is an essential gene.