AI Article Synopsis
- The study aimed to identify a new synthetic nanobody, VH (P-36 tagged with mNeonGreen), that interacts with the Rho GTPase, crucial for controlling the actin cytoskeleton in living cells.
- Researchers developed a library of synthetic VH nanobodies, transformed them into yeast, and analyzed their interactions and effects on cellular processes using various methods like live cell imaging and statistical analysis.
- The findings showed that VH (P-36) does not affect yeast growth, maintains cell polarity, interacts strongly with Rho1GTPase, and indicates a role in actin production during cytokinesis, especially when influenced by the compound latrunculin-A.
Article Abstract
The Rho guanosine triphosphatase hydrolase enzyme (GTPase) is required for the control of the actin cytoskeleton, but its activation in vivo condition is unknown. The study's goal was to find a new synthetic nanobody VH (P-36 tagged with mNeonGreen) that interacts strongly with the Rho GTPase. We present the first novel synthetic nanobody, VH (P-36 tagged with mNeonGreen), tested in fission yeast cells and found to have a particular interaction with Rho1GTPase. Plasmids were constructed by using of certain enzymes to digest the pDUAL-pef1a vector plasmid to produce a protein that was encoded by cloned genes. A varied VH library was created synthetically, then transformed into yeast cells, and positive clones were chosen using chemical agents. To investigate protein interactions and cellular reactions, several studies were carried out, such as live cell imaging, growth curve analysis, coimmunoprecipitation, structural analysis, and cell therapies. Prism and RStudio were used for the statistical analysis. The presence of VH (P-36) has no effect on the growth pattern making it an appropriate model for studying cytokinesis in vivo. According to a computational biological study, its affinity to interact with Rho1GTPase with all the complementarity-determining region (CDR) regions found on VH (P-36) is extremely strong. We were able to track its subcellular target by localization using a fluorescent confocal microscope, ensuring the maintenance of cell polarity and morphology. Spheroplast analysis revealed a circular-shaped cell with an even distribution of Rho1 tagged VH (P-36), indicating that the interaction occurs near the plasma membrane. The introduction of latrunculin-A (Lat-A) disrupted Rho GTPase localization, demonstrating the control over actin production, and the cell did not show evidence of mitotic phase commencement while Lat-A was present. Finally, this important biological tool can aid in our understanding of the mechanics and dynamics of cytokinesis in relation to Rho1GTPase.
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