Optimization of Immunopurification of the Exocyst Complex in Moss

Vitellaro, Lauren

Student Work

Optimization of Immunopurification of the Exocyst Complex in Moss

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Plant tip cell growth (root hairs, pollen tubes, and caulonema) requires a balance of inter-osmotic pressure, expanding the cell outwards and the constant delivery of cell wall loosening enzymes and new wall materials to prevent the cell from bursting. To make these deliveries, the cell uses exocytosis, where myosin XI motor proteins cluster exocytic vesicles along F-actin strands to the apical tip of the cell. Other spatial and temporal controls of vesicle binding include Rho and Rab GTPases, SNAREs, signaling lipids, and the exocyst complex. This multisubunit complex assists with tethering and fusion of the vesicle. The exocyst complex structure, assembly, and binding partners are largely unknown in plants. Physcomitrium patens is used as a model organism due to its rapid polarized tip growth and easy genetic editing. This project tested an immunopurification method where epoxy Dynabeads were derivatized with anti-GFP Lag94-15 nanobodies to isolate moss exocyst. To allow for immunodetection in Western blots and for GFP-based immunopurification, the project used a transgenic moss line with mEGFP endogenously tagged to exocyst subunit Sec6 via a linker with a PreScission protease (PSP) cut site. The assays determined that the appropriate amount of moss tissue to buffer was 1 gram in 2.5 mL of buffer to saturate 50 µL of Dynabeads. Assays using the control mEGFP-HaloTag were run to confirm that the PSP was cleaving appropriately. Although large-scale cleavage-based elution of the exocyst complex was not successful, the Sec6 subunit appears to be present in the eluted fraction in addition to very faint bands near the size of Sec8 (112-120). Because the Sec6 was eluted, it’s possible other subunits and complexes were eluted but are below the detection limit of the Krypton stain. Future experiments should determine if mEGFP is actually cleaved using a Western blot, in addition to testing varying buffer conditions that may prevent any electrostatic interactions between the bead and exocyst.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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