Inducible Knockdown of Dynein’s Cargo-specification Domain

Undergraduate #56
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Session: 1
Room: Exhibit Hall A

Gabriella Hahn - SUNY at Fredonia
Co-Author(s): Dr. Trina A. Schroer, Johns Hopkins University, Baltimore MD

Dynein and dynactin interact to perform many cellular functions. Dynein is a microtubule motor protein, while dynactin is a cofactor that increases dynein?s efficiency. Dynein and dynactin cannot bind together without activating adapters: proteins that facilitate the dynein-dynactin interaction and recruit them to cargo. Most adapters remain unknown, and it is unclear how dynein/dynactin differentiate among different adapters. The pointed end of the dynactin complex, particularly p25/p27, has been implicated in targeting cargo and is a prime target for identifying new adapters. Both proteins direct dynein/dynactin to a subset of cargoes. In order to identify new adaptors, we must create a cell line that has a depletion of p25/p27. The goal of this project was to identify an shRNA that could yield a depletion comparable to siRNAs that are known to work in the lab. Two different shRNA plasmids were electroporated into Cos7 cells, and cells were assayed using Western blots and immunocytochemistry. Control plasmids and siRNA were used as negative controls, and Arp11 siRNA was used as a positive control. One shRNA plasmid did not yield a comparable knockdown, while the second plasmid yielded inconclusive results. The plasmid needs to be further purified and post-transfection conditions need to be optimized to obtain conclusive results. If this second shRNA yields sufficient knockdown, it can be used to deplete p25/p27 levels in cells, allowing us to identify adaptors that lose their ability to bind to dynactin in the absence of p25/p27.

Funder Acknowledgement(s): Support provided to GH through the National Science Foundation REU Site Award DBI-1757708 and NIH grant HL137076 awarded to Dr. Trina Schroer.

Faculty Advisor: Dr. Nicholas Quintyne, nicholas.quintyne@fredonia.edu

Role: Background information/research and materials were given to me by the Schroer lab, however this was my own project in the lab. All experiments were done my me and the results are mine.