Abstract
TCL (or RhoJ) is a plasma membrane localized Rho‐GTPase bearing an additional eighteen N‐terminal amino acids in comparison to its close homologue Cdc42. Previous experiments in the lab have demonstrated that deleting the N‐terminal extension shifts TCL localization to vesicular membranes. To determine how this deletion may influence membrane trafficking of TCL, HeLa cells were transfected with plasmids encoding YFP‐tagged TCL and an N‐terminal deletion mutant (ΔN), and live‐cell imaging was performed using an FV10i fluorescence microscope. The live‐cell imaging demonstrated that wild‐type YFP‐TCL localization was present at the plasma membrane, and it also revealed an organized movement of YFP‐TCL labeled intracellular vesicles. Cells expressing the YFP‐TCL ΔN deletion mutant displayed more of a vesicular localization of the fusion protein, and vesicular movement within the cell was more erratic and random. Previous research has used fluorescent‐labeled transferrin as a marker to investigate a role for TCL in endocytic pathways; therefore, cells were exposed to Alexa 647‐labeled holo‐transferrin to determine if TCL colocalizes with vesicles involved with transferrin receptor uptake and recycling. Colocalization of the two fluorescent labels in time‐lapse images was measured using CellSens software, and initial results show significant colocalization of YFP‐TCL ΔN with transferrin‐labeled vesicles in comparison to wild‐type TCL. However, it is unclear if the alterations in TCL localization are influencing transferrin transport kinetics or if YFP‐TCL ΔN is inappropriately localized to transferrin‐labeled vesicles. Together, these results show TCL has a potential role in vesicle transport, and future experiments will be aimed at determining if it influences vesicle transport kinetics.Support or Funding InformationSupport was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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