Citation
- Authors: Turn RE. et al.
- Year: 2022
- Journal: Mol Biol Cell 33 ar13
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: MEF
Description: Murine embryonic fibroblast cells
Method
Transient transfections of WT or KO MEFs for rescue experiments were performed using jetOPTIMUS (VWR; 76299-634). Cells seeded at 90% confluence were transfected with a ratio of 4 µg DNA:4 µl jetOPTIMUS transfection reagent: 400 µl jetOPTIMUS buffer adhering to the protocol provided by the manufacturer. The DNA/jetOPTIMUS mixture was added dropwise to each respective well, and samples were returned to 37°C to incubate for 24 h. Cells were maintained in normal growth serum (i.e., 10% FBS in DMEM). The next day, cells were replated as needed for different experiments.
Abstract
ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.