The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and cilia to regulate ciliogenesis and ciliary protein traffic

Abstract

ELMODs are a family of three mammalian paralogs that display GTPase activating protein (GAP) activity towards 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 paralogs ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing 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.

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E

“…Our findings of INPP5E and IFT140 accumulation at the Golgi in Arl16 KOs and INPP5E but not IFT140 at the Golgi in Pde6d KOs support our model of ARL16 regulating two novel, independent traffic pathways from the Golgi to cilia. Finally, in a parallel study carried out in our lab we found phenotypes very similar to those reported here in Arl16 KO lines when MEFs were deleted for either of the ARF/ARL GAPs ELMOD1 or ELMOD3 ( Turn et al. , 2022 ).…”

“…, 2022 ). In addition, expression of ARL16-myc in Elmod1 or Elmod3 KO cells was able to reverse those phenotypes ( Turn et al. , 2022 ), providing strong support for a model that includes these two proteins as acting in concert with ARL16 at the Golgi and in ciliary biology.…”

“…, 2013 ; Turn et al. , 2021 , 2022 ). Figure 2B shows double labeling of ARL16 and centrin 3, a marker for the connecting cilium as well as the mother (basal body) and daughter centrioles of photoreceptor cells in human retinas.…”

“…, 2019 ; Turn et al. , 2020 , 2021 , 2022 ). Guide RNA sequences targeting the coding region of the gene were designed using Benchling (benchling.com/academic).…”

A Phylogenetic Profiling Approach Identifies Novel Ciliogenesis Genes InDrosophilaAndC. elegans

“…In contrast, ELMOD family members are thought to act as ARF/ARL GAPs, with broad cellular effects, including on the actin cytoskeleton (Li et al, 2019b). All three vertebrate ELMOD proteins have recently been localized to basal bodies and linked to ciliogenesis, albeit with seemingly opposing effects: While loss of ELMOD1 and 3 reduced ciliation in mouse embryonic fibroblasts, loss of ELMOD2 actually resulted in an increase, with those cilia displaying abnormal morphology, including apparent splaying of axonemes and ciliary rootlet defects (Turn et al, 2022; Turn et al, 2021) (ultrastructural analysis has so far not been performed). Common to all three perturbations is a mislocalization of ciliary proteins, suggesting ciliary trafficking is at least partly responsible for the observed defects (Turn et al ., 2022; Turn et al ., 2021).…”