The ARF GAP ELMOD2 acts with different GTPases to regulate centrosomal microtubule nucleation and cytokinesis

Abstract

<jats:p> ELMOD2 is a ∼32 kDa protein first purified by its GAP activity towards ARL2 and later shown to have uniquely broad specificity toward ARF family GTPases in in vitro assays. To begin the task of defining its functions in cells, we deleted ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) and discovered a number of cellular defects, which are reversed upon expression of ELMOD2-myc. We show that these defects, resulting from the loss of ELMOD2, are linked to two different pathways and two different GTPases: with ARL2 and TBCD to support microtubule nucleation from centrosomes and with ARF6 in cytokinesis. These data highlight key aspects of signaling by ARF family GAPs which contribute to previously under-appreciated sources of complexity, including GAPs acting from multiple sites in cells, working with multiple GTPases, and contributing to the spatial and temporal control of regulatory GTPases by serving as both GAPs and effectors. </jats:p>

“Roles for ELMOD2 and Rootletin in Ciliogenesis”

“…Thus, the GAP activity of ELMOD2 is required at centrosomes/microtubules but not at cilia. In addition, we showed that expression of activated ARF6 reversed the centrosome amplification phenotype in ELMOD2 KO MEFs, consistent with it being a consequence of failed abscission (Turn et al, 2020). Expression of the same ARF6 construct, though, had no effect on ciliary phenotypes (Figure 6).…”

“…Though it was far more common to see 2-3 cilia per cell, some cells had 9 or more cilia (see Figure 1D). We previously reported (Turn et al, 2020) that ELMOD2 KO cells have supernumerary centrosomes at an increased frequency, which may enable this increase in ciliation. To examine whether the increased ciliation is solely a consequence of cell cycle defects, we repeated the scoring in cells with no evidence of cell cycle defects (i.e., one nucleus of normal size and morphology, only 1-2 centrosomes).…”

“…Failure to regulate centrosome cohesion can lead to cell cycle defects, such as centrosome amplification or aneuploidy. ARL2 has also been linked to aspects of breast cancer biology (Beghin et al, 2009; Beghin et al, 2008), and we showed that ELMOD2 KO MEFs take on properties of cell transformation, including loss of contact inhibition and anchorage independent growth (Turn et al, 2020).…”

“…Among its first known cellular functions was in mediating mitochondrial fusion as an ARL2 effector (Newman et al, 2017a; Newman et al, 2017b; Newman et al, 2014; Schiavon et al, 2019). Recent studies from our lab, though, revealed that ELMOD2 also acts with ARL2 on aspects of microtubule biology and with ARF6 in cytokinesis/abscission (Turn et al, 2020). This recent study also provided evidence that ELMOD2 localizes to centrosomes and Flemming bodies, consistent with its effects on microtubules and abscission.…”

“…These novel and unexpected roles were found in cells deleted for ELMOD2 using the CRISPR/Cas9 system in immortalized mouse embryonic fibroblasts (MEFs). These lines were generated in part due to our inability to document its knockdown by siRNA because of its low abundance in cultured cells (Turn, et al 2020). Interestingly, deletion of neither ELMOD1 nor ELMOD3 in MEFs resulted in any of the phenotypes described previously or below (manuscript in preparation), suggesting a high degree of specificity of ELMOD2 within this small family.…”