“Roles for ELMOD2 and Rootletin in Ciliogenesis”

Abstract

ELMOD2 is a GTPase activating protein (GAP) with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway support a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and Cilia to Regulate Ciliogenesis and Ciliary Protein Traffic

“…In efforts to identify the cellular functions of ELMOD1 and ELMOD3, we used CRISPR/Cas9 in MEFs to introduce frameshift mutations in Elmod1 , Elmod3 , or both Elmod1/Elmod3 . We previously used the same procedure to generate Elmod2 KO MEFs and described multiple resulting phenotypes (25,34,89). We used two or more guides to target exons (see Fig.…”

“…We refer to such lines as CRISPR WT (CWT) to distinguish from the parental WT lines. The use of more than one guide, multiple alleles, and independent clones helps alleviate concerns regarding off-target effects, alternative splicing, and downstream initiation (89,90). As an additional protection against off-target effects, we also performed rescue experiments in which we transiently re-expressed the disrupted gene to test for reversal of key phenotypes.…”

“…S2C), microtubule networks (Fig. S2D), or centrosome numbers (25,35). Phalloidin staining for actin was not overtly altered among Elmod1 or Elmod3 KO cell lines (Fig.…”

“…While the ELMODs are GAPs with broad substrate specificities towards ARFs and ARLs (18,20,25,26), the ELMOs primarily function in complex with DOCK proteins as unconventional GEFs for RHO/RAC GTPases (which act at the leading edge of migrating cells (21,27–30)) and lack any GAP activity. In MEFs, ELMOD2 serves critical roles in the regulation of cytokinesis, ciliation, microtubule stability, mitochondrial fusion, ciliogenesis, anchoring of rootlets to centrosomes, and lipid metabolism at lipid droplets (25,31–35). Effects of ELMOD2 deletion on mitochondrial fusion and microtubules are linked to ARL2 (25,34), while effects on cell division/abscission are specifically suppressed by expression of activated ARF6 (25), demonstrating that one ELMOD can regulate multiple pathways through distinct GTPases.…”

“…Here we describe the use of a number of approaches, relying heavily on CRISPR/Cas9 edited MEFs, to begin defining the cellular functions of Elmod1 and Elmod3 and comparing the degree of functional overlap/specificity among the family members (25,34,35). ELMOD1 and ELMOD3 studies to date have focused on in vitro biochemical activities (18,20,26) and analyses of mutations in mammals (38–45,88), so the cellular functions remain largely uncharacterized.…”