ELMOD2 regulates mitochondrial fusion in a mitofusin-dependent manner, downstream of ARL2

Abstract

Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.

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

“…We screened ELMOD2 null lines using markers of various cellular compartments both to assess potential global changes in organelle morphology as well as to specifically look for changes in compartments to which ELMOD2 had been localized (i.e., mitochondria and lipid droplets). As predicted and demonstrated earlier, we observed mitochondrial defects (fragmentation) consistent with ELMOD2 acting with ARL2 in mitochondrial fusion ( Schiavon et al. , 2019 ).…”

“…, 2012 ) GAP dead mutant, ELMOD2[R167K], rescues the cold-sensitive phenotype that we described above. We note that the mitochondrial fragmentation that occurs in MFN1 null MEFs is reversed upon expression of either WT or GAP dead ELMOD2, which supports the model that ELMOD2 acts as an effector of ARL2 inside mitochondria ( Schiavon et al. , 2019 ).…”

“…With the goal of identifying cellular functions for ELMOD2, we generated cell lines specifically deleted for ELMOD2. We used CRISPR-Cas9 genome editing to introduce frame-shifting mutations of both alleles in MEFs, as described earlier ( Schiavon et al. , 2019 ).…”

“…Here, we used CRISPR-Cas9 to generate frameshifting/null mutations of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs). These lines not only proved useful earlier for gaining mechanistic insight into ELMOD2’s role(s) in mitochondrial fusion ( Schiavon et al. , 2019 ), but they also revealed a host of unexpected phenotypes that were not apparent from previous knockdown or overexpression studies.…”

“…, 2014 ) and has been localized to mitochondria, the endoplasmic reticulum (ER), and lipid droplets. Yet, functions at these locations have been shown for only ELMOD2 and ARL2 acting from the intermembrane space to regulate mitochondrial fusion ( Schiavon et al. , 2019 ).…”