No abstract

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.

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 over-expressing 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 over-expression. 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 supports 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.

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.

Mitochondria are essential, dynamic organelles that regularly undergo both fusion and fission in response to cellular conditions, though mechanisms of the regulation of their dynamics are incompletely understood. We provide evidence that increased activity of the small GTPase ARL2 is strongly correlated with an increase in fusion, while loss of ARL2 activity results in a decreased rate of mitochondrial fusion. Strikingly, expression of activated ARL2 can partially restore the loss of fusion resulting from deletion of either mitofusin 1 (MFN1) or mitofusin 2 (MFN2), but not deletion of both. We only observe the full effects of ARL2 on mitochondrial fusion when it is present in the intermembrane space (IMS), as constructs driven to the matrix or prevented from entering mitochondria are essentially inactive in promoting fusion. Thus, ARL2 is the first regulatory (small) GTPase shown to act inside mitochondria or in the fusion pathway. Finally, using high-resolution, structured illumination microscopy (SIM), we find that ARL2 and mitofusin immunoreactivities present as punctate staining along mitochondria that share a spatial convergence in fluorescence signals. Thus, we propose that ARL2 plays a regulatory role in mitochondrial fusion, acting from the IMS and requiring at least one of the mitofusins in their canonical role in fusion of the outer membranes.

ARL2 is among the most highly conserved proteins, predicted to be present in the last eukaryotic common ancestor, and ubiquitously expressed. Genetic screens in multiple model organisms identified ARL2, and its cytosolic binding partner cofactor D (TBCD), as important in tubulin folding and microtubule dynamics. Both ARL2 and TBCD also localize to centrosomes, making it difficult to dissect these effects. A growing body of evidence also has found roles for ARL2 inside mitochondria, as a regulator of mitochondrial fusion. Other studies have revealed roles for ARL2, in concert with its closest paralog ARL3, in the traffic of farnesylated cargos between membranes and specifically to cilia and photoreceptor cells. Details of each of these signaling processes continue to emerge. We summarize those data here and speculate about the potential for cross-talk or coordination of cell regulation, termed higher order signaling, based upon the use of a common GTPase in disparate cell functions.

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.

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.