The small GTPase ARL2 is a novel regulator of mitochondrial fusion Open Access

Newman, Laura Elizabeth (2016)

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ARL2 is a small GTPase and member of the ARF family of GTPases. ARL2 is ubiquitous, conserved across eukaryotes, predicted to be present in the last eukaryotic common ancestor, and essential in three model organisms. ARL2 is unusual among regulatory GTPases in that it localizes to several places in the cell, including cytosol, centrosomes, the nucleus, and mitochondria. Though its actions at the other locations have been documented, its functions in mitochondria are poorly described. In this dissertation, I document our research that has expanded our knowledge of mitochondrial functions of ARL2. We show that ARL2 is required for mitochondrial morphology, motility, and ATP production. In addition, we show that the ARL2 GAP ELMOD2 is also required for mitochondrial morphology and motility, but not ATP production. Based on these data, we model two pathways in mitochondria regulated by ARL2: one regulating morphology and motility with ELMOD2 as the likely ARL2 effector, and the other regulating ATP production without ELMOD2. To further study regulation of morphology by ARL2, we developed plasmids for targeting exogenous proteins to either the mitochondrial intermembrane space or matrix, under the control of variable strength CMV promoters. I then used these plasmids to demonstrate that ARL2 regulates mitochondrial morphology from the intermembrane space. Additionally, I show that ARL2 regulates mitochondrial fusion upstream of the mitofusins. We expand upon these studies to show that the amount of ARL2 and ELMOD2 in mitochondria is responsive to the fusogenic activity of MFN2, as well as cellular stressors leading to mitochondrial hyperfusion. These observations further support a role for ARL2 and ELMOD2 in mitochondrial fusion. Finally, I provide preliminary data that ARL2 regulates ATP levels via cristae morphology, that ARL2 also regulates mitochondrial motility, and that ARL2 is phosphorylated in cells. From this research, I conclude that ARL2, and likely ELMOD2, are novel regulators of mitochondrial fusion. Future research will focus on identifying novel mitochondrial binding partners of ARL2 and ELMOD2, with the goal of elucidating the mechanism by which ARL2 regulates mitochondrial fusion.

Table of Contents

Chapter 1: Introduction 1


Figure 1. Schematic of mitochondrial structure 3

Mitochondrial dynamics 4

Figure 2. Mitochondrial shape varies as a result of fission and fusion. 5

Figure 3. Summary of mitochondrial fusion. 9

Regulation of mitochondrial fusion 10

ARL2 and ELMOD2 11

References 13

Chapter 2: The ARL2 GTPase is required for mitochondrial morphology,26

motility, and maintenance of ATP levels

Abstract 27

Introduction 27

Materials and methods 30

Results 36

Discussion 50

References 55

Figures 65

Figure 1. Mitochondria are fragmented in cells where ARL2 activity 65

is compromised.

Figure 2. Expression of GFP-DRP1[K38A] reverses fragmentation 67

caused by ARL2[T30N].

Figure 3. Mitochondria cluster in the perinuclear region in cells 68

depleted of ARL2.

Figure 4. Loss of ARL2 activity compromises mitochondrial motility. 69

Figure 5. Microtubules are not lost with expression of ARL2[K71R] 70

or ARL2 siRNA.

Figure 6. ATP levels are lower in cells depleted of ARL2 by siRNA. 71

Figure 7. A pool of ARL2 localizes to the mitochondrial matrix. 72

Figure 8. ELMOD2 localizes to mitochondria. 73

Figure 9. ELMOD2 knockdown alters mitochondrial morphology 74

and distribution.

Figure S1. ARL2 and ARL2[T30N] are expressed when 75

co-transfected with GFP-DRP1[K38A].

Figure S2. Mitochondrial ARL2 staining is retained after cBid 76


Figure S3. ELMOD2 mitochondrial staining is competed by 77

purified, recombinant ELMOD2.

Figure S4. ELMOD2 localizes to the mitochondrial matrix. 78

Chapter 3: Plasmids for variable expression of proteins targeted to the79

mitochondrial matrix or intermembrane space

Abstract 80

Introduction 80

Materials and methods 84

Results and discussion 88

References 96

Figures 102

Figure 1. Schematic of constructs. 102

Figure 2. Neither OCT-HA-GFP nor SMAC-HA-GFP 103

alter mitochondrial morphology.

Figure 3. SMAC-HA-ARL2 and OCT-HA-ARL2 are correctly 104

localized to the IMS or matrix, respectively.

Figure 4. CCCP treatment prevents the import and cleavage of 106


Figure 5. OCT-HA-GFP is efficiently imported/cleaved and levels 107

of expression decrease with decreasing strength CMV promoters.

Figure 6. SMAC-HA-GFP is expressed to similar levels as GFP and 108

processed less completely than OCT-HA-GFP, particularly at higher


Figure 7. SMAC-HA-ARL2 is expressed to lower levels than is ARL2 109

and cleavage appears to be complete at every level of expression.

Figure 8. Antigen competition confirms the identity of ARL2 fusion 110

proteins and specificity of the ARL2 antibody.

Figure 9. Different proteins expressed off the same promoters and leader 111

sequences are expressed to different levels and with differing extents

or kinetics of import/processing.

Chapter 4: The ARL2 GTPase regulates mitochondrial fusion from the113

intermembrane space upstream of mitofusins

Abstract 114

Introduction 114

Materials and methods 118

Results 125

Discussion 140

References 147

Figures 162

Figure 1: Activating and inactivating mutants of ARL2 have 162

opposing effects on mitochondrial morphology.

Figure 2: Mitochondrial fusion is impaired in cells expressing 163


Figure 3: ARL2[T30N] acts from the IMS to fragment mitochondria 165

Figure 4: ARL2 or ARL2[Q70L] expression reverses fragmentation 167

in mfn2-/- MEFs.

Figure 5: ARL2[Q70L] expression reverses fragmentation in mfn1-/-169


Figure 6: ARL2 or ARL2[Q70L] expression does not reverse 170

fragmentation in MEFs deleted for both MFNs.

Figure 7: ARL2 or ARL2[Q70L] expression does not reverse 171

fragmentation in MEFs deleted for OPA1.

Figure 8: ARL2 localizes to puncta within mitochondria that display 172

regularity in spacing.

Figure 9: Mitofusins localize to puncta that align with ARL2 puncta. 173

Figure 10: OPA1 shows no clear association with ARL2 puncta. 174

Figure S1: ARL2 is present in a complex with HSP60. 175

Figure S2: SMAC-HA-ARL2 and OCT-HA-ARL2 are imported into 176 mitochondria and cleaved.

Figure S3: SMAC-HA-ARL2 and OCT-HA-ARL2 are targeted to the 177

appropriate compartments.

Chapter 5: Recruitment of the ARL2 GTPase and its GAP, ELMOD2, to178

mitochondria is modulated by the fusogenic activity of mitofusins

Abstract 179

Introduction 179

Materials and methods 181

Results 186

Discussion 202

References 207

Figures 215

Figure 1: Mitochondrial staining of ARL2 increases with days 215

after plating but diminishes at high cell densities.

Figure 2: Mitochondrial staining of ELMOD2 diminishes upon 216

approaching confluence but is unchanged with days after plating.

Figure 3: Mitochondrial staining of ARL2 and ELMOD2 is increased 218

in MEFs deleted for MFN2.

Figure 4: Increased ARL2 and ELMOD2 mitochondrial staining is 221

reversed with expression of MFN2-myc, but not MFN2[K109A]-myc

or MFN1-myc, in mfn1-/-mfn2-/- MEFs.

Figure 5: Increased ARL2 and ELMOD2 mitochondrial staining is 223

reversed with expression of MFN2-myc and MFN2[K109A]-myc

in mfn2-/- MEFs.

Figure 6: Mitochondrial staining of ARL2 increases in cells cultured 224

in medium with no glucose or low serum.

Figure 7: Mitochondrial staining of ELMOD2 increases in cells 225

cultured in medium with no glucose or low serum.

Figure 8: Glucose or serum deprivation increases ARL2 and 226

ELMOD2 staining in wild type, mfn2-/-, or mfn1-/-mfn2-/- MEFs, but not mfn1-/- MEFs.

Figure 9: Mitochondrial staining of ARL2 is increased by treatment 227

with 2-deoxyglucose. COS7 cells were cultured in normal medium

or medium in which the 25 mM glucose was replaced with 25 mM


Figure 10: Mitochondrial staining of ELMOD2 does not change with 228

2-deoxyglucose treatment.

Figure 11: Both ARL2 and HA staining increase in stressed cells 229

expressing ARL2-HA.

Figure 12: Neither ARL2 nor HA staining increases in stressed 230

cells expressing SMAC-HA-ARL2.

Table I. Summary of the effects of energetic stressors on 231

mitochondrial staining of ARL2 or ELMOD2.

Table II. Summary of effects of MFN deletions on ARL2/ELMOD2 232

staining in MEF lines.

Chapter 6: Studies of mitochondrial functions of ARL2233

Abstract 234

Materials and methods 234

Results 238

Discussion 249

References 253

Figures 256

Figure 1. The ARL2/HSP60 complex is unchanged by knockdown 256

of ARL2.

Figure 2. The ARL2/HSP60 complex does not change with 257

metabolic stressors.

Figure 3. Basal cell respiration is increased in HeLa cells depleted 258

of ARL2.

Figure 4. Knockdown of ARL2 causes a loss in cristae density. 259

Figure 5. ARL2 siRNA leads to fewer number and length of cristae 260

per mitochondrion.

Figure 6. Expression of ARL2[T30N] does not affect cristae density. 261

Figure 7. Expression of ARL2[Q70L] does not affect cristae density. 262

Figure 8. Summary of mutations that reverse phenotypes caused by 263

expression of dominant ARL2 mutants.

Figure 9. [L3A] and [F50A] mutations reverse microtubule destruction 264

caused by ARL2[Q70L].

Figure 10. [L3A] and [I6R] do not prevent import of ARL2 into 266


Figure 11: MIROs localize to puncta that align with ARL2 puncta. 267

Chapter 7: Studies of phosphorylation of ARL2269



Materials and methods271





Figure 1. Serine-45, a predicted site of phosphorylation, interfaces with 286


Figure 2. Immunoprecipitation (IP) of ARL2. 287

Figure 3. ARL2 is phosphorylated in cells. 288

Figure 4. Both ARL2 and ARL2[S45A] are phosphorylated in cells. 289

Figure 5. Optimized IP of overexpressed ARL2 for mass spectrometry. 290

Figure 6. Treatment with metabolic inhibitors diminishes 32Pi 291

incorporation into ARL2.

Chapter 8: Discussion292


Figure 1. ARL2 and ELMOD2 are novel regulators of mitochondrial 294


Future directions299

Concluding remarks301


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