ARL2 and ELMOD2: Roles in mitochondrial morphology and localization to Rods and Rings Restricted; Files Only

Schiavon, Cara (Fall 2018)

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The Kahn lab studies the role of ARF family GTPases in the regulation of basic cellular processes. One of my projects focused on ELMOD2, and by extent, ARL2 and their role in mitochondrial dynamics. While studying ARL2, ELMOD2, and mitochondria, I came across an unexpected cellular localization of ARL2 and ELMOD2 which we eventually identified as Rods and Rings (RR). As a result, I developed a second project focusing on the role of ARL2, ELMOD2, and RRs. The results of both projects are described herein.

Mitochondria are essential, dynamic organelles that respond to a number of stressors with changes in morphology that are linked to several mitochondrial functions, though the mechanisms involved are poorly understood. Laura Newman and I worked together to develop a set of plasmids for the expression of ARL2 and other proteins in specific compartments of the mitochondria under varying levels of expression. We also documented changes in the staining intensity of endogenous, mitochondrial ARL2 and ELMOD2 under a variety of growth conditions and stressors. Following up on Laura’s work with ARL2 and mitochondria, I determined that ELMOD2 functions in a pathway downstream of ARL2 and upstream of the mitofusins promoting mitochondrial fusion. I also demonstrated evidence of a novel complex at mitochondria composed of ARL2, ELMOD2, the mitofusins, and the miros.

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis, however, the function and regulation of RRs is poorly understood. I have demonstrated that ARL2, a subset of its binding partners, and several resident proteins at the ER, also localize to RRs. I also have identified two new inducers of RR formation, AICAR and glucose deprivation. I demonstrated that RRs can be disassembled if guanine nucleotides can be generated by salvage synthesis regardless of the inducer. Finally, I showed that there is an ordered addition of components as RRs mature, with IMPDH first forming aggregates, followed by ARL2, and only later calnexin, a marker of the ER. 

Table of Contents

Chapter 1: Introduction 1

ARL2 and ELMOD2 2

ARL2 and mitochondria 3

Figure 1. MEFs lacking MFN1 or 2 have fragmented mitochondria 3

ARL2 and disease 4


Figure 2. The canonical GTPase cycle 5

Mitochondrial dynamics 6

Figure 3. Examples of variation in mitochondrial morphology 6

Figure 4. Simplified schematic of mitochondrial fusion 7

Regulation of mitochondrial dynamics 8

Mitochondria and disease 8

Rods and Rings 9

Figure 5. RR localization, size, and shape variability 9


Figure 6. IMPDH catalyzes the rate-limiting step in de novo guanine nucleotide synthesis 10

Inducers of RRs 11

Figure 7. MPA robustly triggers the formation of RRs 11

Cytoophidia 12

References 12

Chapter 2: Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space 25

Abstract 26

Introduction 26

Methods 28

Results and Discussion 33

Acknowledgements 40

References 40

Figure 1. Schematic of constructs 46

Figure 2. Neither OCT-HA-GFP nor SMAC-HA-GFP alter mitochondrial morphology 47

 Figure 3. SMAC-HA-ARL2 and OCT-HA-ARL2 are correctly localized to the IMS or matrix, respectively 48

Figure 4. CCCP treatment prevents the import and cleavage of SMAC-HA-GFP 49

Figure 5. Comparing OCT-HA-GFP and SMAC-HA-GFP expressed with decreasing strength CMV promoters 50

Figure 6. SMAC-HA-ARL2 is expressed to lower levels than is ARL2 and cleavage appears to be complete at every level of expression 51

Figure 7. Antigen competition confirms the identity of ARL2 fusion proteins and specificity of the ARL2 antibody 52

Figure 8. Different proteins expressed off the same promoters and leader sequences are expressed to different levels and with differing extents or kinetics of import/processing 53

Chapter 3: The abundance of the ARL2 GTPase and its GAP, ELMOD2, at mitochondria are modulated by the fusogenic activity of the mitofusins and stressors 55

Abstract 56

Introduction 56

Methods 58

Results 65

Discussion 79

Acknowledgements 84

References 84

Figure 1. Mitochondrial staining of ARL2 and ELMOD2 vary in intensity with cell density and days after plating 95

Figure 2. Mitochondrial ARL2 and ELMOD2 are increased in mfn2-/- and mfn1-/-mfn2-/- MEFs 97

Figure 3. Elevated mitochondrial ARL2 and ELMOD2 is reversed in mfn1-/-mfn2-/- MEFs with restoration of fusion by MFN2 99

Figure 4. Elevated mitochondrial ARL2 and ELMOD2 are reversed with expression of MFN2-myc or MFN2[K109A]-myc in mfn2-/- MEFs 100

Figure 5. Expression of MFN1-myc or MFN2-myc does not affect ARL2 or ELMOD2 staining intensity inwild type MEFs 101

Figure 6. Mitochondrial ARL2 and ELMOD2 increase in cells cultured in 0 glucose or 2% serum 102

Figure 7. ARL3 staining is unchanged with MFN2 deletion, glucose starvation, or serum starvation 103

Figure 8. Glucose or serum deprivation increases mitochondrial ARL2 in wild type, mfn2-/-, or mfn1-/-mfn2-/-, but not mfn1-/- MEFs 104

Figure 9. Mitochondrial ARL2, but not ELMOD2, is increased by 2-deoxyglucose 105

Figure 10. ARL2 and HA staining increase in stressed cells expressing ARL2-HA, but not in cells expressing SMAC-HA-ARL2 106

Table I. Summary of effects of MFN deletions on ARL2/ELMOD2 staining in MEF lines 108

Table II. Summary of the effects of energetic stressors on mitochondrial staining of ARL2 or ELMOD2 109

Chapter 4: ELMOD2 regulates mitochondrial fusion in a mitofusin-dependent manner downstream of ARL2 110

Abstract 111

Introduction 111

Methods 113

Results 119

Discussion 131

Acknowledgements 135

References 136

Figure 1. Knock-out of ELMOD2 causes mitochondrial fragmentation 141

Figure 2. Loss of ELMOD2 decreases mitochondrial fusion 142

Figure 3. Expression of ELMOD2 or ELMOD2[R167K] partially reverses mitochondrial fragmentation in MFN2-null MEFs 143

Figure 4. Expression of ELMOD2 or ELMOD2[R167K] partially reverses mitochondrial fragmentation in MFN1-null MEFs 144

Figure 5. Expression of ELMOD2 or ELMOD2[R167K] does not affect the morphology of DKO, OPA1-null, or WT MEFs 145

Figure 6. ARL2[Q70L] does not promote mitochondrial elongation in the absence of ELMOD2 146

Figure 7. Expression of ELMOD2 or ELMOD2[R167K] increases mitochondrial fusion in MFN2-null MEFs 147

Figure 8. Mitochondrial ELMOD2 and ARL2 staining show the same periodic, punctate staining pattern when imaged by gSTED 148

Figure 9. ELMOD2 puncta have a similar staining pattern when compared to MFN1-myc and MFN2-myc puncta at mitochondria 149

Figure 10. ELMOD2, myc-Miro1, and myc-Miro2 have similar punctate staining patterns at mitochondria 150

Figure S1. Mouse ELMOD2 expresses to higher levels than human ELMOD2 151

Figure S2. Lentiviral expression of ELMOD2 also partially reverses mitochondrial fragmentation in MFN1-null and MFN2-null MEFs 152

Figure S3. The periodicity of ELMOD2 and ARL2 staining at mitochondria is consistent 153

Figure S4. ELMOD2 and ARL2 puncta are less defined but still visible by confocal microscopy 154

Figure S5. Cytochrome c and HSP60 do not share the same staining pattern as ELMOD2 155

Figure S6. TOM20 and mitoPLD-GFP do not share the same staining pattern as ELMOD2 156

Figure S7. ARL2 puncta have a similar staining pattern when compared to MFN1-myc and MFN2-myc puncta at mitochondria 157

Figure S8. There is no correlation between ELMOD2 and several resident mitochondrial proteins 158

Figure S9. There is a moderate positive correlation between ELMOD2, ARL2, the mitofusins, and the miros 159

Chapter 5: Compositional complexity of rods and rings 160

Abstract 161

Introduction 161

Methods 164

Results 178

Discussion 180

Acknowledgements 185

References 185

Figure 1. ARL2 localizes to IMPDH2-positive structures that are inducible with MPA 197

Figure 2. AICAR induces RR formation 198

Figure 3. AICAR is capable of inducing RR formation in AMPK-null MEFs 199

Figure 4. Glucose starvation increases the percentage of cells with RRs 200

Figure 5. Guanosine does not prevent RR formation in LND fibroblasts 201

Figure 6. ARL2 does not localize to CTPS1-positive structures which are induced with DON but does co-localize with IMPDH2-positive RRs 202

Figure 7. A subset of ARL2 binding partners localize to RRs 203

Figure 8. Three different ER membrane proteins also co-localize with RRs 204

Figure 9. The filamentous nature of RRs is evident by EM staining and ARL2 and IMPDH2 co-localize to RRs as seen by immunogold staining 205

Figure 10. RRs increase in size and quantity over time after induction with MPA 206

Figure 11. Calnexin recruits to RRs well after IMPDH2 and ARL2 207

Table I. Summary of results from multiple different cell lines and their responses to different inducers of RRs, reversal by guanosine, and co-localization of different antigens with IMPDH2 or ARL2 208

Figure S1. ARL2 localizes to nuclear RRs 209

Figure S2. IMPDH2 and ARL2 staining at RRs is lost with antigen competition 210

Figure S3. ARL2 monoclonal and polyclonal antibodies display different staining intensities for RRs and other organelles 211

Figure S4. Guanosine prevents MPA-induced RR formation 212

Figure S5. Guanosine reverses RR formation induced by AICAR or glucose-starvation 213

Figure S6. ARL3, ELMOD1, and Cofactor E do not co-localize with ARL2 at RRs 214

Figure S7. mCherry-Sec61β co-localizes with RRs 215

Figure S8. Calnexin staining at RRs is more apparent after glucose starvation than MPA-treatment 216

Figure S9. Calreticulin, TGN46, and α-tubulin do not co-localize with ARL2 or IMPDH2 at RRs 217

Chapter 6: Discussion 218

Summary 219

Mitochondria 219

Rods and Rings 221

Future Directions 223

Mitochondria 223

Rods and Rings 224

Concluding remarks 226

Figure 1. Currently established cellular locations of ARL2 and ELMOD2 228

References 229

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