ARL2 and ELMOD2: Roles in mitochondrial morphology and localization to Rods and Rings Open Access
Schiavon, Cara (Fall 2018)
Abstract
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
ELMOD2 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
IMPDH 10
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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