The small GTPase ARL2 is a novel regulator of mitochondrial fusion Pubblico
Newman, Laura Elizabeth (2016)
Abstract
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
Mitochondria2
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
treatment.
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
SMAC-HA-GFP.
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
levels.
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
ARL2[T30N].
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
MEFs.
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
2-deoxyglucose.
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
mitochondria.
Figure 11: MIROs localize to puncta that align with ARL2 puncta. 267
Chapter 7: Studies of phosphorylation of ARL2269
Abstract270
Introduction270
Materials and methods271
Results275
Discussion279
References282
Figures286
Figure 1. Serine-45, a predicted site of phosphorylation, interfaces with 286
effectors.
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
Summary293
Figure 1. ARL2 and ELMOD2 are novel regulators of mitochondrial 294
fusion.
Future directions299
Concluding remarks301
References302
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