The ELMOD Family of ARF GAPs: Proposed Roles in Inter-pathway Communication 公开
Turn, Rachel (Spring 2021)
Abstract
The ARF family of small GTPases are regulators of diverse cellular pathways. Because of the
numerous functions that a single GTPase can mediate (e.g vesicular traffic, cytoskeletal dynamics, ciliary
function), we propose that the ARFs are drivers of inter-pathway communication, or “higher order
signaling.” The mechanisms by which ARF GTPases would regulate higher order signaling, though, remain
unclear. The focus of my dissertation work has been on the ELMODs, a 3-member family of ARF GAPs
that regulate the on/off state of ARF GTPases. I have discovered new, diverse functions for the ELMODs
in cells, lending to our model that the regulators of the GTPases themselves may also drive inter-pathway
communication to mediate essential cell functions.
The majority of my dissertation work has been with one of the family members, ELMOD2.
Previous lab members and I worked to probe for functions of ELMOD2 in mitochondrial fusion. I generated
ELMOD2 KO cells using CRISPR-Cas9 to probe for mechanism. This model system served as a
springboard for my own project, as I discovered that the loss of ELMOD2 led to the disruption of a number
of cellular processes. As detailed in my first-author manuscript, I discovered numerous phenotypes
consistent with defects in microtubules and cell cycle. I discovered that ELMOD2 works in two additional
pathways: with ARL2 to regulate microtubule anchoring at centrosomes, and with ARF6 to regulate
cytokinesis from the Flemming body. Another large portion of my dissertation work has been exploring
functions for ELMDO2 in yet another pathway, acting in concert with ciliary rootlets and ARL2 to inhibit
spurious ciliogenesis. Altogether, these findings shed light on the mechanisms by which ARFs and their
GAPs regulate cell signaling and pave the way for understanding how such discrete cell functions as
microtubule anchoring, ciliogenesis, and cytokinesis coordinate to ensure cell survival. My findings will
pave the way for future study of other ELMOD family members as well as ARF GTPases, with the ultimate
goal of understanding how the vast network of signaling pathways communicate with one another.
Table of Contents
Table of Contents
Chapter 1: Introduction 1
ARF GTPases and their regulators 2
Figure 1: ARF GTPases are molecular switches that provide spatial and temporal control of signaling 4
ARF GTPases and their regulators drive diverse signaling pathways 5
Figure 2: ARF GTPases and their regulators coordinate diverse cellular compartments 8
ARF GAPs 9
Figure 3: Summary of known ARF GAP specificities, localizations, and functions 11
ELMOD family of ARF GAPs 12
Figure 4: Summary of the mammalian ELMOD family of 6 ELMO-domain containing family members 14
ELMOD2 15
References 17
Chapter 2: The ARF GAP ELMOD2 acts with different GTPases to regulate centrosomal microtubule nucleation and cytokinesis 22
Abstract 23
Introduction 23
Materials and methods 28
Results 38
Discussion 63
References 73
Figures 89
Figure 1. Loss of ELMOD2 leads to decreased microtubule stability. 89
Figure 2. ELMOD2 nulls are slow to recruit γ-TuRC, ARL2, and TBCD to centrosomes during recovery from cold. 91
Figure 3. Deletion of ELMOD2 causes multinucleation, supernumerary centrosomes, and polyploidy. 93
Figure 4. ELMOD2 null cells display a prolonged cytokinesis and both early and late cytokinesis defects. 95
Figure 5. ARF6, RAB11, and FIP3 are specifically altered in localization in ELMOD2 null cells. 97
Figure 6. ELMOD2 KO cells display decreased recruitment of FIP3-GFP, along with loss of RAB11 and increases in ARF6 at recycling endosome clusters. 99
Figure 7. ELMOD2 nulls have higher mitotic index at high densities and increased anchorage independent growth. 102
Figure 8. Model of ELMOD2’s role in microtubules and cytokinesis. 104
Figure S1. Summary of ELMOD2 alleles in 10 KO MEF lines. 106
Figure S2. Lipid droplet sizes and abundance, with or without oleic acid treatment, are unchanged in ELMOD2 null cells 107
Figure S3. Flemming body markers FIP3, RAB11, and ARL2 are unchanged in ELMOD2 null cells. 108
Figure S4. Binning cells into loss or no loss of MT network. 109
Figure S5. Measuring the diameter of asters. 110
Figure S6. Scoring microtubules at asters during recovery from cold (4°C). 111
Figure S7. Workflow for scoring mitotic indices. 112
Chapter 3: Roles for ELMOD2 and Rootletin in Ciliogenesis 114
Abstract 115
Introduction 115
Materials and methods 120
Results and discussion 129
Discussion 153
References 168
Figures 189
Figure 1. Deletion of ELMOD2 causes ciliation defects. 189
Figure 2. Ciliary signaling is disrupted in ELMOD2 KO lines. 191
Figure 3. ELMOD2 localizes to rootlets and its deletion causes rootlet defects. 193
Figure 4. ELMOD2 localizes to the base of the connecting cilium of both human and mouse retinal epithelium. 195
Figure 5. Rootletin KO lines phenocopy ELMOD2 null ciliary and centrosomal cohesion defects. 196
Figure 6. ELMOD2-myc and ELMOD2[R167K]-myc rescue ciliation and centrosomal cohesion defects in ELMOD2 KO but not rootletin KO cells. 198
Figure 7. ARL2 and ARL2[V160A] reverse increased ciliation, rootlet fragmentation, and increased centrosome separation defects in ELMOD2 and rootletin KO cells. 199
Figure 8. ARL2 localizes to ciliary rootlets in WT MEFs and human/mouse photoreceptor cells. 200
Figure 9. ELMOD2 KO causes misregulation of markers of different steps in the ciliogenesis process. 201
Figure 10. ELMOD2, ARL2, and rootletin work together to prevent spurious ciliogenesis. 203
Figure S1. ELMOD2 localizes to cilia in WT MEFs upon ciliobrevin treatment. 205
Figure S2. Summary of Crocc frame shifting alleles in KO and CroccΔ239 MEFs. 206
Figure S3. Western blot for rootletin in WT, Rootletin KO, ELMOD2 KO, and CroccΔ239 MEFs. 208
Figure S4. ELMOD2 still localizes to mitochondria, Flemming bodies, and centrosomes in rootletin KO cells. 209
Figure S5. ELMOD2 does not localize to non-centrosomal rootlets. 210
Figure S6. Cep44 localization to centrosomes is unaltered in ELMOD2 KO or rootletin KO cells. 211
Figure S7. ARL3 localizes to cilia and centrosomes but not rootlets in WT MEFs. 212
Figure S8. ARL2 localizes along the length of cilia in human (multiciliated) bronchial epithelial cells,
while ELMOD2 localizes to the tips of cilia and rootlets in human bronchial cells. 213
Figure S9. ELMOD2 KO does not alter the localization of IFT or transition zone (TZ) markers. 215
Figure S10. Centrin and ARL2 localize to rootletin KO cilia. 216
Figure S11. Live cell imaging of GFP-rootletin transfected WT MEFs reveal that serum starvation induces rootlet tendrils to fall off. 217
Chapter 4: Discussion 218
Summary 219
Figure 1. Summary of contributions to the field 225
Future directions 226
Concluding remarks 232
References 233
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