Ivan Robert Nabi

Caveolin-1 (Cav1) is a membrane protein that plays important structural and signalling roles in several mammalian cell types. While best known for the formation of flask-shaped membrane structures known as caveolae, which have been implicated in endocytic, mechanoprotective, and signalling roles, Cav1 also readily oligomerizes within lipid rafts to form Cav1 domains known as scaffolds. Disrupting Cav1 expression promotes tumour survival and migration, and Cav1 knockout animals suffer from pulmonary and cardiovascular conditions. While Cav1 is well-known to interact with multiple cell signalling partners, notably its inhibition of endothelial nitric oxide synthase (eNOS), it remains unclear if this interaction is caveolae- or scaffold-dependent, of if it involves the caveolin scaffolding domain or the tyrosine-14 residue of Cav1.Here, we used confocal and single molecule localization microscopy (SMLM) network analysis of Cav1 and eNOS localization in a transiently transfected Cav1 knockout MDA-MB-231 cell line, as well as whole-mount labeling of Cav1 and eNOS in mouse pulmonary arteries to study Cav1 interaction with eNOS. We found that phosphomimetic Cav1-Y14D mutant caveolae and scaffolds had similar features to caveolin scaffolding domain (CSD) mutants, while phosphorylation-deficient Cav1-Y14F mutant Cav1 structures closely resembled Cav1-WT blobs. Using binary mask analyses of Cav1 and eNOS, we identified that eNOS interacts with caveolae and scaffolds approximately equally, and that CSD mutant and Y14D mutant Cav1 had a slight but non-significant increase in Cav1-eNOS co-occurrence across all Cav1 domains.Additionally, to investigate changes in Cav1 and eNOS localization and behaviour with age, we used confocal microscopy to image Cav1, eNOS, and the Golgi marker GM130 in the pulmonary artery endothelium young and old mice. We observed that eNOS expression decreases with age (p
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Selective autophagic degradation of the mitochondria, or mitophagy, is a quality control mechanism through which mitochondria that are dysfunctional or no longer required are degraded. Dysregulation of mitophagy has been associated with oncogenesis and neurodegenerative diseases. Gp78 is an E3 ubiquitin ligase involved in ER-associated degradation and previously implicated in damage-induced mitophagy. Here we demonstrate that CRISPR Cas9 KO of Gp78 results in impaired basal and damage-induced mitophagy using both mtKeima and LC3-GFP-RFP assays which report on mitochondrial delivery to autolysosomes and autophagic flux, respectively. We demonstrate that impaired autophagic flux results in decreased mitochondrial potential and the accumulation of mitochondrial derived superoxide. Induction of mitochondrial fission through the overexpression of Drp1 fails to rescue decreased membrane potential in Gp78 KO cells suggesting that Gp78 regulation of mitophagy extends beyond the induction of mitochondrial fission. In our HT-1080 cell model we demonstrate that autophagosomes selectively localize to mitochondria exhibiting low membrane potential and that the regulation of membrane potential by Gp78 extends to additional cell lines other than our HT-1080 model. Mitochondria-endoplasmic reticulum (ER) contacts (MERCs) are regions of close apposition between the ER and mitochondria. These sites act as signaling hubs regulating lipid transfer, calcium signaling and mitochondrial dynamics. These structures are classified based on the type of ER (rough/smooth) associating with the mitochondria forming ribo-MERCs and smooth MERCs, respectively. Dysregulation of MERCs has been demonstrated in neurodegenerative diseases, cancer, and metabolic disorders. Here we demonstrate that COS7 and HT-1080 preferentially form smooth MERCs and rough MERCs, respectively. We have developed a novel Laplacian-based algorithm to detect MERCs in 3D super-resolution microscopy data capable of detecting endogenous and induced MERCs. We demonstrate that Gp78 and RRBP1 selectively regulate ribo-MERCs independent of one another. MERC regulation through the expression of Gp78 is dependent on GP78’s E3 ligase activity as RING-mut Gp78 failed to induce ribo-MERCs. MERCs generated through Gp78 expression exhibit convoluted tubular morphologies distinct from planar MERCs induced through artificial tether expression.

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Cancer cells often exhibit altered metabolisms that support aerobic glycolysis and reduced mitochondrial function to promote cell survival. Caveolin-1 (Cav1) is an integral membrane protein and the main structural component of caveolae. Caveolae formation requires the adaptor protein Cavin1 and without it, Cav1 forms scaffolds at the plasma membrane. Cav1 can be phosphorylated at tyrosine-14 (pCav1) and is involved in signal transduction, cell migration, and mitochondrial function. Cav1 plays contrasting roles in cancer progression and its precise role in metabolism and, specifically, mitochondrial function remains poorly defined. Using a potential- dependent mitochondrial reporter, MitoView 633, and a mitochondrial reactive oxygen species (ROS) reporter, MitoSOX Red, we report that CRISPR/Cas9 knockout of Cav1 in MDA-MB- 231 triple-negative breast cancer cells increases mitochondrial potential and reduces ROS. These effects are reversed upon reintroduction of Cav1 by stably expressing Cav1 in KO cells. Further, introduction of the Cav1 Y14D phosphomimetic mutant decreased mitochondrial potential in Cav1 KO cells while the Y14F non-phosphorylatable mutant did not. pCav1 is a known effector of Rho-associated kinase (ROCK) signaling. Treatment of MDA-MB-231 cells with Y-27632, a ROCK1/2 inhibitor, or with ROCK1/2 siRNA increased mitochondrial potential and decreased ROS in Cav1-expressing cells. Similar results were observed in PC3 prostate cancer cells that lack caveolae suggesting this is a caveolae-independent effect. Use of the mito-Keima mitophagy probe reported increased basal mitophagy in Cav1 KO MDA-MB-231 and PC3 cells and induction of mitophagy by ROCK inhibition in Cav1-expressing cells. Knockdown of the autophagy protein ATG5 and mitophagy regulator PINK1 in Cav1 KO MDA-MB-231 cells reduced potential and increased ROS while Cav1-expressing cells were not significantly affected, demonstrating that mitophagy promotes mitochondrial health and reduces ROS in Cav1 KO cells. Finally, we show that Cav1 KO MDA-MB-231 cells have more active AMP-activated protein kinase (AMPK), a major mitochondrial homeostasis protein whose activity is inhibited by ROCK. Knockdown of AMPK resulted in decreased mitochondrial potential, increased ROS, and decreased mitophagy in Cav1 KO cells while the Cav1-expressing cells were not affected. Overall, our results suggest that Cav1 expression in cancer cells reduces mitochondrial potential and increases ROS via ROCK/AMPK regulated mitophagy.

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Caveolin-1 (CAV1) is an integral membrane protein with previous roles defined in regulating both cancer cell migration and metabolism. Aberrant metabolism and migration are two of the many pathways that are associated with tumorigenesis and function to promote tumor cell growth, survival, and metastasis. Importantly, cell migration is energy-dependent and as a result, cancer metabolism and migration are closely linked. Using wildtype and CAV1 KO clonal cell lines, we show support that CAV1 regulates metabolism in MDA-MB-231 triple-negative breast cancer cells, a subtype of breast cancer that overexpresses CAV1. Compared to CAV1 KO cells, we found that wildtype cells expressing CAV1 displayed higher glycolytic activity and decreased mitochondrial respiration, correlating with a decrease in mitochondria abundance and exclusion of mitochondria from the cell periphery. Using metabolic inhibitors to target glycolysis and mitochondrial respiration, we next looked at the association of these metabolic pathways with actin remodeling and focal adhesion formation, which are closely associated with cell migration. Inhibition of glycolysis but not inhibition of mitochondrial respiration induced loss of F-actin, cell spreading, and significant cell death in both wildtype and CAV1 KO cells. Compared to CAV1 KO cells, wildtype cells expressing CAV1 displayed increased sensitivity to inhibition of glycolysis demonstrated by a more drastic loss of F-actin and increased cell spreading. Overall, these results suggest a role for CAV1 in promoting glycolytic metabolism in MDA-MB-231 cells. Additionally, it is shown here that MDA-MB-231 cells depend primarily on glycolysis and not mitochondrial respiration to maintain cell shape and cell survival and that CAV1 may be implicated in this dynamic.

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Mitochondrial degradation through the lysosomal delivery pathway, otherwise known as mitophagy, is a strongly conserved evolutionary process that cells and tissues use to adapt to various cellular environments. Defects in mitophagy have been linked to neurodegenerative pathologies, aging phenotypes, as well as cancer progression. Here, we show that CRISPR/Cas9 knockout (KO) of Gp78 in the HT-1080 cell line results in an approximately two-fold increase in mitochondrial volume. Knockdown of the essential autophagy gene ATG5 in the wildtype HT-1080 cells showed similarly increased levels of mitochondrial mass as present in the Gp78 KO clones. Therefore, the increased mitochondrial mass exhibited by the Gp78 KO clones is because of its reduced capacity for autophagy upon loss of Gp78. The increase of mitochondria phenotype upon loss of Gp78 was recapitulated in vivo, where xenograft tumors in Rag2 immunodeficient mice showed increased mitochondrial staining in tumor sections for all Gp78 knockout clones relative to the parental HT-1080 cells. To directly assess Gp78-mediation of basal mitophagy, we developed a sensitive assay using the HT-1080 and Gp78 KO cells stably transfected with the GFP-mRFP-LC3 (tfLC3) autophagy reporter and assessed mitophagic flux upon the chemical inhibition of autophagy with bafilomycin A1. We applied SPECHT, a novel Laplacian-based image analysis algorithm, to quantify tfLC3 puncta association with mitochondria, and found that loss of Gp78 results in impaired autophagic and mitophagic flux. We also show that loss of Gp78 results in the accumulation of defective mitochondria and increased levels of reactive oxygen species (ROS) that are closely associated with mitochondria. Collectively, the data presented provide evidence that Gp78 mediates basal mitophagy, and that impaired basal mitophagy due to loss of Gp78 results in increased ROS and the accumulation of defective mitochondria.

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The Zika virus (ZIKV) is a positive-sense RNA flavivirus that has been determined as a causative agent of severe neurological diseases including Guillain-Barré syndrome and microcephaly. A common characteristic of flavivirus infection is the re-organization of host cell membranes, usually endoplasmic reticulum (ER) derived, to promote viral replication. As ZIKV-induced ER structures, being vesicular replication factories (RF) and convoluted membranes (CM), are smaller than the diffraction limit of light, studies to characterize these structures and their protein compositions have been limited. In this thesis, Stimulated Emission Depletion (STED) super resolution microscopy is utilized to study the whole cell organization of ZIKV-induced ER morphologies and several viral proteins involved in replication.Generally in cells, the ER can be divided into two regions, central ER sheets and peripheral ER tubules. Here we report the formation of an ZIKV-induced, organized crescent-shaped, dense central ER region of ER tubules. ZIKV RFs localize to this dense, central ER region in a similar crescent-shaped organization. Computational 3D reconstruction of these 3D STED imaged ZIKV-induced ER structures, along with validation by electron microscopy of ZIKV infected cerebral organoids, revealed that these dense ER regions are composed of novel smooth ER tubular matrices. Moreover, we have found that ZIKV NS4B, a viral encoded integral membrane protein predicted to play a role in ER membrane re-organization during ZIKV replication, is enriched in ZIKV-induced tubular matrices. Additionally, a subpopulation of ZIKV NS4B localizes to active RFs suggesting that NS4B plays a role in the formation of both ZIKV-induced tubular matrices and RFs among other functions. Overall, the application of super resolution microscopy to study ZIKV replication has led to the identification of novel ZIKV-induced, RF-containing tubular matrices and characterization of a viral protein involved in this process.

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Mitophagy is an evolutionarily conserved process by which cells remove damaged mitochondria. Defects in mitophagy lead to mitochondrial dysfunction and are associated with pathological states such as cancer and neurodegenerative disorders. PINK1 protein accumulates in damaged mitochondria, recruits the Parkin ubiquitin ligase and induces mitophagy. We showed in the past that Gp78, an endoplasmic reticulum (ER) membrane E3 ligase localizes to ER-mitochondria contact sites. We also showed that Gp78 induces mitophagy independently of Parkin which is dependent on the presence of an intact Gp78 RING finger domain. We now show that Gp78 mitophagy of damaged mitochondria, induced by the mitochondrial poison CCCP, also requires PINK1. Further, the Gp78 cytoplasmic domain also contains a ubiquitin-binding Coupling of Ubiquitin to ER degradation (CUE) domain. Unlike wild-type Gp78 that induces mitophagy only upon mitochondrial damage, Gp78 with CUE-domain mutation constitutively performs mitophagy regardless of mitochondrial damage and independently of PINK1. This suggests that PINK1 acts upon the CUE domain of Gp78 to promote its mitophagy. The USP13 deubiquitinase has been shown to bind to Gp78 and by co-immunoprecipitation analysis, we found that the USP13-Gp78 interaction is CUE domain-dependent. USP13 knock-down (KD) enhances Gp78 ubiquitination of mitochondria, and like the CUE-mutant Gp78, promotes mitophagy even without induction of mitochondrial damage. We then tested the interdependence of USP13 and PINK1 for Gp78-dependent mitophagy. While PINK1 siRNA KD alone prevents Gp78-mediated mitophagy of damaged mitochondria, knockdown of both PINK1 (siRNA) and USP13 (shRNA), restores the Gp78-dependent mitophagy. This suggests that USP13 inhibition of Gp78-mediated mitophagy is relieved upon mitochondrial damage by PINK1.

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Caveolae, a special type of lipid-raft, are cave-like invaginations of plasma-membrane maintained and formed by Caveolins (Cav1, 2 and 3) and Cavin 1 and 2. Caveolae regulate various trafficking and signaling pathways. Cav1, a 178 amino acid protein has a Src-dependent tyrosine-14 phosphorylation site that regulates integrin signaling and focal adhesion dynamics, and a Caveolin Scaffolding Domain (CSD) that physically interacts with multiple proteins. Glutathione-S-transferase (GST) pull-downs and quantitative proteomics analysis (Maxquant) were performed using lysates from the DU145 prostate cancer cell line and GST-beads tagged with the N-terminal polypeptide of Cav1 (amino acids 1-101) incorporating phosphomimeitic (Y14D) and non-phosphorylatable (Y14F) mutations. Proteomic analysis showed 1.5 fold increased interaction of 196 and 78 proteins with Cav1(1-101)Y14D and Cav1(1-101)Y14F, respectively. Gene Ontology (GO) analysis revealed that Cav1(1-101)Y14D interacted more with proteins that regulate cell stress, proliferation, signal transduction, metabolic processes, apoptosis (Heat shock protein-90 (HSP90)) and focal adhesions, whereas Cav1(1-101)Y14F interacted more with proteins that regulate actin cytoskeleton and RNA processing. Pseudopod-enriched proteome list from DU145 cells revealed 84 proteins overlapping with the Cav1(1-101)Y14D interactome. Comparative proteomics analysis of the CSD mutants (F92A/V94A) suggested that one-third binding proteins of Cav1(1-101)Y14D and Cav1(1-101)Y14F were influenced by this mutation. Binding specificity of Y14 phosphorylation on Cav1 is partially affected by these CSD mutations. Pseudopod enriched HSP90 was one of the top hits in the Cav1(1-101)Y14D interactome. Inhibition of HSP90 with 17-N-allylamino-17-demethoxygeldanamycin (17AAG) increased expression of Protein Kinase B (also known as AKT) and Cav1 in pCav1 (Y14 phosphorylatedCav1) expressing DU145 and PC3 prostate cancer cell lines. However, there was no effect of HSP90 inhibition on pCav1 lacking DU145 and Cav1 knocked-down PC3 cells. Reduced cell migration and viability was observed after 17AAG treatment of DU145 (stably expressing various Cav1 mutants) and PC3 in pCav1 dependent manner. This suggests the HSP90 function in regulating cell migration rely on pCav1. This study reveals that Y14 phosphorylation impacts Cav1 interaction with different proteins and is partially affected by CSD mutants in our study. Also, pCav1 specific interaction with HSP90 decreases prostate cancer cell migration.

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A ligand-receptor pair, autocrine motility factor (AMF) and Gp78, have been discovered to play multiple roles in mammalian cells. AMF functions as the essential glycolytic enzyme phosphoglucose isomerase in the cytoplasm, but when secreted acts a cytokine that stimulates cell motility, growth and survival. Gp78 serves as the cell surface receptor of AMF, and thus it is also known as AMFR. However, Gp78 can localize to the ER membrane and functions as an E3 ubiquitin ligase in the endoplasmic reticulum associated degradation (ERAD) pathway where it targets a wide variety of proteins for degradation. The concerted actions of AMF and Gp78 contribute to multiple aspects of cancer progression, and thus elevated levels of both proteins have been found in many types of cancers. Recently, it was discovered that AMF and Gp78 alter mitochondrial morphology and ER-mitochondria calcium coupling, processes that are essential in regulating mitochondrial metabolism and apoptosis. Furthermore, Gp78 has also been localized to ER-mitochondria contact sites where it targets the mitochondrial fusion proteins, mitofusin 1 and 2 (Mfn1/2), for degradation. In this dissertation, I show that during Gp78 induced mitophagy, autophagosome marker LC3 is recruited to mitochondria associated ER membrane. Moreover, I show that Gp78-dependent degradation of the mitofusins leads to diminished mitochondrial fusion and a perturbation of mitochondrial dynamics. I also report the ability of AMF to inhibit Gp78-induced mitochondrial fission. In my study of ER-mitochondrial association, I observed two types of ER-mitochondria contacts in HT-1080 fibrosarcoma cells: the rough and the smooth. Gp78 ubiquitin ligase activity selectively promotes rough ER-mitochondria association through the degradation of Mfn2. AMF treatment inhibits Gp78-dependent Mfn2 degradation and decreases rough ER-mitochondria contact sites. By dissecting the functions of AMF and Gp78 at the ER mitochondria contact sites, my thesis not only expands our understanding of the relationship between AMF and Gp78, it also provides novel insights into the intimate connection between the ER and mitochondria.

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