Cell death is fundamental in animal development, tissue homeostasis, and disease. Approximately 50 billion cells die by apoptosis every day in an adult human. During apoptosis, mitochondrial permeabilization results in the activation of caspase proteases. Caspases cleave hundreds of protein targets killing the cell and fragmenting it into apoptotic bodies for immune clearance. Cell death research has long suggested that once mitochondrial dysfunction and caspase activation are observed, death is inevitable. However, a growing body of research challenges this long-standing assumption. A variety of normal and cancer cell types can survive transient activation of apoptosis in a process termed anastasis.

I study anastasis using human cancer cell lines, representing some of the most prevalent cancer types, as a model. I first document major phenotypic changes following transient treatment of the apoptosis inducer staurosporine (STS). I observed cancer cells recovering from STS induced mitochondrial collapse and damage. From these observations, I discovered that anastatic cancer cells survive with low mitochondrial membrane potential (ΔΨm) and that low ΔΨm correlated with nuclear health. This dissertation provides a cell biological description of anastasis in cervical cancer HeLa cells and reveals a previously unknown connection between anastasis and metabolic reprogramming.

Collective cell migration is critical for development, wound healing, and tumor metastasis. Moving cells can sense and respond to physical features of the microenvironment; however, in vivo, the significance of tissue topography is mostly unknown. My research focused on Drosophila border cells in the Drosophila ovary, an established model for in vivo cell migration, to study how chemical and physical information influences path selection. Although chemical cues were thought to be sufficient, live imaging, genetics, modeling, and simulations show that microtopography is also important. Chemoattractants promote predominantly posterior movement, whereas tissue architecture presents orthogonal information, a path of least resistance concentrated near the center of the egg chamber. E-cadherin supplies a permissive haptotactic cue. The results provide insight into how cells integrate and prioritize topographical, adhesive, and chemoattractant cues to choose one path among many.An advantage of the border cell model is that it is amenable to large-scale genetic screens. In a screen for mutations that cause border cell migration defects in mosaic clones, the gene Catsup was identified. The Drosophila ortholog of ZIP7 (SLC39A7), Catsup encodes a multifunctional endoplasmic reticulum (ER) transmembrane protein reported to negatively regulate catecholamine biosynthesis, to be required for Notch and EGFR trafficking, to function as a Zn2+ transporter, and to reduce ER stress. However, the relationship between these functions was unclear. Here we report that Catsup knockdown caused abnormal accumulation of Notch and EGFR proteins and induced ER stress in border cells. Ectopic expression of a folding-defective rhodopsin mutant protein, Rh1G69D, also induced ER stress, inhibited Notch transcriptional responses, and blocked border cell migration, even in the absence of abnormal Notch or EGFR accumulation in the ER. Remarkably, simultaneous overexpression of Catsup and Rh1G69D was sufficient to degrade Rh1G69D, resolve ER stress, and rescue border cell migration. Mutant forms of Catsup predicted to disrupt the Zn2+ transport were nonfunctional, indicating a requirement for Zn2+ transport in resolving ER stress. We propose a model for Catsup/ZIP7 function: local ZIP7-mediated Zn2+ transport at the ER/cytosol interface is rate-limiting for Zn2+-binding ubiquitin ligases that promote ER-associated degradation (ERAD). Accumulation of misfolded proteins in the absence of functional ERAD triggers ER stress, which inhibits Notch transcriptional activity independently of trafficking or proteolytic activation. This proposed mechanism may be evolutionarily ancient, accounting for observations in multiple cell types, tissues, and organisms and suggests a novel treatment strategy for retinitis pigmentosa. Cell movement depends heavily on cytoskeletal dynamics. Intermediate filaments are one of the major cytoskeletal networks involved in cell migration. Until recently, intermediate filaments were unknown in Drosophila. In the last chapter, I identified a new isoform of Tropomyosin1: Tm1-X, which has a domain architecture similar to intermediate filament proteins and forms filaments in vitro. In vivo, Tm1-X promotes border cell migration. Together this work provides new insights into the intracellular and extracellular mechanisms regulating cell migration in vivo.

Apoptosis is the process of highly regulated programmed cell death that utilizes caspase-3 to initiate the executioner phase of apoptosis. However, under certain circumstances, cells are able to survive its activation through a process called “anastasis”. Anastasis has been documented to occur in several cell lines and in Drosophila melanogaster. However, the purpose of anastasis and how it is regulated are currently unknown. In Drosophila, anastasis can be visualized using a previously created biosensor, CasExpress, that permanently marks cells that have survived caspase-3 activation with GFP. CasExpress can detect anastasis in Drosophila wing disc during normal development. Since Drosophila wing discs have been used as a model for studying regeneration, it would be beneficial to use this model to investigate if anastasis occurs during tissue regeneration. Using the CasExpress system, we found that anastasis occurs during regeneration in response to reaper-induced tissue damage. To determine if anastasis is required for regeneration, overexpression of reaper in cells that have undergone caspase-3 activation results in a decrease in the number of CasExpress positive cells. We found that the removal of cells that survive caspase-3 activation during tissue ablation does not manifest in a significant change in wing size, possibly due to the incomplete killing of anastatic cells. To start unraveling how anastasis is regulated, CasExpress was utilized in screening the requirement of several genes known to function in wound healing.

Proteotoxic stress drives the progression of numerous degenerative diseases including Type I diabetes, Huntingtin’s Disease, Parkinson’s Disease, and retinitis pigmentosa, a degenerative blinding disease. Cells respond to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated-protein-degradation (ERAD) and proteasomal degradation, but persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. From plants to humans, loss of the zinc transporter ZIP7 causes ER stress; however, the mechanism is unknown. We used Drosophila melanogaster to genetically manipulate ZIP7 and investigate its function in vivo. Here, we show that ZIP7-mediated Zn2+ transport enhances ERAD and that cytosolic Zn2+ is limiting for deubiquitination of client proteins by the Rpn11 Zn2+ metalloproteinase as they enter the proteasome. We then decided to test ZIP7’s effects in a fly model of retinitis pigmentosa. Interestingly, we found ZIP7 overexpression rescues defective vision and photoreceptor degeneration caused by misfolded rhodopsin in Drosophila. We have begun a collaboration looking into whether ZIP7 can prevent or suppress mutant Rhodopsin-mediated neurodegeneration in human retinal organoids. Additionally, these results prompted a screen for potential ZIP7 rescue of other aggregate-prone proteins associated with neurodegenerative diseases. So far, we found that ZIP7 rescues photoreceptor degeneration caused by Aβ42 and Vap33. Further study of ZIP7, a novel component of ERAD, holds significant promise for therapeutic advancements in protein-folding disorders.

Diapause is a protective trait used by many invertebrates to overcome unfavorable environmental conditions, such as cold temperature and starvation. Drosophila melanogaster undergoes a shallow reproductive diapause, characterized by metabolic reprogramming, reduced locomotion, increased stress tolerance, and delayed tissue senescence, ultimately, resulting in an increase in overall organism lifespan. Several pathways implicated in longevity also regulate diapause. This, along with the prevention of normal degeneration seen during this process, offers a unique way in which to study aging and its associated degenerative diseases. Most studies of this complex trait have utilized the pause of ovary development at pre-vitellogenic stages as the main determinant for categorizing diapause inducibility. This approach ignores the complexity of this trait by only relying on reproductive development as the defining characteristic. In this study, the power of the Drosophila Genetic Reference Panel is implemented in order to conduct a genome-wide association study of diapause traits. By quantifying two phenotypes, fecundity and longevity, this tool has led to the identification of a total of 151 genes associated with this trait, and 52 intergenic regions, which may be involved in the regulation of the diapause program. Enrichment analyses suggest the involvement of metabolic pathways, the innate immune system, and GPCR signaling, components of which have also been shown in C. elegans. Further studies should aim to better combine the two mechanisms of quantifying this phenotype, and functionally confirm the involvement of the candidate genes.