Necrosis, originally considered chaotic, has been found to be regulated by distinct molecular pathways. To identify novel regulators of plasma membrane rupture, a hallmark of necrotic cell death, a genome-wide shRNA loss-of-function screen was performed. We identified SNARE complex members, mediators of canonical membrane fusion events, as necessary for Ca2+ and ROS-induced plasma membrane rupture andnecrosis. We targeted N-ethylmaleimide sensitive factor (NSF) due to its requirement in SNARE recycling, lack of gene homologs, and redox sensitivity. Deletion of Nsf protected against membrane rupture induced by various necrotic stimuli, yet did not influence apoptosis, suggesting specificity in programmed necrosis. We discovered that NSF localizes to sites of membrane blebbing and rupture. Additionally, SNARE-binding, ATPase activity, and redox modification are necessary for NSF’s role in necrosis. We generated conditional Nsf knockout mice and induced skeletal muscle injury via cardiotoxin injection. Loss of NSF protected against sarcolemmal rupture and myocyte death. Complementarily, cardiomyocyte-specific deletion of Nsf protected against ischemia-reperfusion injury. The SNARE complex regulator, NSF, is a key mediator of membrane rupture in necrotic cell death and a promising therapeutic target for numerous diseases. / Biomedical Sciences

No description available.

No description available.

In mammalian cells, the formation of a bipolar spindle is an essential process as any mistake in the segregation of chromosomes can result in aneuploidy, an outcome that can be detrimental to the organism. Microtubules are the key structures required for the success of this operation, as they are the main constituents of mammalian bipolar spindles. Centrosomes also play an important role, being the primary source of microtubules. Although centrosomes are dispensable for the formation of bipolar spindles, the fidelity of this process decreases when they are experimentally removed. One way to explain how a bipolar spindle assembles has been the ‘search and capture’ model. According to this model, microtubules emanating from centrosomes search the cytoplasm for kinetochores, which capture microtubules laterally. Once captured, the chromosomes move towards the spindle equator, ultimately resulting in a bipolar spindle. In the research presented here, our aim is to understand the role of microtubule sources other than centrosomes in centrosome-containing mammalian cells. We use different techniques and manipulations to bypass the presence of the centrosomes in order to identify the origin of these sources and their importance. We mainly use LLCPK1, pig kidney epithelial, cells stably expressing GFP tagged proteins, such as alpha-tubulin, to follow microtubules and their associated proteins in live cells. Two main sources of microtubules other than centrosomes have been documented: peripheral microtubules and kinetochores. Peripheral, non-centrosome-associated microtubules were originally thought to disassemble at the beginning of mitosis. However, we found that they form bundles and contribute to the forming spindle. Our model for spindle assembly, which incorporates these peripheral microtubules, and the ‘search and capture’ model are not mutually exclusive; instead, they compliment each other in the formation of bipolar spindles. Lastly, we develop a spindle assembly assay in which centrosomes and kinetochores can be observed separately in mammalian cells. The data documented here demonstrate that kinetochores also contribute to the formation of bipolar spindles by nucleating and organizing microtubules. In addition, TPX2, a microtubule associated protein, is revealed as one of the requirements for microtubule formation and organization at the kinetochores.

The zebrafish protein Mosaic eyes (Moe), and the mammalian orthologue Erythrocyte protein band 4.1-like 5 (Epb4.1l5), are FERM (for Protein 4.1, Ezrin, Radixin, Moesin) domain containing proteins that have important roles during embryonic development. Zebrafish with loss-of-function mutations in moe exhibit defects in retinal lamination, brain ventricle formation, and heart and body morphology. The mammalian epb4.1l5 locus encodes at least two alternately spliced transcripts, the protein products (Epb4.1l5long and Epb4.1l5short) of each contain the FERM domain, but have unique C’ termini. Mice with loss-of-function mutations in epb4.1l5 have early developmental defects in germ layer morphogenesis during the Epithelial to Mesenchymal Transition (EMT), and these mutants fail to properly form a gut and neural tube. Our lab has shown that Moe functions in rod photoreceptors as a negative regulator of outer segment size, and directly binds to members of the Crumbs (Crb) family of proteins, which are apical polarity determinants. Little is known of how Moe, or the Epb4.1l5 isoforms, perform their roles in cell or tissue morphogenesis. The collective aim of my studies was to elucidate the role of Moe and Epb4.1l5 isoforms in development and retinal function. I show that Moe and the apical polarity determinant Crb2a require reciprocal protein function for their respective localization at the eye and brain ventricle surface in zebrafish. I have identified multiple tissues and developmental stages wherein Moe and Crb proteins colocalize, and thus are likely to interact in vivo. I have identified specific sequences shared by Moe and Epb4.1l5 long that are important for function in specific tissues during zebrafish development, and I have investigated morphological, ultrastructural, and behavioral consequences of losing Moe (Epb4.1l5long) function in the patterned zebrafish retina. I describe the interaction of Moe with the Ca2+ binding protein Calmodulin (CaM). I identify three novel binding partners of Epb4.1l5long; Casein Kinase II, Moesin and Radixin, in mammalian retina and retinal pigmented epithelial (RPE) tissue homogenate. And lastly, I integrate the results of my studies to provide a model for Moe function, wherein Moe, together with Moesin and Radixin, link the vesicular transport Crb proteins to the actin cytoskeleton.

Calcium ([Ca2+]i) oscillations, a hallmark of mammalian fertilization, are essential to induce egg activation and embryonic development. As the zygotes transition through the cell cycle, these [Ca 2+]i oscillations progressively diminish until they cease in interphase zygotes. While the mechanism(s) underlying the regulation of [Ca2+]i oscillations may be multi-layered, inositol 1,4,5-trisphosphate receptors (IP3Rs) emerge as a focal point in this regulation. IP3Rs, the calcium channels expressed in all cell types and abundantly expressed in mammalian eggs, contain consensus sequences for phosphorylation by various kinases and interact with members of the cytoskeleton, serving as an integrator of regulatory signals. This dissertation highlights the impact of biochemical and cellular changes in IP3R-1 on fertilization-induced [Ca2+]i oscillations. Together with the changes in the cellular distribution of the sperm factor, these cell cycle-dependent modifications in IP3R-1 may underlie the regulation of [Ca 2+]i oscillations in fertilized mammalian eggs.

The nuclear receptor superfamily is a large group of related receptors that bind steroid hormones, signaling molecules, or xenobiotic chemicals and are expressed across many mammalian tissues. The impact of nuclear receptor activation using two different mouse model systems is explored in this thesis: (1) in utero exposure of the environmental xenoestrogen and proposed endocrine disruptor bisphenol A (BPA) and (2) short adult exposures to the mouse constitutive androstane receptor (CAR) specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). First, experiments involving the impact of in utero BPA exposure on the male mouse reproductive tract are described. Minimal changes to long-term mouse testis morphology and function were observed as mice treated with BPA in utero did not show significant changes in spermatozoa production or testis histopathology. Microarray analysis showed few persistently dysregulated genes, none of which were validated using qPCR due to high variability among biological replicates. Next, nuclear RNA-seq was used to characterize global changes in the mouse liver transcriptome following exposure to TCPOBOP, including changes in novel long non-coding RNAs that may contribute to xenobiotic-induced pathophysiology. Dysregulated protein coding genes were associated with a striking male-biased pro-tumor response, including activation of pro-tumor upstream regulators such as cyclin D1 and inhibition of tumor suppressors such as p21 and p53, consistent with the reported male-biased susceptibility to CAR-dependent mouse liver tumorigenesis. Novel long non-coding RNAs were identified in livers of mice exposed to TCPOBOP, including lncRNAs proximal to the CAR target genes like Cyp2b10. Then, DNase-seq was used to identify DHS in male and female mouse liver that open or close following TCPOBOP treatment proximal to CAR responsive coding and non-coding genes. Finally, a series of ChIP-seq experiments targeting the activating histone modifications H3K4me1, H3K4me3 and H3K27ac, and the repressive chromatin modification H3K27me3 were performed in male mice to characterize the corresponding changes in local chromatin environment around DHS and responsive genes. Using a combination of DNase-seq and ChIP-seq, several classes of DNA regulatory elements have been identified, including active enhancers and promoter regions that may play a function role in regulating nearby CAR-responsive protein-coding and lncRNA genes. / 2020-10-08T00:00:00Z

There are various novel approaches to cancer immunotherapy. These potential treatments for malignant cancers include the adoptive transfer of natural killer cells, T cell receptor T cells, chimeric antigen receptor T cells, and many more. It is important to note that conducting these revolutionizing treatments became possible due to the humanized mouse models, which provide the experimental base models for investigating anti-tumor research. To further enhance the measure of treatment for malignant cancers, it is vital to understand how the humanized mouse models work and represent the human immune microenvironment. Here, I review the history of the humanized mouse models, their variations and advantages and limitations that follow with the different types of models, their roles in evaluating the potential cancer immunotherapy treatments, and their anticipated roles in the future to further assist in developing a more personalized antitumor treatment.

As the principal cellular agents initiating thrombus (clot) formation, the primary function of platelets is maintenance of blood hemostasis. These anucleate cell fragments lack nuclear DNA and so provide a unique model for investigating post-transcriptional regulation of gene expression via RNA interference (RNAi). Although platelets are not capable of transcribing nascent mRNA, they are able to maintain protein homeostasis throughout their ~10-day lifespan in circulation; how platelets are able to do so, considering they contain a functional proteasome, remain unclear. We hypothesized that platelet protein translation and homeostasis is driven by ongoing plasma-derived growth factor signaling balanced with degradation, which is regulated by Argonaute 2 (Ago2)-mediated RNAi. In nucleated cells, RNAi occurs via RNA-induced silencing complexes (RISC), in which a guide RNA such as a microRNA (miRNA) docked in an effector protein, typically Ago2, recruits a target mRNA via guide:target sequence complementarity and results in suppression of mRNA translation via multiple mechanisms. Though RNAi has not yet been fully explored in the context of anucleate cells, platelets contain the machinery and proteins necessary for RNAi as well as being highly enriched in a diverse array of miRNAs. Additionally, variations in platelet miRNAs in humans are associated with variable platelet reactivity and risk of thrombosis.   We have found platelets to be capable of taking up exogenous double-stranded miRNA and have published on their subsequent ability to utilize the miRNA to modulate signal-activated translation and platelet function (Aim 1). In order to investigate the role of constitutive translation in circulating platelets (Aim 2) and the effect of Ago2 in regulation of platelet reactivity (Aim 3) we generated a novel platelet-specific Ago2 deleted (Ago2fl/fl/Pf4-Cre) mouse line and compared the proteome of knockout (KO) mice to wild-type (WT), by sex. Based on Ago2 roles in RNAi and miRNA enrichment in platelets, we predicted large-scale increases in platelet protein expression with megakaryocytic Ago2 deletion. However, only a small subset of genes was significantly upregulated. Among these was Ago1, which was significantly increased in both male and female Ago2 KO platelets. These results strongly suggest that Ago-mediated RNAi is essential for platelet development. Interestingly, deletion of platelet Ago2 resulted in decreased protein expression of a different subset of genes in platelet from female mice only; this sex-specific effect may be due to hormonal differences, namely estrogen, as many of these regulated proteins are encoded by estrogen-responsive genes. The gene set downregulated in Ago2 KO female platelets vs WT, but not males, corresponded to genes known to be coordinately regulated at the transcriptional level by ERβ in conjunction with Ago2 in breast cancer cells. These results point to a novel role for Ago2 in modulating the platelet transcriptome in megakaryocytes, affecting the mRNAome in the platelets they generate, in a hormone-dependent manner.   Platelets from both male and female KO mice had significantly increased mean platelet volumes (MPV), not explained by changes in platelet production, lifespan, or protein content; however, KO megakaryocytes (MKs) had significantly increased size & ploidy, accounting for increased MPV, although femurs in the KO mice had fewer MKs. Furthermore, KO mice exhibited faster clot retraction and increased surface expression of integrin αIIbβ3. Platelets from male KO mice showed increased αIIbβ3 integrin activity and P-selectin expression following thrombin and U46619 stimulation, a thromboxane analogue, but these effects were not seen with collagen or ADP, pointing to agonist pathway-specific effects. Bioinformatics analysis revealed sex-specific changes in the platelet proteome of KOs to account for this change in reactivity; a cluster of mitochondrial enzymes modulated in male KOs only, involved in ROS metabolism and respiration, may be responsible for the observed difference in the thromboxane response. Taken together, our findings suggest Ago2-mediated RNAi to be an important regulator of megakaryopoeisis and platelet reactivity. / Biomedical Sciences

The work undertaken in this thesis was directed towards understanding the organization of lipids in cell membranes and the lipid specificity of perforin. Three aspects of the organization of membrane lipids were investigated, (1) the asymmetrical distribution of lipids between the outer and inner leaflets of the membrane, (2) the bobbing motion of lipids perpendicular to the plane of the membrane, and (3) the spacing of lipids in the external leaflet of the membrane. The binding of perforin to membranes was studied by (1) comparing the ability of liposomes constructed from defined lipids to bind perforin, and (2) the ability of perforin to bind to and lyse lipid symmetric and lipid asymmetric erythrocyte membranes. The asymmetrical distribution of lipids in the plasma membrane of erythrocytes was investigated by examining two mechanisms for maintenance of lipid asymmetry: binding of internal lipids to cytoskeletal proteins, and pumping of internal lipids from the outside to the inside of the cell. Analysis of the kinetics of lipid internalization suggested the presence of an ATP-dependent lipid flip/flop catalyst. The thermal energy of lipids results in oscillatory motion perpendicular to the plane of the membrane, and in movement in the plane of the membrane. The vertical distribution of lipids arising from this oscillatory motion was calculated. The motion of lipids in the plane of the membrane results in an average spacing between the lipid molecules. Previous reports, had demonstrated that the fluorescent dye merocyanine 540 bound preferentially to widely spaced phosphatidylcholine lipids. MC540 was shown to have little headgroup, sidechain or cholesterol specificity per se, but bound preferentially to membranes whose lipids are widely spaced, suggesting its use as a probe of lipid spacing. Comparison of perforin binding to liposomes constructed from defined lipids revealed that perforin bound preferentially to liposomes whose lipids were widely spaced. Using erythrocytes as a model, it was observed that increasing the spacing of lipids in the outer leaflet of the plasma membrane resulted in increased perforin binding and susceptibility to perforin-mediated lysis, confirming the conclusions derived from the liposome studies and further suggesting that increased lipid spacing increases overall susceptibility to the more complex processes involved in cellular lysis by perforin. The external phospholipids of the plasma membrane of the CTL were shown to be more tightly packed than the lipids of the more-susceptible target cell, suggesting that the resistance of CTL to perforin is at least in part due to the presence of closely spaced lipids in its membrane.