Browsing by Subject "Calcium-Binding Proteins"
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Item Crosstalk Between Calcium Signaling and Lipid Metabolism at Endoplasmic Reticulum-Plasma Membrane Junctions(2014-04-14) Chang, Chi-Lun; Moe, Orson W.; Roth, Michael G.; Yin, Helen L.; Liou, JenReceptor-induced Ca2+ signaling is the key to many cellular functions, such as secretion, migration, differentiation, and proliferation. The increase in cytosolic Ca2+ signals is dependent on the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane (PM). To enable subsequent signaling activation and maintain cellular homeostasis, it is necessary to replenish the consumed PIP2. However, the molecular mechanisms underlying PM PIP2 replenishment after hydrolysis remain elusive. PIP2 is generated at the PM by sequential phosphorylation of phosphatidylinositol (PI) originating from the endoplasmic reticulum (ER). Delivering PI from the ER to the PM by PI transfer proteins (PITPs) is therefore postulated to support PM PIP2 replenishment. Such transfer is more likely to take place at ER-PM junctions, since the close apposition of the ER and the PM enables PITPs to efficiently interact with two heterologous membranes. To study ER-PM junctions and their roles in PM PIP2 replenishment, I generated a genetically-encoded fluorescent marker to selectively label ER-PM junctions. With this marker, minute ER-PM junctions were easily observed in live cells using multiple imaging techniques. At the resting state, approximately two hundred stable ER-PM junctions were detected at the adhesion surface of a single HeLa cell. Photo-activated localization microscopy (PALM) super-resolution imaging further demonstrate that ER-PM junctions labeled by this marker were remarkably uniform in size and slightly elongated in shape with a long axis of 255.5 nm and a short axis of 157.7 nm. Furthermore, analysis of the distance to nearest neighbor of individual ER-PM junctions show that these junctions are distributed uniformly in the cells. Following the activation of Ca2+ signaling, I observed an enhanced ER-to-PM connection resulting from new junction formation and a decrease in the gap distance of ER-PM junctions. The enhanced ER-to-PM connection depends on cytosolic Ca2+ levels and extended synaptotagmin-like protein 1 (E-Syt1), a C2 domain-containing ER membrane protein. E-Syt1 detects the increase in cytosolic Ca2+ via its C2C domain and translocates from the bulk of ER to ER-PM junctions to enhance ER-to-PM connection. This in turn facilitates the recruitment of Nir2, an ER-associated PITP, to ER-PM junctions to promote PM PIP2 replenishment. In summary, these results indicate a feedback loop for PM PIP2 replenishment via E-Syt1 and Nir2 at ER-PM junctions. Disruption of this feedback mechanism by knockdown of E-Syt1 or Nir2 abolished PM PIP2 replenishment and therefore, impaired receptor-induced Ca2+ signaling. This work reveals the long-sought mechanism of PM PIP2 replenishment following hydrolysis and sheds light on the functional roles of poorly characterized ER-PM junctions. Furthermore, given the fact that PIP2 and Ca2+ are pivotal signaling molecules for many cellular functions, these findings are of significance for providing new mechanistic insights into the signaling crosstalk and may have a broader impact on fields beyond cell signaling, organelle dynamics, and lipid trafficking.Item Molecular and Ocular Characterization of Novel Fibulin-3 Variants Involved in Retinal Degeneration(2021-12-10) Woodard, Danae Rochelle; Kohler, Jennifer J.; Petroll, W. Matthew; Tu, Benjamin; Hulleman, John D.Distinct mutations in fibulin-3 (F3), a secreted extracellular matrix glycoprotein, have been associated with various ocular diseases including Malattia Leventinese (ML) and the most common macular degenerative disease, age-related macular degeneration (AMD), which ultimately lead to vision loss. AMD is a late onset disease characterized by the progressive loss of photoreceptors and retinal pigment epithelial (RPE) cells that result in irreversible blindness. Although AMD is an etiologically complex disease due to a variety of genetic and environmental risk factors, insight into its pathogenesis can be gained by studying phenotypically similar early-onset monogenic macular diseases. One such disease is ML, a rare macular dystrophy caused by an autosomal dominant Arg345Trp (R345W) mutation in the F3 protein. Previous research has demonstrated that the R345W mutation leads to protein misfolding, inefficient secretion, and accumulation at higher intracellular steady state levels in cultured cells. However, it remained unclear whether other potentially pathogenic or clinically-identified F3 variants recently reported in the human population also share features similar to that of R345W. We hypothesized that secretion defects in one or more F3 mutants may be a shared mechanism that ultimately contributes to ocular disease. First, I characterized 15 clinically-identified F3 mutations, some of which were identified in patients with AMD, primary open-angle glaucoma (POAG), or had non-discript retinal abnormalities. I found that of the mutants tested, only a single F3 variant, L451F, presented with a significant secretion defect as well as similarities in its biochemical and molecular properties to that of R345W. Subsequently, I generated a retinal disease mouse model of the L451F mutant utilizing recombinant adeno-associated virus (rAAV) in order to robustly evaluate disease phenotypes and uncover how L451F and other F3 mutations (i.e. R345W) are involved in retinal degeneration.Item The Role of the WWTR1(TAZ)-CAMTA1 Gene Fusion in Epithelioid Hemangioendothelioma(2021-05-01T05:00:00.000Z) Driskill, Jordan Harrison; Dellinger, Michael T.; Cleaver, Ondine; McFadden, David G.; Pan, DuojiaEpithelioid hemangioendothelioma (EHE) is a devastating and mysterious vascular cancer which has no known definitive treatment. Due to a lack of valid animal or cell-based models of EHE, progress toward understanding and treating this cancer has been severely limited. However, recent studies have determined that 90% of patients exhibit a lone, characteristic in-frame gene fusion, TAZ(WWTR1)-CAMTA1. While expression of the TAZ-CAMTA1 fusion protein has been validated as a biomarker of EHE, it remains unknown whether this genetic abnormality is a passenger or a driver of EHE. In this project, I present the first genetically-engineered mouse model (GEMM) of EHE, showing that the expression of the TAZ-CAMTA1 protein in endothelial cells is sufficient to drive the formation of EHE-like tumors in the lungs of mice. Furthermore, I demonstrate that the cessation of TAZ-CAMTA1 expression leads to the regression of these vascular tumors. I also demonstrate that TAZ-CAMTA1 transforms the MS1 endothelial cell line and that subcutaneous transplantation of these cells into nude mice leads to the formation of solid, progressive EHE-like vascular tumors that have the capacity to metastasize to the lung. Utilizing these two novel models of EHE, I unravel the gene program of TAZ-CAMTA1 and demonstrate that TAZ-CAMTA1 drives a gene signature similar to TAZ, the key effector of the Hippo pathway. Expression of an activated TAZ in endothelial cells is also sufficient to drive EHE-like vascular tumors in mice, and genetic blockade of the transcriptional partners of TAZ, the TEAD family of transcription factors, prevents the formation of TAZ-CAMTA1-induced vascular tumors. Next, I show that TAZ-CAMTA1 induces an angiogenic and regenerative-like gene program in endothelial cells. I validate that TAZ-CAMTA1 exhibits gain-of-function activities by having increased resistance to proteasomal degradation and increased nuclear enrichment over TAZ. Lastly, I show that TAZ-CAMTA1 still maintains its binding to the Hippo pathway proteins which are known to negatively regulate TAZ. In summary, I generate two novel models that pinpoint TAZ-CAMTA1 as the key driver of EHE and utilize these models to suggest several new lines of investigation for the treatment of patients with EHE.