OBJECTIVE: One of the major issues in current radiotherapy (RT) is the normal tissue toxicity. A smart combination of agents within the tumor would allow lowering the RT dose required while minimizing the damage to healthy tissue surrounding the tumor. We chose gold nanoparticles (GNPs) and docetaxel (DTX) as our choice of two radiosensitizing agents. They have a different mechanism of action which could lead to synergistic effect. Our first goal was to assess the variation in GNP uptake, distribution, and retention in the presence of DTX. Our second goal was to assess the therapeutic results of the triple combination, RT/GNPs/DTX. METHODS: We used HeLa and MDA-MB-231 cells for our study. Cells were incubated with GNPs (0.2nM) in the absence and presence of DTX (50nM) for 24 hrs for determination of uptake, distribution, and retention of NPs. For RT experiment, treated cells were given a 2 Gy dose of 6 MV photons using a linear accelerator. RESULTS: Concurrent treatment of DTX and GNPs resulted in over 85% retention of GNPs in tumor cells. DTX treatment also forced GNPs to be closer to the most important target, the nucleus, resulting in a significant decrease in cell survival with the triple combination of RT, GNPs, and DTX vs. RT plus DTX alone. Our experimental therapeutics results are supported by Monte Carlo simulations. CONCLUSION: The ability to not only trap GNPs at clinically feasible doses but also to retain them within the cells could lead to meaningful fractionated treatments in future combined cancer therapy. Furthermore, the suggested triple combination of RT/GNPs/DTX may allow lowering the RT dose to spare surrounding healthy tissue. ADVANCES IN KNOWLEDGE: This is the first study to show intracellular GNP transport disruption by DTX, and its advantage in radiosensitization. / Graduate / 2020-10-31

8 ABSTRACT Rab GTPases (Rabs) are the most populous branch of eukaryotic Ras GTPase superfamily. In active GTP-binding conformation, they serve as key instruments in defining transient membrane identity and through various effectors regulate formation, transport, conversion, and fusion of membrane vesicles. This is important for upkeep of compartmentalized structure of eukaryotic cells and for facilitating both endo- and exocytic processes. Rabs are converted into GDP-binding conformation by interactions with Rab GTPase activating proteins (Rab GAPs) that possess ability to significantly speed up weak intrinsic GTP hydrolytic activity of Rabs. Through this process, Rab GAPs can limit scope of the Rabs' activity and lay out spatiotemporal boundaries for varying Rab populations. In this thesis, I tried to characterize a Rab GAP, GAP2, seemingly necessary for standard development of thale cress plants. Besides TBC catalytic domain, GAP2 (product of At2g39280 gene) possesses a C-terminal coiled-coil motif, which was previously found to interact with Rab GTPases. Experiments aiming to complement T-DNA insertion mutant in GAP2, elucidate GAP2 intracellular localization, novel interacting partners, and character of interaction with the Rabs discovered in the pilot study were undertaken. The results suggest that...

Complex Exocyst consists of eight proteins and it is known as a Sec6/8. Its composition is evolutionarily highly conserved amongst all the species. This complex is involved in vesicle trafficking as a part of attaching mechanism to a specific place on the plasma membrane. EXO70 subunit has been found in 23 copies in Arabidopsis thaliana genome. In this study we have been examine paraloges EXO70H7 and EXO70H8. There have been suggestions that these genes are important in development of roots according to the previous studies. We have not been able to identify any significant phenotype within the mutant plants in these genes. There has been studied other mutant appearance during the stress experiments. Most of these experiments did not identify any divergence. Only experiments with germination during stress conditions revealed significantly worse germination of exo70H7 mutant seeds on the medium containing sorbitol. This suggests that mutant seeds have a worse protection against osmotic stress. Significantly worse germination of exo70H8 seeds on the medium with excess NaCl indicates that these seeds incriminate to higher absorption of sodium ions. Analysis of the cell localization of GFP constructs brought knowledge of appearance EXO70H7 and EXO70H8 proteins. EXO70H7 locates in the cytoplasm and in...

Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes involves defects in glucose-stimulated insulin secretion (GSIS) from the pancreatic beta cells in combination with defects in peripheral (muscle and adipose) tissue glucose uptake. Both GSIS and glucose uptake are regulated by Syntaxin 4 (Syn4)-Munc18c complexes. Importantly, reports link obesity and Type 2 diabetes in humans with changes in protein levels of Munc18c and Syn4; yet the molecular mechanisms underlying this requirement remain unclear. The central hypothesis proposed is that Syn4-Munc18c complexes are modulated by post-translational modifications and novel interactions. Toward this, we found that Syn4-Munc18c complexes are regulated by tyrosine phosphorylation of Munc18c at Y219 in beta cells. Munc18c tyrosine phosphorylation disrupts Syn4-Munc18c complexes, which leads to an increase in Munc18c associating with the double C2 domain protein Doc2β. Disruption of Syn4-Munc18c upon tyrosine phosphorylation results in an increase in Syn4-SNARE complex formation and GSIS from beta cells. Similarly, tyrosine phosphorylation of Munc18c at Y219 and also Y521, disrupts its association with Syn4 in insulin-stimulated 3T3L1 adipocytes and skeletal muscle. In vitro kinase assays further suggested that the insulin receptor tyrosine kinase targeted Y521 of Munc18c. Further investigations using 3T3L1 adipocytes and skeletal muscle extracts indicate that Munc18c interacts with the insulin receptor tyrosine kinase in an insulin-dependent manner, resulting in phosphorylation of Munc18c, coordinate with the timing of its dissociation from Syn4. Finally, we found that stimulus-induced changes occurred also with Syn4, most notably in the islet beta cells. Syn4-mediated insulin release requires F-actin remodeling to mobilize insulin granules to the plasma membrane. Our studies reveal that Syn4 directly associates with F-actin in MIN6 beta cells, and that the disruption of this complex correlates with increases in glucose-stimulated insulin secretion. Future studies will focus upon the potential link between Syn4, F-actin remodeling with Munc18c, to further gain understanding of the requirements for Syn4-Munc18c complexes in insulin secretion. In sum, given the parallels of Munc18c tyrosine phosphorylation in regulating Syn4-Munc18c interaction and exocytosis in beta cells and peripheral tissues, manipulations of this complex may have therapeutic potential as a strategy to treat Type 2 diabetes.

Biological membranes form the structural boundaries and compartments of cells, owing to their robustness and impermeability facilitated by phospholipid bilayers. The strength of biological membranes is intricately linked to the behavior of membrane pores, whose formation and expansion can lead to membrane rupture. However, processes essential for drug delivery, gene editing via genetic material transfer, and antimicrobial peptide action necessitate controlled membrane disruption for efficient cellular entry. Likewise, fundamental phenomena such as exocytosis, including neurotransmitter release between neurons and hormone secretion for physiological responses, rely on membrane breach to release cargo beyond cell confines. Exocytosis involves the fusion of cargo-contained vesicle membranes with the cell's plasma membrane, resulting in the release of cargo into the extracellular milieu. Post-release, these fused vesicles may either integrate with the plasma membrane, remain stationary, enlarge, or depart the release site through fusion pore closure, which, in turn, can modulate exocytosis rate through site availability. However, the precise mechanism of membrane rupture remains elusive. Similarly, the pathway of membrane fusion facilitated by SNARE proteins, pivotal in cellular fusion machinery, remains a subject of debate. Additionally, the mechanisms governing exocytosis remain incompletely understood. To address these inquiries, we employ ultra-coarse-grained molecular dynamics simulations which can explore these phenomena in physiological timescale. These simulations explore membrane rupture mechanisms via pore formation and expansion under varying membrane tension. Furthermore, the research addresses how SNARE proteins drive membrane fusion. In addition, we also rigorously analyze confocal microscopy data from Ling-Gang Wu's research group and develop a quantitative model to elucidate exocytosis rate regulation. Furthermore, the research verifies the robustness of a mathematical model outlining Ca2+-mediated membrane fusion and establishes that hemifusion diaphragms (HDs), where only the outer leaflets of membranes fuse, act as hubs in the Ca2+-mediated fusion network. This finding casts new light on the role of membranes in SNARE-mediated fusion. In the extra study, we analyzed fission yeast contractile ring behavior based on z-stack confocal microscopy data from Mohan Balasubramanian's research group, offering insights into the mechanism behind a critical step in cytokinesis. Chapter one examines membrane pore energetics and bilayer rupture times through highly coarse-grained simulations operating at submillisecond time scales. No metastable states are detected during pore formation. At lower tensions, small hydrophobic pores mature into large hydrophilic pores that ultimately rupture from reversible hydrophilic pores, aligning with classical tension-dependent rupture times. At higher tensions, membranes rupture directly from small hydrophobic pores, with rupture times exhibiting exponential tension dependence. Upon reaching a minimum hydrophobic pore size, a critical tension threshold prompts immediate rupture. This analysis corroborates established experimental findings but reveals that the high-tension exponential regime is not related to long-lived pre-pore defects but rather to the instability of hydrophilic pores beyond a critical tension, leading to significant changes in pore dynamics and rupture kinetics. Chapter two describes utilizing ultra-coarse-grained simulations to dissect the core requirements of membrane fusion and unravel the intricacies of SNARE-mediated fusion. Remarkably, simulations conducted on a millisecond timescale expose the inefficiency of fusion through simple body forces pushing vesicles together. Successful inter-vesicle fusion hinges on the rod-like structure of fusogens, ensuring their sufficient length for effective fusion and subsequent clearance from the fusion site via entropic forces. Simulations featuring rod-shaped fusogens and SNARE proteins demonstrate the fusion of 50-nanometer vesicles in submilliseconds, propelled by entropic forces that direct a predictable fusion pathway. The entropic force hypothesis of SNARE-mediated membrane fusion garners strong support from these findings, emphasizing the necessity of the rod-like configuration of the SNARE complexes for entropic force generation and fusion. Chapter three focuses on the spatiotemporal dynamics of dense-core vesicle exocytosis events in chromaffin cells, deducing a novel mechanism for exocytosis regulation based on the availability of release sites. Repeated fusion supports membrane reservoir comprising incompletely merged or closed vesicles, occupying release sites and dampening exocytosis frequency. Mathematical modeling suggests reservoir formation relies on locally reduced membrane tension, eliminating the driving force for vesicle merging. Endocytosis facilitates the clearance of unmerged vesicles from the reservoir, ultimately restoring release site availability for subsequent exocytosis events. Chapter four introduces a mathematical model pinpointing the hemifusion diaphragm (HD) as the decision nexus dictating the outcomes of pathways and the fate of final products during multivalent cation-mediated membrane interactions. Transient formation of a high-tension hemifusion interface between membrane-enclosed compartments underscores the model's prediction of fusion, dead-end hemifusion, or vesicle lysis. This comprehensive framework offers predictive insights into interactions mediated by cationic fusogens within membrane-enclosed compartments. Chapter five offers a unique exploration of writhing contractile rings in fission yeast cell ghosts, resulting from controlled digestion of the cell wall and subsequent membrane permeabilization. This innovative approach unveils the intricate dynamics of contractile rings under exceptional circumstances. Writhing of rings is attributed to the detachment of sections from the weakened membrane, followed by their coiling due to apparent twisting torques at anchoring points. Iterative rotations give rise to multiple coils within the rings.

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<p dir="ltr">Exocytosis is crucial for delivering proteins, lipids, and cell wall polysaccharides to the plasma membrane and extracellular spaces, playing a vital role in normal plant development as well as responses to biotic and abiotic stresses. One key molecular player, the exocyst, is an octameric protein complex that tethers secretory vesicles to the plasma membrane (PM). Chapter 1 is a literature survey that introduces the function of the exocyst, as well as the characterization of Endosidin2 (ES2), a synthetic molecule that targets the EXO70 subunit of exocyst. This chapter also defines existing knowledge gaps in the profiling of cargo proteins trafficked by the exocyst and the identification of novel modulators of exocytosis. Chapter 2 employs a comparative proteomics approach to examine the changes of PM proteome of root cells following ES2-treatment. Proteins with decreased abundance at the PM were considered candidate cargo proteins of ES2-targeted trafficking and several were validated with quantitative live-cell imaging. Chapter 3 describes the use of ES2 as a tunable and reversible chemical genetics tool as demonstrated by the development and deployment of a large-scale mutant screen in Arabidopsis that identified 70 <u>ES2</u>-hyper<u>s</u>ensitive mutants (<i>es2s</i>). Among these, candidate mutations for 14 non-allelic lines were mapped and reported. T-DNA insertion lines were subsequently screened as alternative alleles to identify causal mutations. In Chapter 4, the causal mutation of <i>es2s-15-12</i> was confirmed as <i>ArgJ</i> with a second T-DNA insertion mutant allele as well as genetic and chemical complementation. <i>ArgJ</i> encodes an enzyme in the arginine biosynthesis pathway. It was demonstrated that arginine biosynthesis deficiency synergizes with ES2 to inhibit root growth in Arabidopsis. Root growth in <i>argj</i> mutants was not hypersensitive to other inhibitors with different modes of action, such as LatB, ES9-17, and BFA. Additionally, roots of <i>argj-1</i> displayed a reduced abundance of PIN2 at the apical PM in epidermal cells; however, PIN2 polar distribution was not further reduced by ES2 treatment. Our findings point to a functional connection between arginine metabolism and exocytosis. Chapter 5 discusses potential future directions and experiments, including technological advances and the testing of new hypotheses. Overall, this study presents a detailed application of chemical genetics to dissect the exocytosis process in Arabidopsis and uncovers novel modulators of exocytosis in plants.</p>

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Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the identity of the functional syntaxin has been controversial. Previous studies, using anti-syntaxin antibodies in permeabilized platelets, suggested roles for both syntaxin-2 and -4. These conclusions were experimentally tested using platelets from syntaxin knockout mice and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient that lacks syntaxin-11. Platelets from syntaxin-2 and syntaxin-4 single or double knockout mice had no significant secretion defect. However, platelets from the FHL4 patient had a robust defect, though their morphology, activation, and cargo levels appeared normal. Semi-quantitative western blotting showed that syntaxin-11 is the most abundant syntaxin in both human and murine platelets. Co-immunoprecipitation experiments showed that syntaxin-11 forms SNARE complexes with VAMP-8 and SNAP-23. These data conclusively demonstrate that syntaxin-11, but not syntaxin-2, or -4, is required for platelet exocytosis. We also show that a syntaxin binding protein, tomosyn-1, is important for platelet exocytosis and hemostasis. Tomosyn-1 was identified from platelet extracts using affinity chromatography, RT-PCR analysis, and western blotting analysis. Tomosyn-1 was co-immunoprecipitated with syntaxin-11/SNAP-23 from both resting and activated platelet extracts. Platelets from tomosyn-1-/- mice displayed a secretion defect, but their morphology and activation appeared normal. Tomosyn-1-/- mice showed impaired thrombus formation in two different injury models. Given the importance of platelet secretion to hemostasis, it is hoped that the insights gained from these studies in this dissertation will help to identify new and more valuable therapeutic targets to control clot formation.

Cells in the body communicate with each other in order to cooperate efficiently. This communication is in part achieved by regulated secretion of signaling molecules, which when released from a cell may activate receptors present at the plasma membrane of an adjacent cell. Such signals affect both cell fate and behavior. Dysregulated signaling may lead to disease, including cancer. This thesis is focused on how exocytosis and subsequent activation and trafficking of receptors can be regulated, and what the consequences of this regulation may be for cell migration. Actin filaments are important transport structures for secretory vesicle trafficking. In Paper 1, actin polymerization was shown to induce formation of ordered lipid domains in the plasma membrane. Accordingly, actin filaments may thus create and stabilize specific membrane domains that enable docking of vesicles containing secretory cargo. The RhoGEF FGD5 regulates Cdc42 which can result in cytoskeletal rearrangements. In Paper II, FGD5 was shown to be selectively expressed in blood vessels and required for normal VEGFR2 signaling. FGD5 protected VEGFR2 from proteasome-mediated degradation and was essential for endothelial cells to efficiently respond to chemotactic gradients of VEGFA. The exocyst component EXOC7 is essential for tethering secretory vesicles to the plasma membrane prior to SNARE-mediated fusion. In Paper III, EXOC7 was required for trafficking of VEGFR2-containing vesicles to the inner plasma membrane and VEGFR2 presentation at the cell surface. The ability of tumor cells to escape the primary tumor and establish metastasis is in part dependent on their capacity to migrate. In Paper IV, a method based on time-lapse microscopy and fluorescent dyes was created to analyze single cancer cell migration in mixed cancer cell cultures, and in particular the influence of different types on neighboring cells was assessed. In conclusion, these studies have enhanced our understanding of the mechanisms behind cellular trafficking, and may be applied in the future to develop more specific therapeutics to treat cancer and other diseases associated with abnormal angiogenesis and cellular migration.