Regulation of cell morphogenesis by targeted noise suppression and trafficking of a G protein alpha subunit

G proteins and their associated receptors form the largest class of proteins that receive and transduce chemical and sensory signals. Regulation of G proteins therefore, is critical to appropriate cellular responses. The work in this thesis evaluates a new function for a known regulator of signaling and identifies a new class of previously unknown regulators that mediate G protein trafficking. Using a yeast model system we demonstrate a novel role for the Regulator of G protein Signaling (RGS) protein in suppression of cell-to-cell variability. Furthermore we identify a novel cascade of ubiquitin-binding domain (UBDs) proteins that serve to deliver the Gα protein from the plasma membrane to the vacuole. Through biochemical assays and single cell analysis we find that the RGS and UBD proteins regulate cellular morphogenesis during signaling. More broadly through this work we were able to uncouple signal and noise in a prototypical stimulus-response pathway and demonstrate for the first time the consequences of G protein trafficking in a non-visual system. This work is important because a thorough understanding of how G protein signaling is spatially and temporally regulated will eventually lead to new drug targets, and more effective or targeted therapeutics.Doctor of Philosoph

Assessment of total phenolic and flavonoid contents and potential biological efficacy of few Pinus species growing in Northern Himalayas

Environmental interventions and ecological adaptations harbor millions of valued substances and metabolites in plants which can be employed and commercialized for human benefits. Present study encompasses the untapped potential of pine needles of Indo-Himalayan region for the production of different metabolites and their pharmacological significance in terms of antioxidant and antimicrobial activity. Total phenolic and flavonoid content from the needles of ten pine species was quantified using three different solvent systems. Results revealed that out of 10 different selected Pinus species, Pinus taeda L. showed the highest concentration of total phenolics, Soluble-F phenolics and flavonoids content (approx. 147.02 mg/g, 141.08 mg/g and 21.91 mg/g, respectively) as compared to other species. On the other hand, Pinus greggii Engelm. ex Parl. showed the highest Bound-W phenolic content (approx. 3.62 mg/g). Among all the selected plant species, the needles of Pinus echinata Mill. exhibited the highest and Pinus thunbergii Parl. had the lowest ratio of total flavonoids to total phenolics. Most of these compounds were found to have effective antioxidant activities as well as antimicrobial activity, as estimated by oxygen radical absorbance capacity (ORAC) and disk diffusion test, respectively

RGS Proteins and Septins Cooperate to Promote Chemotropism by Regulating Polar Cap Mobility

Background—Septins are well known to form a boundary between mother and daughter cells in mitosis, but their role in other morphogenic states is poorly understood. Results—Using microfluidics and live cell microscopy, coupled with new computational methods for image analysis, we investigated septin function during pheromone-dependent chemotropic growth in yeast. We show that septins colocalize with the regulator of G-protein signaling (RGS) Sst2, a GTPase-activating protein that dampens pheromone receptor signaling. We show further that the septin structure surrounds the polar cap, ensuring that cell growth is directed toward the source of pheromone. When RGS activity is abrogated, septins are partially disorganized. Under these circumstances the polar cap travels toward septin structures and away from sites of exocytosis, resulting in a loss of gradient tracking. Conclusion—Septin organization is dependent on RGS protein activity. When assembled correctly, septins promote turning of the polar cap and proper tracking of a pheromone gradient

Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast

Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization

Cellular Noise Suppression by the Regulator of G Protein Signaling Sst2

G proteins and their associated receptors process information from a variety of environmental stimuli to induce appropriate cellular responses. Generally speaking, each cell in a population responds within defined limits despite large variation in the expression of protein signaling components. Therefore we postulated that noise suppression is encoded within the signaling system. Using the yeast mating pathway as a model we evaluated the ability of a regulator of G protein signaling (RGS) protein to suppress noise. We found that the RGS protein Sst2 limits variability in transcription and morphogenesis in response to pheromone stimulation. While signal suppression is a result of both the GAP (GTPase accelerating) and receptor binding functions of Sst2, noise suppression requires only the GAP activity. Taken together our findings reveal a hitherto overlooked role of RGS proteins as noise suppressors, and demonstrate an ability to uncouple signal and noise in a prototypical stimulus-response pathway

Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK

MAPKs are activated by dual phosphorylation. Much of the MAPK Fus3 is monophosphorylated and acts to inhibit signaling in vivo. Computational models reveal how a kinase scaffold and phosphatase act together to dynamically regulate dual-phosphorylated and monophosphorylated MAPKs and the downstream signal.Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5ND) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or “synthetic,” supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling

Modulation of receptor dynamics by the regulator of G protein signaling Sst2

G protein–coupled receptor (GPCR) signaling is fundamental to physiological processes such as vision, the immune response, and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. After pheromone stimulation, the GPCR Ste2 is removed from the cell membrane, and new receptors are delivered to the growing edge. The regulator of G protein signaling (RGS) protein Sst2 acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 together with its interaction with the receptor function to reestablish a receptor pool at the site of polarized growth. To validate the model, we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and its induction by pheromone. Together our results reveal that a regulator of G protein signaling can also regulate the G protein–coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge

Reporting trends, practices, and resource utilization in neuroendocrine tumors of the prostate gland: a survey among thirty-nine genitourinary pathologists

Background: Neuroendocrine differentiation in the prostate gland ranges from clinically insignificant neuroendocrine differentiation detected with markers in an otherwise conventional prostatic adenocarcinoma to a lethal high-grade small/large cell neuroendocrine carcinoma. The concept of neuroendocrine differentiation in prostatic adenocarcinoma has gained considerable importance due to its prognostic and therapeutic ramifications and pathologists play a pivotal role in its recognition. However, its awareness, reporting, and resource utilization practice patterns among pathologists are largely unknown. Methods: Representative examples of different spectrums of neuroendocrine differentiation along with a detailed questionnaire were shared among 39 urologic pathologists using the survey monkey software. Participants were specifically questioned about the use and awareness of the 2016 WHO classification of neuroendocrine tumors of the prostate, understanding of the clinical significance of each entity, and use of different immunohistochemical (IHC) markers. De-identified respondent data were analyzed. Results: A vast majority (90%) of the participants utilize IHC markers to confirm the diagnosis of small cell neuroendocrine carcinoma. A majority (87%) of the respondents were in agreement regarding the utilization of type of IHC markers for small cell neuroendocrine carcinoma for which 85% of the pathologists agreed that determination of the site of origin of a high-grade neuroendocrine carcinoma is not critical, as these are treated similarly. In the setting of mixed carcinomas, 62% of respondents indicated that they provide quantification and grading of the acinar component. There were varied responses regarding the prognostic implication of focal neuroendocrine cells in an otherwise conventional acinar adenocarcinoma and for Paneth cell-like differentiation. The classification of large cell neuroendocrine carcinoma was highly varied, with only 38% agreement in the illustrated case. Finally, despite the recommendation not to perform neuroendocrine markers in the absence of morphologic evidence of neuroendocrine differentiation, 62% would routinely utilize IHC in the work-up of a Gleason score 5 + 5 = 10 acinar adenocarcinoma and its differentiation from high-grade neuroendocrine carcinoma. Conclusion: There is a disparity in the practice utilization patterns among the urologic pathologists with regard to diagnosing high-grade neuroendocrine carcinoma and in understanding the clinical significance of focal neuroendocrine cells in an otherwise conventional acinar adenocarcinoma and Paneth cell-like neuroendocrine differentiation. There seems to have a trend towards overutilization of IHC to determine neuroendocrine differentiation in the absence of neuroendocrine features on morphology. The survey results suggest a need for further refinement and development of standardized guidelines for the classification and reporting of neuroendocrine differentiation in the prostate gland

The role of "penumbra sign" and diffusion-weighted imaging in adnexal masses : do they provide a clue in differentiating tubo-ovarian abscess from ovarian malignancy?

Purpose: To evaluate the role of "penumbra sign", diffusion-weighted imaging (DWI), and the apparent diffusion coefficient (ADC) value in differentiating tubo-ovarian abscess (TOA) from ovarian malignancy. Material and methods: Thirty-six patients with 50 adnexal masses (tubo-ovarian abscess, n = 24; ovarian malignancy, n = 26), who underwent magnetic resonance imaging (MRI) with DWI, were retrospectively evaluated. "Penumbra sign" (hyperintense rim on T1W images), diffusion restriction, and mean apparent diffusion coefficient (ADC) values from cystic (c-ADC) and solid (s-ADC) components were evaluated for all the masses. Results: "Penumbra sign" on T1W images was significantly more common in the TOA group (n = 21, 87.5%) than in the ovarian malignancy group (n = 2, 7.7%) (p < 0.001). Similarly, diffusion restriction in the cystic component was more frequent in the TOA group (n = 24, 100% vs. n = 2, 10.5%; p < 0.001). In contrast, diffusion restriction in the solid component was more common in the ovarian malignancy group (n = 5, 20.8% vs. n = 26, 100%; p < 0.001). The mean c-ADC value was significantly lower in TOAs (p < 0.001). A c-ADC value of 1.31 × 10-3 mm2/s may be an optimal cut-off in distinguishing TOAs from ovarian malignancies. Conversely, the mean s-ADC value was significantly lower in the ovarian malignancy group (p < 0.001). An s-ADC value of 0.869 × 10-3 mm2/s may be an optimal cut-off in differentiating ovarian malignancies from TOAs (p < 0.001). ROC curve analysis showed that c-ADC values had a higher diagnostic accuracy than s-ADC values. Conclusions: "Penumbra sign" on T1W images, diffusion characteristics, and ADC values provide important clues in addition to conventional MR imaging features in differentiating TOA from ovarian malignancy