Turnover and bypass of p21-activated kinase during Cdc42-dependent MAPK signaling in yeast.

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular behaviors, including the response to stress and cell differentiation, and are highly conserved across eukaryotes. MAPK pathways can be activated by the interaction between the small GTPase Cdc42p and the p21-activated kinase (Ste20p in yeast). By studying MAPK pathway regulation in yeast, we recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is regulated in a similar manner and is turned over by the 26S proteasome. This turnover did not occur when Ste20p was bound to Cdc42p, which presumably stabilized the protein to sustain MAPK pathway signaling. Although Ste20p is a major component of the fMAPK pathway, genetic approaches here identified a Ste20p-independent branch of signaling. Ste20p-independent signaling partially required the fMAPK pathway scaffold and Cdc42p-interacting protein, Bem4p, while Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p. Interestingly, Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p, Rga1p, which unexpectedly dampened basal but not active fMAPK pathway activity. These new regulatory features of the Rho GTPase and p21-activated kinase module may extend to related pathways in other systems.

Intratumoral PD1+CD38+Tim3+ CD8+ T Cells in Pre-BCG Tumor Tissues Are Associated with Poor Responsiveness to BCG Immunotherapy in Patients with Non-Muscle Invasive Bladder Cancer.

Intravesical immunotherapy with Bacillus Calmette-Guerin (BCG) is a standard of care therapy for non-muscle invasive bladder cancer (NMIBC), which accounts for about 75% of newly diagnosed urothelial cancer. However, given the frequent recurrence and progression, identification of a pre-treatment biomarker capable of predicting responsiveness to BCG in NMIBC is of utmost importance. Herein, using multiparametric flow cytometry, we characterized CD8+ T cells from peripheral blood and tumor tissues collected from 27 pre-BCG patients bearing NMIBC to obtain immune correlates of bladder cancer prognosis and responsiveness to BCG therapy. We observed that intratumoral CD8+ T cell subsets were highly heterogenous in terms of their differentiation state and exist at different proportions in tumor tissues. Remarkably, among the different CD8+ T cell subsets present in the tumor tissues, the frequency of the terminally exhausted-like CD8+ T cell subset, marked as PD1+CD38+Tim3+ CD8+ T cells, was inversely correlated with a favorable outcome for patients and a responsiveness to BCG therapy. Moreover, we also noted that the intratumoral abundance of the progenitor exhausted-like PD1+CD8+ T cell subset in pre-BCG NMIBC tumor tissues was indicative of better recurrence-free survival after BCG. Collectively, our study led to the identification of biomarkers that can predict the therapeutic responsiveness of BCG in NMIBC.

Detection of interface flaws in Concrete-FRP composite structures using linear and nonlinear ultrasonics based techniques.

Timely and appropriate retrofitting of existing structures holds paramount importance to ensure the structural integrity and sustainability. Fiber Reinforced Polymer (FRP) composites with high corrosion resistance, strength and durability, have been increasingly used in recent years for retrofitting of concrete infrastructure. The effectiveness of retrofitting is primarily dependent on the appropriate integrity at the interface between FRP and concrete substrate. Presence of any interface flaw can jeopardize the structural performance. In the present study, investigations are carried out to detect the early stage flaws at the FRP-concrete interface using ultrasonic waves. Artificial flaws of different size are introduced in the adhesive (epoxy) layer of carbon based FRP composite concrete beam. Rayleigh waves (at different frequencies) are generated for measuring the response from different FRP composite-concrete specimens. The specimens consist of three different types of materials, namely, concrete, epoxy and FRP. Two different input excitation frequencies, i.e., 75 KHz and 250 KHz, are tried out during the experimental investigations. The output signals are processed using different linear and nonlinear ultrasonic methods. Numerical simulations are also performed to better understand the wave signals' interactions with the multi-layer composite medium. The results showed that the linear ultrasonic methods are not able to provide a consistent information on presence and extent of flaws. Nonlinear ultrasonic methods showed significantly better performance for characterizing both small and large flaws considered in this investigation. Sensitivity analysis reveals that relatively new and promising nonlinear ultrasonic technique, namely, the Sideband Peak Count-Index (SPC-I) performs remarkably well for detection of flaws in FRP-concrete interface.

New Features Surrounding the Cdc42-Ste20 Module that Regulates MAP Kinase Signaling in Yeast.

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular responses, including the response to stress and cell differentiation, and are highly conserved across eukaryotes from yeast to humans. In yeast, the canonical activation of several MAPK pathways includes the interaction of the small GTPase Cdc42p with the p21-activated kinase (PAK) Ste20p. We recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is turned over by the 26S proteasome. Ste20p was stabilized when bound to Cdc42p, presumably to sustain MAPK pathway signaling. Ste20p is a major conduit by which signals flow through the fMAPK pathway; however, by genetic approaches we also identified a Ste20p-independent branch of the fMAPK pathway. Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p, while Ste20p-independent signaling required the fMAPK pathway adaptor and Cdc42p-interacting protein, Bem4p. Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p in the fMAPK pathway, Rga1p, which also dampened basal but not active fMAPK pathway activity. Finally, the polarity adaptor and Cdc42p-interacting protein, Bem1p, which also regulates the fMAPK pathway, interacts with the tetra-span protein Sho1p, connecting a sensor at the plasma membrane to a protein that regulates the GTPase module. Collectively, these data reveal new regulatory features surrounding a Rho-PAK module that may extend to other pathways that control cell differentiation.

Slow-cycling (dormant) cancer cells in therapy resistance, cancer relapse and metastasis.

It is increasingly appreciated that cancer cell heterogeneity and plasticity constitute major barriers to effective clinical treatments and long-term therapeutic efficacy. Research in the past two decades suggest that virtually all treatment-naive human cancers harbor subsets of cancer cells that possess many of the cardinal features of normal stem cells. Such stem-like cancer cells, operationally defined as cancer stem cells (CSCs), are frequently quiescent and dynamically change and evolve during tumor progression and therapeutic interventions. Intrinsic tumor cell heterogeneity is reflected in a different aspect in that tumors also harbor a population of slow-cycling cells (SCCs) that are not in the proliferative cell cycle and thus are intrinsically refractory to anti-mitotic drugs. In this Perspective, we focus our discussions on SCCs in cancer and on various methodologies that can be employed to enrich and purify SCCs, compare the similarities and differences between SCCs, CSCs and cancer cells undergoing EMT, and present evidence for the involvement of SCCs in surviving anti-neoplastic treatments, mediating tumor relapse, maintaining tumor dormancy and mediating metastatic dissemination. Our discussions make it clear that an in-depth understanding of the biological properties of SCCs in cancer will be instrumental to developing new therapeutic strategies to prevent tumor relapse and distant metastasis.

Spatiotemporal control of pathway sensors and cross-pathway feedback regulate a differentiation MAPK pathway in yeast.

Mitogen-activated protein kinase (MAPK) pathways control cell differentiation and the response to stress. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the pathway that regulates the response to osmotic stress (HOG). Here, we show that the two pathways exhibit different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding mucin sensors that regulate the pathways. Cross-pathway regulation from the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. We also show that the shared tetraspan protein Sho1p, which has a dynamic localization pattern throughout the cell cycle, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway regulation, control a MAPK pathway that regulates cell differentiation in yeast.

Nonlinear ultrasonics-based technique for monitoring damage progression in reinforced concrete structures.

In reinforced concrete (RC), material nonlinearity is evident even in its undamaged state due to the inherent microstructure. In the present work, damage progression in RC structure at different levels of damage is investigated using linear and nonlinear ultrasonic techniques. The primary focus of this study is to monitor the structure from its initiation stage(s) of damage to advanced stages. Ultrasonic velocity tomography is first implemented to identify the weaker regions and map any damage occurring at various levels of loading. Two critical regions are identified from ultrasonic tomography and further damage characterization is carried out using various ultrasonic techniques to quantitatively assess the progression of damage in these two regions. The linear ultrasonic techniques such as time-of-flight (TOF) and attenuation, and the nonlinear ultrasonic techniques such as sub- and super- harmonic, energy distribution, etc. are employed to detect the damage progression. It is found that the changes in linear parameters due to damage progression in RC structure are often insignificant and inconsistent. However, some of the nonlinear ultrasonics-based techniques are found to be very efficient to monitor the damage progression. A relatively new and promising nonlinear ultrasonic technique, namely the sideband peak count-index (or SPC-I) provides a very clear and consistent indication of damage at the early stage. The present study shows that during the initial stages of damage, SPC-I based nonlinear technique performs significantly better (at both regions as identified through ultrasonic tomography) than other linear and nonlinear techniques, whereas at higher damage stage the superiority of this nonlinear ultrasonic technique slowly diminishes. The present study also shows that out of all nonlinear ultrasonics-based techniques considered here, SPC-I technique provides the highest sensitivity to the damage progression and can be effectively used as a very robust nonlinear ultrasonic tool for identifying the onset and progression of damage in RC structures.

Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways.

Reactive oxygen species (ROS) that exceed the antioxidative capacity of the cell can be harmful and are termed oxidative stress. Increasing evidence suggests that ROS are not exclusively detrimental, but can fulfill important signaling functions. Recently, we have been able to demonstrate that a NADPH oxidase-like enzyme (termed Yno1p) exists in the single-celled organism Saccharomyces cerevisiae. This enzyme resides in the peripheral and perinuclear endoplasmic reticulum and functions in close proximity to the plasma membrane. Its product, hydrogen peroxide, which is also produced by the action of the superoxide dismutase, Sod1p, influences signaling of key regulatory proteins Ras2p and Yck1p/2p. In the present work, we demonstrate that Yno1p-derived H2O2 regulates outputs controlled by three MAP kinase pathways that can share components: the filamentous growth (filamentous growth MAPK (fMAPK)), pheromone response, and osmotic stress response (hyperosmolarity glycerol response, HOG) pathways. A key structural component and regulator in this process is the actin cytoskeleton. The nucleation and stabilization of actin are regulated by Yno1p. Cells lacking YNO1 showed reduced invasive growth, which could be reversed by stimulation of actin nucleation. Additionally, under osmotic stress, the vacuoles of a ∆yno1 strain show an enhanced fragmentation. During pheromone response induced by the addition of alpha-factor, Yno1p is responsible for a burst of ROS. Collectively, these results broaden the roles of ROS to encompass microbial differentiation responses and stress responses controlled by MAPK pathways.

Functions for Cdc42p BEM adaptors in regulating a differentiation-type MAP kinase pathway.

Ras homology (Rho) GTPases regulate cell polarity and signal transduction pathways to control morphogenetic responses in different settings. In yeast, the Rho GTPase Cdc42p regulates cell polarity, and through the p21-activated kinase Ste20p, Cdc42p also regulates mitogen-activated protein kinase (MAPK) pathways (mating, filamentous growth or fMAPK, and HOG). Although much is known about how Cdc42p regulates cell polarity and the mating pathway, how Cdc42p regulates the fMAPK pathway is not clear. To address this question, Cdc42p-dependent MAPK pathways were compared in the filamentous (Σ1278b) strain background. Each MAPK pathway showed a unique activation profile, with the fMAPK pathway exhibiting slow activation kinetics compared with the mating and HOG pathways. A previously characterized version of Cdc42p, Cdc42pE100A, that is specifically defective for fMAPK pathway signaling, was defective for interaction with Bem4p, the pathway-specific adaptor for the fMAPK pathway. Corresponding residues in Bem4p were identified that were required for interaction with Cdc42p and fMAPK pathway signaling. The polarity adaptor Bem1p also regulated the fMAPK pathway. Versions of Bem1p defective for recruitment of Ste20p to the plasma membrane, intramolecular interactions, and interaction with the GEF, Cdc24p, were defective for fMAPK pathway signaling. Bem1p also regulated effector pathways in different ways. In some pathways, multiple domains of the protein were required for its function, whereas in other pathways, a single domain or function was needed. Genetic suppression tests showed that Bem4p and Bem1p regulate the fMAPK pathway in an ordered sequence. Collectively, the study demonstrates unique and sequential functions for Rho GTPase adaptors in regulating MAPK pathways.

Colloidal Gels for Guiding Endothelial Cell Organization via Microstructural Morphology.

One of the fundamental challenges in vascular morphogenesis is to understand how the microstructural morphology of a 3D matrix can provide the spatial cues to organize the endothelial cells (ECs) into specific vascular structures. Colloidal gels can provide well-controlled distinct morphological matrices because these gels are formed by the aggregation of particles. By altering the aggregation mode, the spatial organization of the particles can be controlled to yield different microstructural morphology. To demonstrate this, colloidal aggregates and gels were developed by electrostatic interaction-mediated aggregation of cationic polyurethane (PU) colloidal particles by using low molecular weight electrolyte and polyelectrolyte to develop microstructurally different colloidal gels without altering their bulk elasticity. Compact dense colloidal aggregates with constricted voids were developed via electrolyte-mediated aggregation, whereas stranded branched networks with interconnected voids were formed via polyelectrolyte-mediated bridging interactions. Results show that the microstructure of aggregated colloids and gels can regulate EC organizations. Within endothelial matrices, ECs track the microstructure of particulate phase to interconnect with stranded colloidal network but cluster around compact colloidal aggregate. Similarly, in colloidal gels, ECs formed capillary-like structures by interconnecting along the stranded networks with enhanced cell-matrix interactions and increased cell extension but aggregated within the constricted voids of compact dense gel with enhanced cell-cell interaction. Both morphometric analysis and expression of EC markers corroborated the cell organizations in these gels. Using these colloidal gels, we demonstrated the role of 3D microstructural morphology as an important regulator for spatial guidance of ECs and simultaneously established the significance of colloidal gels as 3D matrix to regulate cellular morphogenesis.

Colloidal Gels with Tunable Mechanomorphology Regulate Endothelial Morphogenesis.

Endothelial morphogenesis into capillary networks is dependent on the matrix morphology and mechanical properties. In current 3D gels, these two matrix features are interdependent and their distinct roles in endothelial organization are not known. Thus, it is important to decouple these parameters in the matrix design. Colloidal gels can be engineered to regulate the microstructural morphology and mechanics in an independent manner because colloidal gels are formed by the aggregation of particles into a self-similar 3D network. In this work, gelatin based colloidal gels with distinct mechanomorphology were developed by engineering the electrostatic interaction mediated aggregation of particles. By altering the mode of aggregation, colloidal gels showed either compact dense microstructure or tenuous strand-like networks, and the matrix stiffness was controlled independently by varying the particle fraction. Endothelial Cell (EC) networks were favored in tenuous strand-like microstructure through increased cell-matrix and cell-cell interactions, while compact dense microstructure inhibited the networks. For a given microstructure, as the gel stiffness was increased, the extent of EC network was reduced. This result demonstrates that 3D matrix morphology and mechanics provide distinct signals in a bidirectional manner during EC network formation. Colloidal gels can be used to interrogate the angiogenic responses of ECs and can be developed as a biomaterial for vascularization.

Inositol polyphosphates regulate and predict yeast pseudohyphal growth phenotypes.

Pseudohyphal growth is a nutrient-regulated program in which budding yeast form multicellular filaments of elongated and connected cells. Filamentous growth is required for virulence in pathogenic fungi and provides an informative model of stress-responsive signaling. The genetics and regulatory networks modulating pseudohyphal growth have been studied extensively, but little is known regarding the changes in metabolites that enable pseudohyphal filament formation. Inositol signaling molecules are an important class of metabolite messengers encompassing highly phosphorylated and diffusible inositol polyphosphates (InsPs). We report here that the InsP biosynthesis pathway is required for wild-type pseudohyphal growth. Under nitrogen-limiting conditions that can induce filamentation, InsPs exhibit characteristic profiles, distinguishing the InsP7 pyrophosphate isoforms 1PP-InsP5 and 5PP-InsP5. Deletion and overexpression analyses of InsP kinases identify elevated levels of 5PP-InsP5 relative to 1PP-InsP5 in mutants exhibiting hyper-filamentous growth. Overexpression of KCS1, which promotes formation of inositol pyrophosphates, is sufficient to drive pseudohyphal filamentation on medium with normal nitrogen levels. We find that the kinases Snf1p (AMPK), Kss1p, and Fus3p (MAPKs), required for wild-type pseudohyphal growth, are also required for wild-type InsP levels. Deletion analyses of the corresponding kinase genes indicate elevated InsP3 levels and an absence of exaggerated 5PP-InsP5 peaks in trace profiles from snf1Δ/Δ and kss1Δ/Δ mutants exhibiting decreased pseudohyphal filamentation. Elevated 5PP-InsP5:1PP-InsP5 ratios are present in the hyperfilamentous fus3 deletion mutant. Collectively, the data identify the presence of elevated 5PP-InsP5 levels relative to other inositol pyrophosphates as an in vivo marker of hyper-filamentous growth, while providing initial evidence for the regulation of InsP signaling by pseudohyphal growth kinases.

Spatial landmarks regulate a Cdc42-dependent MAPK pathway to control differentiation and the response to positional compromise.

A fundamental problem in cell biology is to understand how spatial information is recognized and integrated into morphogenetic responses. Budding yeast undergoes differentiation to filamentous growth, which involves changes in cell polarity through mechanisms that remain obscure. Here we define a regulatory input where spatial landmarks (bud-site-selection proteins) regulate the MAPK pathway that controls filamentous growth (fMAPK pathway). The bud-site GTPase Rsr1p regulated the fMAPK pathway through Cdc24p, the guanine nucleotide exchange factor for the polarity establishment GTPase Cdc42p. Positional landmarks that direct Rsr1p to bud sites conditionally regulated the fMAPK pathway, corresponding to their roles in regulating bud-site selection. Therefore, cell differentiation is achieved in part by the reorganization of polarity at bud sites. In line with this conclusion, dynamic changes in budding pattern during filamentous growth induced corresponding changes in fMAPK activity. Intrinsic compromise of bud-site selection also impacted fMAPK activity. Therefore, a surveillance mechanism monitors spatial position in response to extrinsic and intrinsic stress and modulates the response through a differentiation MAPK pathway.

Regulation of Toll signaling and inflammation by ß-arrestin and the SUMO protease Ulp1.

The Toll signaling pathway has a highly conserved function in innate immunity and is regulated by multiple factors that fine tune its activity. One such factor is ß-arrestin Kurtz (Krz), which we previously implicated in the inhibition of developmental Toll signaling in the Drosophila melanogaster embryo. Another level of controlling Toll activity and immune system homeostasis is by protein sumoylation. In this study, we have uncovered a link between these two modes of regulation and show that Krz affects sumoylation via a conserved protein interaction with a SUMO protease, Ulp1. Loss of function of krz or Ulp1 in Drosophila larvae results in a similar inflammatory phenotype, which is manifested as increased lamellocyte production; melanotic mass formation; nuclear accumulation of Toll pathway transcriptional effectors, Dorsal and Dif; and expression of immunity genes, such as Drosomycin. Moreover, mutations in krz and Ulp1 show dosage-sensitive synergistic genetic interactions, suggesting that these two proteins are involved in the same pathway. Using Dorsal sumoylation as a readout, we found that altering Krz levels can affect the efficiency of SUMO deconjugation mediated by Ulp1. Our results demonstrate that ß-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation.