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 b-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 b-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation.
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.
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.
How Rho GTPases are directed to effector pathways is an important question. We show here that BEM-type adaptors play unique roles in sequentially directing Cdc42 to an effector MAPK pathway. Our study may provide insight into Rho GTPase specification in other systems.
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