FKS1 and FKS2 are alternative subunits of the glucan synthase complex, which is responsible for synthesizing 1,3-␤-glucan chains, the major structural polymer of the Saccharomyces cerevisiae cell wall. Expression of FKS1 predominates during growth under optimal conditions. In contrast, FKS2 expression is induced by mating pheromone, high extracellular [Ca 2؉ ], growth on poor carbon sources, or in an fks1 mutant. Induction of FKS2 expression in response to pheromone, CaCl 2 , or loss of FKS1 function requires the Ca 2؉ /calmodulindependent protein phosphatase calcineurin. Therefore, a double mutant in calcineurin (CNB1) and FKS1 is inviable due to a deficiency in FKS2 expression. To identify novel regulators of FKS2 expression, we isolated genes whose overexpression obviates the calcineurin requirement for viability of an fks1 mutant. Two components of the cell integrity signaling pathway controlled by the RHO1 G protein (MKK1 and RLM1) were identified through this screen. This signaling pathway is activated during growth at moderately high temperatures. We demonstrate that calcineurin and the cell integrity pathway function in parallel, through separable promoter elements, to induce FKS2 expression during growth at 39°C. Because RHO1 also serves as a regulatory subunit of the glucan synthase, our results define a regulatory circuit through which RHO1 controls both the activity of this enzyme complex and the expression of at least one of its components. We show also that FKS2 induction during growth on poor carbon sources is a response to glucose depletion and is under the control of the SNF1 protein kinase and the MIG1 transcriptional repressor. Finally, we show that FKS2 expression is induced as cells enter stationary phase through a SNF1-, calcineurin-, and cell integrity signalingindependent pathway.The cell wall of the budding yeast Saccharomyces cerevisiae is required to maintain cell shape and integrity (4,20). Vegetative proliferation requires that the cell remodels its wall to accommodate growth. The main structural components responsible for the rigidity of the yeast cell wall are 1,3-␤-linked glucan polymers with some branches through 1,6-␤ linkages. The biochemistry of the yeast enzyme complex that catalyzes the synthesis of 1,3-␤-glucan chains has been studied extensively (15, 29), and three genes that encode components of this complex have been identified. A pair of closely related genes, FKS1 and FKS2, encode alternative subunits of the 1,3-␤-glucan synthase (GS) (8,15,28,36). Either FKS1 or FKS2 function is sufficient for GS activity and cell viability. Additionally, the Rho1 GTPase is an essential regulatory subunit of the GS complex, serving to stimulate GS activity in a GTP-dependent manner (9, 35).A second essential function of RHO1 is to regulate the cell integrity signaling pathway by binding and activating protein kinase C (19, 33), which is encoded by PKC1 (25). Loss of PKC1 function results in a cell lysis defect that is attributable to a deficiency in cell wall construction (23,24,34 ...

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Near-infrared (NIR) fluorescent nanostructured materials have emerged as novel contrast agents for non-invasive bioimaging. Here we report a class of polymer-silica nanoparticles doped with a NIR fluorescent dye prepared through a facile one-pot strategy. Hydrophobic NIR fluorescent dyes such as IR 780 iodide could be easily encapsulated into the micellar core by self-assembly of amphiphilic triblock copolymer Pluronic F127. When subsequently adding silane in aqueous solution, nanoparticles with a cross-linked core and a hydrophilic PEG shell were formed. The structure of the as-obtained nanoparticles was confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The nanoparticles exhibited a well-defined spherical structure with a mean diameter of approximately 30 nm, and excellent monodispersity and stability in aqueous solution. In addition, the photo-stability of IR 780 was significantly improved by encapsulation into the nanoparticles. In vitro MTT assay with cell lines HEK293 and A431 demonstrated that the IR 780 loaded nanoparticles (termed as IR780@NPs) were biocompatible. In vivo sentinel lymph node imaging revealed that the fluorescent intensity and retention time of the IR780@NPs were clearly superior to its constituent free dye, making it amenable to in vivo bioimaging. Further in vivo tumor imaging indicated that IR780@NPs have a longer retention time and much higher accumulation on the tumor site compared to free dye after intravenous administration. Overall this hydrophilic NIR fluorescent contrast agent exhibits excellent photophysical characteristics and low cytotoxicity, and holds a strong promise for a variety of applications including bioimaging and therapy.

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