Edited by Berend WieringaKeywords: Hsf1 Msn2/4 Phosphorylation PKA Rim15 a b s t r a c t Rim15 kinase, a downstream effector of PKA and TORC1 signaling pathways, initiates the quiescent program upon nutrient starvation via induction of genes whose expression depends on transcription factors Msn2, Msn4, and Gis1. Here, we demonstrate that Rim15 also induces expression of Hsf1 target genes upon glucose depletion by both transcriptional activation and stabilization of the transcripts. Rim15 phosphorylates Hsf1 in vitro, suggesting that Rim15 might directly activate Hsf1. In addition, Igo1 and Igo2, Rim15 substrate proteins involved in mRNA stabilization, regulate mRNA levels of Hsf1 target genes. We also show that Rim15 can phosphorylate Msn2, but not Gis1, in vitro, implying different mechanisms for the activation of these transcription factors. Structured summary of protein interactions:Rim15 phosphorylates Msn2 by protein kinase assay (View interaction) Rim15 phosphorylates Igo1 by protein kinase assay (View interaction) Rim15 phosphorylates Hsf1 by protein kinase assay (View interaction) Yak1 phosphorylates Hsf1 by protein kinase assay (View interaction)

SummaryYak1 is a member of an evolutionarily conserved family of Ser/Thr protein kinases known as dualspecificity Tyr phosphorylation-regulated kinases (DYRKs). Yak1 was originally identified as a growth antagonist, which functions downstream of Ras/PKA signalling pathway. It has been known that Yak1 is phosphorylated by PKA in vitro and is translocated to the nucleus upon nutrient deprivation. However, the regulatory mechanisms for Yak1 activity and localization are largely unknown. In the present study, we investigated the role of PKA and Bmh1, a yeast 14-3-3 protein, in regulation of Yak1. We demonstrate that PKA-dependent phosphorylation of Yak1 on Ser295 and two minor sites inhibits nuclear localization of Yak1. We also show that intramolecular autophosphorylation on at least four Ser/Thr residues in the noncatalytic N-terminal domain is required for full kinase activity of Yak1. The most potent autophosphorylation site, Thr335, plays an essential role for Bmh1 binding in collaboration with a yet unidentified second binding site in the N-terminal domain. Bmh1 binding decreases the catalytic activity of Yak1 without affecting its subcellular localization. Since the binding of 14-3-3 proteins to Yak1 coincides with PKA activity, such regulatory mechanisms might allow cytoplasmic retention of an inactive form of Yak1 under high glucose conditions.

There are many proposed mechanisms by which single cells can be trapped; among them is the through-hole membrane for the characterization of individual microorganisms. Due to the small scale of the fabricated pores, the construction of through-hole membranes on a large scale and with relatively large areas faces many difficulties. This paper describes novel fabrication methods for a large-area, freestanding micro/nano through-hole membrane constructed from versatile membrane materials using through-hole membranes on a microfluidic chip (THMMC). This process can rapidly (<20 min) fabricate membranes with high fidelity multiscale hole size without residual layers. The through-hole site was easily customizable from the micro to the nanoscale, with a low or high aspect ratio giving rise to reliable membranes. Also, the rigidity and biocompatibility of the through-hole membrane are easily tunable by simple injection of versatile membrane materials to obtain a large area (up to 3600 mm). Membranes produced in this manner were then applied as a proof of concept for the isolation, cultivation, and quantification of individual micro-algal cells for selection with respect to the growth rate, while controlling the quorum sensing mediated metabolic and proliferative changes.

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Soft lithography and other techniques have been developed to investigate biological and chemical phenomena as an alternative to photolithography-based patterning methods that have compatibility problems. Here, a simple approach for nonlithographic patterning of liquids and gels inside microchannels is described. Using a design that incorporates strategically placed microstructures inside the channel, microliquids or gels can be spontaneously trapped and patterned when the channel is drained. The ability to form microscale patterns inside microfluidic channels using simple fluid drain motion offers many advantages. This method is geometrically analyzed based on hydrodynamics and verified with simulation and experiments. Various materials (i.e., water, hydrogels, and other liquids) are successfully patterned with complex shapes that are isolated from each other. Multiple cell types are patterned within the gels. Capillarity guided patterning (CGP) is fast, simple, and robust. It is not limited by pattern shape, size, cell type, and material. In a simple three-step process, a 3D cancer model that mimics cell-cell and cell-extracellular matrix interactions is engineered. The simplicity and robustness of the CGP will be attractive for developing novel in vitro models of organ-on-a-chip and other biological experimental platforms amenable to long-term observation of dynamic events using advanced imaging and analytical techniques.