The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1. Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we observed phosphorylation of S31 in Dam1p purified from asynchronously growing yeast cells. Cells carrying high-copy TPK2 or a Dam1p phospho-mimetic S31D mutant displayed a reduction in Dam1p localization at the kinetochore, suggesting that PKA phosphorylation plays a role in assembly and/or stability of the Dam1 complex. Furthermore, we observed spindle defects associated with S31 phosphorylation. Finally, we find that phosphorylation of Dam1p on S31 is reduced when glucose is limiting as well as during a-factor arrest, conditions that inhibit PKA activity. These observations suggest that the PKA site of Dam1p participates in regulating kinetochore activity. While PKA is a wellestablished effector of glucose signaling, our work shows for the first time that glucose-dependent PKA activity has an important function in chromosome segregation.

Chemotropism is an essential response of organisms to external chemical gradients that direct the growth of cells toward the gradient source. Chemotropic responses between single cells have been studied using in vitro gradients of synthetically derived signaling molecules and helped to develop a better understanding of chemotropism in multiple organisms. However, dynamic changes including spatial changes to the gradient as well as fluctuations in levels of cell generated signaling molecules can result in the redirection of chemotropic responses, which can be difficult to model with synthetic peptides and single cells. An experimental system that brings together populations of cells to monitor the population-scale chemotropic responses yet retain single cell spatiotemporal resolution would be useful to further inform on models of chemotropism. Here, we describe a microfluidic platform that can measure the chemotropic response between populations of mating yeast A-and α-cells with spatiotemporal programmability and sensitivity by positioning cell populations side by side in calcium alginate hydrogels along semipermeable membranes with micrometer spatial control. The mating phenotypes of the yeast populations were clearly observed over hours. Three distinct responses were observed depending on the distance between the A-and α-cell populations: the cells either continued to divide, arrest, and develop a stereotypical polarized projection termed a "shmoo" toward the cells of opposite mating type or formed shmoos in random directions. The results from our studies of yeast mating suggest that the biofabricated microfluidic platform can be adopted to study population-scale, spatial-sensitive cell-cell signaling behaviors that would be challenging using conventional approaches.