Microfluidics has great potential, but the complexity of fabricating and operating devices has limited its use. Here we describe a method — Freestyle Fluidics — that overcomes many key limitations. In this method, liquids are confined by fluid (not solid) walls. Aqueous circuits with any 2D shape are printed in seconds on plastic or glass Petri dishes; then, interfacial forces pin liquids to substrates, and overlaying an immiscible liquid prevents evaporation. Confining fluid walls are pliant and resilient; they self-heal when liquids are pipetted through them. We drive flow through a wide range of circuits passively by manipulating surface tension and hydrostatic pressure, and actively using external pumps. Finally, we validate the technology with two challenging applications — triggering an inflammatory response in human cells and chemotaxis in bacterial biofilms. This approach provides a powerful and versatile alternative to traditional microfluidics.

SignificanceDespite improvements in our ability to manipulate ever-smaller volumes, most workflows in cell biology still use volumes of many microliters. We describe a method for creating microfluidic arrangements containing submicroliter volumes. It exploits interfacial forces dominant at the microscale to confine liquids with fluid (not solid) walls. We demonstrate many basic manipulations required for cell culture and some widely used downstream workflows. The method eliminates many problems associated with the fabrication of conventional microfluidic devices, thereby providing a simple on-demand approach for fabrication of microfluidic devices using materials familiar to biologists.

Plasma membrane electron transport (tPMET) pathways have been identified in all living cells, and a wide variety of tools have been used to study these processes. In our laboratory we have used the cell-impermeable tetrazolium dye WST-1, together with the mitochondrial gene knockout (rho0) cell model, to investigate one of these pathways. We have shown that growth of HL60rho0 cells is dependent on oxygen, and that these cells consume oxygen at the cell surface. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and WST-1 reduction suggest that both systems share a common tPMET pathway. In support of this, oxygen was shown to compete with the intermediate electron acceptor that mediates WST-1 reduction, for reducing electrons. The observation that tPMET activity is higher in rho0 cells compared to their mitochondrially-competent counterparts was shown to be the result of competition between the mitochondrial and plasma membrane electron transport systems for intracellular reducing equivalents. Elevated rates of dye reduction appear to be mediated through increased expression of the key components of tPMET, which include the cell surface NADH oxidase, CNOX. These findings have played a critical role in shaping our current understanding of the mechanisms of this particular pathway of tPMET.

The S. cerevisiae kinesin Kip2 stabilises astral microtubules (MTs) and facilitates spindle positioning through transport of MT-associated proteins, such as the yeast CLIP-170 homologue Bik1, dynein and the adenomatous-polyposis-coli-related protein Kar9 to the plus ends of astral MTs. Here, we show that Kip2 associates with its processivity factor Bim1, the yeast homologue of the plus-end-tracking protein EB1. This interaction requires an EB1-binding motif in the N-terminal extension of Kip2 and is negatively regulated by phosphorylation through Mck1, the yeast glycogen synthase kinase 3. In addition, Mck1-dependent phosphorylation decreases the intrinsic MT affinity of Kip2. Reduction in Kip2 phosphorylation leads to stabilisation of astral MTs, and accumulation of Kip2, dynein and Kar9 at MT plus ends, whereas loss of Mck1 function leads to defects in spindle positioning. Furthermore, we provide evidence that a subpopulation of Mck1 at the bud-cortex phosphorylates Kip2. We propose that yeast GSK-3 spatially controls astral MT dynamics and the loading of dynein and Kar9 on astral MT plus ends by regulating Kip2 interactions with Bim1 and MTs.

The precise temporal and spatial concentration of microtubule-associated proteins (MAPs) within the cell is fundamental to ensure chromosome segregation and correct spindle positioning. MAPs form an intricate web of interactions among each other and compete for binding sites on microtubules. Therefore, when assessing cellular phenotypes upon MAP up-or downregulation, it is important to consider the protein interaction network between individual MAPs. Here, we show that changes in the amounts of the spindle positioning factor Kar9 specifically affect the distribution of yeast EB1 on spindle microtubules, without influencing other microtubule-associated interacting partners of Kar9, i.e. yeast XMAP215 and CLIP-170. Alterations in the distribution of yeast EB1 explain chromosome segregation defects upon knockout, overexpression or stabilization of Kar9 and provide an example for non-linear effects on MAP behavior after perturbation of their equilibrium.