Abstract
Interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of scientific applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, our microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression with single hyphal compartment resolution in response to carbon source starvation and exchange. Although the microfluidic device is demonstrated on filamentous fungi, the technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth, with applications ranging from bioenergy production to human health.
Highlights
Dynamics of a large cell population and understand cell-to-cell variability, a new approach is needed to confine many hyphae along parallel paths with controlled microenvironments
Combined with advanced imaging technologies, a variety of microfluidic cell trapping and culture systems have been developed for measuring cell proliferation and fate, gene expression, and intracellular signaling pathways of mammalian cells[15,16,17,18], yeasts[19,20,21,22,23] and bacteria[19,24,25,26] at the single cell level
Stanley et al.[29] employed a microfluidic platform to investigate the interactions of filamentous fungi with bacteria
Summary
Dynamics of a large cell population and understand cell-to-cell variability, a new approach is needed to confine many hyphae along parallel paths with controlled microenvironments. Microfluidic systems have been developed for culturing tip-growing cells including neurons[30,31] and plant pollen tubes[32]. These devices lack high-throughput capabilities and efficient compartmentalization of single cells for long-term tracking. Each conidium is trapped at the entrance of long, narrow growth channels of tunable dimensions, allowing individual hypha confinement throughout the experiments. This layout enables different filaments to be imaged in rapid succession by translating the microscope stage along one dimension. Coupled with live-cell imaging and fluorescent fusion protein technologies, we have probed single hypha growth kinetics, nuclear organization and migration, and long-term gene expression dynamics in response to carbon source switching
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