Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

• In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.

Ämnesord

NATURVETENSKAP  -- Fysik (hsv//swe)

NATURAL SCIENCES  -- Physical Sciences (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Läkemedelskemi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Medicinal Chemistry (hsv//eng)

Nyckelord

Microfluidics

Optical manipulation

Signal transduction pathways

Single-cell analysis

Biological techniques

Cell array

Cell signaling

Cell-to-cell variation

Diffusion rate

Dynamic signals

HOG pathway

Inlet channels

Kinase inhibitors

Micro fluidic system

Nuclear localization

Osmolarity

Osmotic stress

Sequential treatments

Single-cell level

Singlecell analysis

Subcellular localizations

Yeast cell

Cells

Cytology

Fluidic devices

Glycerol

Micromanipulators

Optical tweezers

Signal transduction

Yeast

Molecular biology

Microfluidics

Optical manipulation

Signal transduction pathways

Single-cell analysis

Publikations- och innehållstyp

ref (ämneskategori)

kon (ämneskategori)