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  • Light-inducible activation of cell cycle progression in Xenopus egg extracts under microfluidic confinement.

Light-inducible activation of cell cycle progression in Xenopus egg extracts under microfluidic confinement.

Lab on a chip (2019-09-24)
Jitender Bisht, Paige LeValley, Benjamin Noren, Ralph McBride, Prathamesh Kharkar, April Kloxin, Jesse Gatlin, John Oakey
ABSTRACT

Cell-free Xenopus egg extract is a widely used and biochemically tractable model system that allows recapitulation and elucidation of fundamental cellular processes. Recently, the introduction of microfluidic extract manipulation has enabled compartmentalization of bulk extract and a newfound ability to study organelles on length scales that recapitulate key features of cellular morphology. While the microfluidic confinement of extracts has produced a compelling platform for the in vitro study of cell processes at physiologically-relevant length scales, it also imposes experimental limitations by restricting dynamic control over extract properties. Here, we introduce photodegradable polyethylene glycol (PEG) hydrogels as a vehicle to passively and selectively manipulate extract composition through the release of proteins encapsulated within the hydrogel matrix. Photopatterned PEG hydrogels, passive to both extract and encapsulated proteins, serve as protein depots within microfluidic channels, which are subsequently flooded with extract. Illumination by ultraviolet light (UV) degrades the hydrogel structures and releases encapsulated protein. We show that an engineered fluorescent protein with a nuclear localization signal (GST-GFP-NLS) retains its ability to localize within nearby nuclei following UV-induced release from hydrogel structures. When diffusion is considered, the kinetics of nuclear accumulation are similar to those in experiments utilizing conventional, bulk fluid handling. Similarly, the release of recombinant cyclin B Δ90, a mutant form of the master cell cycle regulator cyclin B which lacks the canonical destruction box, was able to induce the expected cell cycle transition from interphase to mitosis. This transition was confirmed by the observation of nuclear envelope breakdown (NEBD), a phenomenological hallmark of mitosis, and the induction of mitosis-specific biochemical markers. This approach to extract manipulation presents a versatile and customizable route to regulating the spatial and temporal dynamics of cellular events in microfluidically confined cell-free extracts.

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Sigma-Aldrich
Anticorpo anti-fosfo-istone H3 (Ser10) marcatore di mitosi, Upstate®, from rabbit
Millipore
Glutathione−Agarose, lyophilized powder
Sigma-Aldrich
Poly(dimethylsiloxane), viscosity 1.0 cSt (25 °C)
General Purpose Viscosity Standard; UKAS ISO/IEC17025 and ISO 17034 certified, viscosity 4599 mPa.s (25 °C)