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. 2019 Oct 9;19(20):3499-3511.
doi: 10.1039/c9lc00569b.

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

Affiliations

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

Jitender Bisht et al. Lab Chip. .

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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Figures

Figure 1.
Figure 1.
Schematic showing device concept and overview of light-mediated control of protein release from hydrogel structures (not drawn to scale). a) Microfluidic chambers are filled with protein-laden hydrogel posts using photopatterned illumination (red). b) Unpolymerized PEGdiPDA-protein solution is flushed out and replaced with interphase extract (green) containing assembled nuclei (blue). c) Illumination with 365 nm UV light is used to degrade the hydrogel structures, releasing the protein into the surrounding interphase extract where it accumulates in nuclei (in the case of GST-GFP-NLS) or initiates NEBD and cycles the extract into biochemical mitosis (in the case of cyclin B ∆90).
Figure 2.
Figure 2.
Retention and light-induced release of a model fluorescent protein (GST-GFP-NLS). a) Plot showing the mean fluorescent signal of GST-GFP-NLS encapsulated in five octagonal hydrogel posts as a function of time. b) Representative images of fluorescent protein retention in hydrogel posts at 0 min (left), 30 min (middle), and 150 min (right). c) Graph of the fluorescent signal from GST-GFP-NLS posts both before and after irradiation with UV light (365 nm; three 1 min pulses separated by 30s intervals using a Black-Ray UV Lamp). The purple dashed line indicates the start of UV exposure. After approximately 50 min, the normalized fluorescent intensity levels off, suggesting diffusion into the surrounding extract has slowed due to homogenizing spatial concentrations. d) An array of GST-GFP-NLS hydrogel posts immediately before exposure to UV light (0 min) and then 5 min, 30 min, and 60 min thereafter. Statistics done using a student T-test with alpha equal to 0.05, * represents p < alpha. Scale bar = 75 µm.
Figure 3.
Figure 3.
Nuclei are able to take up GST-GFP-NLS following its UV-induced release from hydrogel posts. a) Images of nuclei assembled in egg extracts containing GST-mCherry-NLS (red) and treated as indicated. For the “interphase extract only” condition, extract was simply mixed with GST-GFP-NLS in a test tube at t = −10 min and then injected into an empty device. For the remaining two conditions, extracts were introduced into devices containing arrays of GST-GFP-NLS posts and either exposed to UV to degrade the posts and release GST-GFP-NLS (+UV) or not exposed to UV to test for protein retention (−UV). Scale bar = 50 µm. b) Quantification of nuclear import kinetics of GST-GFP-NLS. Mean nuclear GST-GFP-NLS fluorescence intensity (background subtracted) was measured over time for the three different conditions described in (a). Error bars represent standard deviation and data are shown from three independent experiment where n = number of nuclei (25 < n < 40). c) Upper panel shows a schematic of the experiment to determine if UV exposure negatively effects nuclear import kinetics of GST-GFP-NLS (not drawn to scale). Extracts prepared as described in (a) were injected into devices containing GST-GFP-NLS post arrays. Single posts were degraded by UV exposure using an aperture opening large enough to expose the post and proximal nuclei (upper image set) or with an aperture just large enough to expose only the post (lower image set). Scale bar = 100 µm. d) Import of GST-GFP-NLS was quantified as in panel (b) and plotted as a function of time. Error bars represent standard deviation. Data are shown from three independent experiments where n = number of nuclei (9 < n < 21).
Figure 4.
Figure 4.
Optically regulated release of cyclin B Δ90 into cell free egg extracts induces nuclear envelope breakdown (NEBD). a–c) Nuclei are assembled in egg extract containing FITC-labelled 150-kD dextran (green) and mAb414 (an antibody against nuclear pore complex) conjugated with Alexa 568 secondary antibody (red). Extracts with assembled nuclei were injected into microfluidic devices containing either cyclin B Δ90 containing hydrogel posts or blank hydrogel posts swelled in CSF-XB Buffer. a) and b) Cyclin B Δ90 encapsulating hydrogel posts were either left intact (−UV) or degraded (+UV) respectively. c) The blank hydrogel posts swelled in CSF-XB buffer were degraded (+UV). d) Nuclei were monitored with confocal microscopy and the extent of NEBD was quantified as the ratio of nuclear to cytoplasmic integrated fluorescence intensity over the span of 120 min. Data are shown from three independent experiments; shaded error bands represent standard deviation (SD). n = number of nuclei; n minimum = 15, n maximum = 26. Scale bar = 25 µm.
Figure 5.
Figure 5.
Light regulated release of cyclin B Δ90 into interphase extract induces mitosis as characterized by western blots. a) Mitotic induction as detected by phosphorylation of H3 Ser-10 and MAPK, where posts with cyclin B Δ90 are exposed to UV light. b) Bar graph showing the quantification of levels of phosphorylated H3 Ser-10 and MAPK protein normalized to total H3 and β-Tubulin controls, respectively, for six conditions: degraded posts containing cyclin B Δ90 (+UV), non-degraded posts containing cyclin B Δ90 (−UV), and degraded posts containing only CSF-XB buffer (+UV), non-degraded posts containing CSF-XB buffer (−UV), interphase extract alone (no posts, no UV), and positive control (cyclin B Δ90 added to interphase extract). Data are shown from three independent experiments with error bars representing standard deviation (S.D). Statistics done using student T-test, NS (non-significant) represents p > 0.05.

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