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Spontaneous emergence of cell-like organization in Xenopus egg extracts.

ABSTRACT: Every daughter cell inherits two things from its mother: genetic information and a spatially organized complement of macromolecular complexes and organelles. The extent to which de novo self-organization, as opposed to inheritance of an already organized state, can suffice to yield functional cells is uncertain. We used Xenopus laevis egg extracts to show that homogenized interphase egg cytoplasm self-organizes over the course of ~30 minutes into compartments 300 to 400 micrometers in length that resemble cells. Formation of these cell-like compartments required adenosine triphosphate and microtubule polymerization but did not require added demembranated sperm nuclei with their accompanying centrosomes or actin polymerization. In cycling extracts with added sperm, the compartments underwent multiple cycles of division and reorganization, with mother compartments giving rise to two daughters at the end of each mitotic cycle. These results indicate that the cytoplasm can generate much of the spatial organization and cell cycle function of the early embryo.

SUBMITTER: Cheng X

PROVIDER: S-EPMC7839252 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Spontaneous emergence of cell-like organization in <i>Xenopus</i> egg extracts.

Cheng Xianrui X Ferrell James E JE

Science (New York, N.Y.) 20191101 6465

Every daughter cell inherits two things from its mother: genetic information and a spatially organized complement of macromolecular complexes and organelles. The extent to which de novo self-organization, as opposed to inheritance of an already organized state, can suffice to yield functional cells is uncertain. We used <i>Xenopus laevis</i> egg extracts to show that homogenized interphase egg cytoplasm self-organizes over the course of ~30 minutes into compartments 300 to 400 micrometers in len ...[more]

PMID: 31672897

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Project description: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 ?90, 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.

Dataset Information

Spontaneous emergence of cell-like organization in Xenopus egg extracts.

Publications

Spontaneous emergence of cell-like organization in <i>Xenopus</i> egg extracts.

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