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.

Fig. 1.. Homogenized Xenopus laevis egg extracts self-organize into cell-like compartments.

(A) Schematic diagram of experimental procedures. (B) The design of the chamber used to image extracts. (C) Bright-field microscopy showing that homogenized Xenopus laevis egg cytoplasmic extracts spontaneously organized into cell-like compartments in a multi-millimeter scale field. Pattern formation dynamics in bright-field, tubulin and ER channels are presented in movie S1 and S2. (D) Spatial organization of microtubules, endoplasmic reticulum, nuclei and mitochondria in the cell-like compartments, visualized by added HiLyte 647 labeled porcine tubulin, ER-Tracker Red, GFP-NLS, and MitoTracker Red CMXRos. Pattern formation dynamics are presented in movie S3. The still images shown in (D) are from the last frame of movie S3, at 60.6 min after imaging began.

Fig. 2.. The formation of cell-like compartments in egg extracts does not require added demembranated sperm nuclei.

(A) Time lapse montage of cell-like compartment formation in an interphase egg extract with approximately 160 demembranated Xenopus laevis sperm nuclei added per microliter of extract. (B) Time lapse montage of cell-like compartment formation in an extract from the same experiment as (A), but with no sperm nuclei added. Dynamics of pattern formation in (A) and (B) are presented in movie S4. (A) and (B) share the scale bar located at the bottom of (B). (C) Interphase egg extracts supplemented with different concentrations of sperm nuclei, viewed at a lower magnification. Microtubules are shown in green, ER in red, and nuclei in cyan. Dynamics of pattern formation are presented in movie S5. (D) Effective radii of cell-like compartments from the same experiment as (C). Blue symbols indicate radii of nucleated compartments and red symbols non-nucleated ones. For each compartment, its area A, volume V and effective radius r are interconvertible by A = π·r² and V = A·d, where d = 120 μm is the thickness of the spacers (Fig. 1B). For example, an effective radius of 200 μm corresponds to a cross-sectional area of 0.125 mm² and a volume of 15 nL.

Fig. 3.. Formation of cell-like compartments requires microtubule polymerization and a threshold concentration of cytoplasm.

(A) Nocodazole, a microtubule polymerization inhibitor, abolished the formation of cell-like compartments. Dynamics of the process is shown in movie S6. (B) Effect of dilution on pattern formation. Sperm nuclei, fluorescently labeled tubulin, ER-Tracker and mCherry-NLS were added after the indicated dilutions of extracts were performed, thus the final concentrations of these supplements were the same across all extracts. mCh-NLS indicates mCherry-NLS.

Fig. 4.. Cell-like compartments are capable of self-replication.

Time lapse images of cell-like compartments performing a complete mitotic cell cycle in a cycling Xenopus laevis egg extract. Formation of the mother compartments became apparent at 15 min, and was completed by 23 min, when duplicated centrosomes could also be seen. A mitotic spindle subsequently formed at 33 min. The mother compartments completed their divisions at 40 min, each giving rise to two daughter compartments. Detailed dynamics of the process are shown in movie S7.