Size Scaling of Microtubule Assemblies in Early Xenopus Embryos

Abstract

The first 12 cleavage divisions in Xenopus embryos provide a natural experiment in size scaling, as cell radius decreases ∼16-fold with little change in biochemistry. Analyzing both natural cleavage and egg extract partitioned into droplets revealed that mitotic spindle size scales with cell size, with an upper limit in very large cells. We discuss spindle-size scaling in the small- and large-cell regimes with a focus on the "limiting-component" hypotheses. Zygotes and early blastomeres show a scaling mismatch between spindle and cell size. This problem is solved, we argue, by interphase asters that act to position the spindle and transport chromosomes to the center of daughter cells. These tasks are executed by the spindle in smaller cells. We end by discussing possible mechanisms that limit mitotic aster size and promote interphase aster growth to cell-spanning dimensions.

Figure 1.

Spindle-size scaling in Xenopus laevis. A–D show confocal images of eggs and early embryos fixed at different stages, stained for tubulin (red) and DNA (green), cleared and imaged by confocal microscopy. Embryos containing metaphase spindles were selected for analysis. (A) Unfertilized egg with meiosis-II spindle (blue arrow). (B) First mitosis. Note scaling mismatch between the spindle and egg. (C,D) Cleavage stages. (E) Spindle lengths and cell lengths derived from confocal images like A–D. Note spindle length is approximately constant in the large-cell regime and scales with cell size in the small-cell regime. (F) Spindle assembled in a droplet of unfertilized egg extract containing fluorescent probes suspended in oil and imaged live. aNuMA, anti-nuclear mitotic apparatus. (A–E from Wühr et al. 2008; adapted, with permission, from the author; F is an unpublished image provided by Jesse Gatlin, University of Wyoming, which is similar to images in Hazel et al. 2013.)

Figure 2.

Hypothetical roles of component limitation in spindle-size scaling. (A) Small-cell regime. Spindle size is limited by depletion of one or more limiting components, drawn as a red dot, from the whole cell. (B) Large-cell regime hypothesizing spindle size is not component limited. Some spindle-intrinsic mechanisms, drawn as a blue spring, limits length. Candidate mechanisms include bounded single-microtubule lengths, opposed motor activities, and a mechanochemical switch at the pole (see text for references). (C) Large-cell regime hypothesizing spindle size is component limited. The limiting component is locally activated by a reaction–diffusion system centered on chromosomes. The spindle depletes the activated component within the activation radius, leading to component limitation. A candidate reaction–diffusion system is RCC1-Ran-RanGAP1, which activates two potentially limiting spindle components, TPX2 and HURP.

Figure 3.

Aster scaling and centrosome orientation in compressed eggs. (A) In a famous experiment, Hertwig compressed amphibian eggs after fertilization, which oriented the first cleavage plane to cut across the short axis of the cell. (Image from Hertwig 1893.) (B–E) Repeat of Hertwig’s egg compression experiment in Xenopus laevis followed by fixation at different stages and staining for tubulin (yellow–green) and the microtubule nucleation factor γ-tubulin (red). (From Wühr et al. 2010; adapted, with permission, from the authors.) (B,B′) Prophase of first mitosis, just before nuclear envelope breakdown. The remains of the cell-spanning sperm aster can be faintly seen near the egg periphery. Note that centrosomes (red) are already oriented correctly (N–S) to define the future division plane (E–W). (C, C′) Metaphase of first mitosis. The scaling mismatch between spindle and cell is evident. Astral microtubules are poorly visualized, but appear short compared with egg radius, even in the compressed egg. (D) Early interphase. Sister asters from the mitotic spindle are growing out toward the cortex. Their periphery grows outward at 20–30 µm/min, the centrosomes and nuclei at their centers move apart at about half this rate. (E) Early cytokinesis. The expanding sister asters have now grown to touch the cortex. The cleavage furrow has just started to ingress at the overlap zone between the two asters (blue arrowheads). In this example, the furrow is imperfectly aligned relative to the long axis of the compressed cell (by ∼7°) (see Wühr et al. 2010 for alignment statistics).

Figure 4.

Models for aster and microtubule (MT) length scaling. The top row illustrates a model for aster growth in mitosis (A) and two related models for growth in interphase (B,C). A quadrant of the aster is shown. The bottom row illustrates individual microtubule dynamics for each model, in which length fluctuations are caused by dynamic instability of plus ends. (A) Bounded mitotic aster made from bounded microtubules. Nucleation is restricted to the centrosome. Aster radius is determined by the length scale of individual microtubules. A similar model with unbounded microtubules would suffice for interphase asters in small cells. (B) Unbounded interphase aster made from unbounded microtubules. Both the aster and individual microtubules grow to cell-spanning dimensions. Nucleation away from centrosomes maintains a high density of microtubules at the periphery of the growing aster. (C) Unbounded interphase aster made from bounded microtubules. Individual microtubules are short compared with aster radius. Nucleation away from centrosomes is required at a faster rate than in B to promote aster growth.