Size Scaling of Microtubule Assemblies in Early Xenopus Embryos

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.How components and processes within cells scale in size and rate with the size of the cell has become a topic of considerable interest in recent years (reviewed in Chan and Marshall 2012; Goehring and Hyman 2012; Levy and Heald 2012). For molecular machines with precise architectures (e.g., ribosomes), size is invariant, but rates of assembly and function, which depend on regulation and energy, might scale. For assemblies whose dimensions are not hard wired (e.g., cytoskeleton assemblies and organelles), both size and rate might scale. For pathways involving distributed biochemical change (e.g., the cell-cycle oscillator), size is not well defined, but rate might scale in interesting ways. Here, we will address only size scaling, and refer the reader to interesting recent progress on cell-cycle timing in early Xenopus embryos (Chang and Ferrell 2013; Tsai et al. 2014).Size-scaling relationships, which are part of the science of allometry, have long informed on whole organism physiology. Explicitly seeking them at the subcellular level is a newer endeavor, which in our mind holds two kinds of promise. It can inform on mechanism at the level of integrated cell physiology (e.g., on establishment of cleavage plane geometry). It can also inform on molecular processes involved in assembly growth and dynamics, and perhaps help us discern logic in often frustratingly complex molecular architectures. It is not obvious, for example, why ∼100 protein complexes are required to build a mitotic spindle in higher eukaryotes (Hutchins et al. 2010), when bacteria can segregate plasmids with far fewer (Salje et al. 2010). Part of the answer is the need for higher fidelity in the eukaryotic process. Gerhart and Kirschner (1997) also emphasized the need for highly adaptable processes in the evolution of higher eukaryotes. At least part of the complexity of subcellular assemblies might reflect the need for adaptable scaling of size, shape, and timing.Vertebrate embryos derived from large eggs provide a natural experiment in size scaling (Fig. 1). A Xenopus laevis egg, for example, is ∼1.2 mm in diameter. Following fertilization, it cleaves completely ∼12 times at an approximately constant rate of ∼2 divisions/h (most rates in early development are temperature dependent, and can vary up to about eightfold over the tolerated range). These divisions generate a quasispherical array of quasispherical cells that are, on average, smaller by 2¹²-fold in volume, or 2⁴-fold in radius. The first 12 divisions occur with little gene expression and little change in cell physiology, and it may be reasonable to assume approximately constant biochemistry (discussed below), other than periodic cell-cycle regulation. After the 12th division, cell physiology changes dramatically as part of the midblastula transition (MBT) (discussed below), which provides a natural cut-off for size-scaling investigations. An interesting and potentially informative complication is that cleaving amphibian embryos develop a gradient in blastomere sizes, with larger cells at the vegetal pole where yolk is more abundant (evident in Fig. 1C,D). Larger blastomeres tend to divide more slowly, which gradually eliminates division synchrony (Gerhart 1980).Open in a separate windowFigure 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.)Embryos from different species have pros and cons for experimental analysis of size scaling during early divisions. Amphibian eggs provide a large dynamic range in cell size, complete division, and quasispherical geometry of both cells and embryos. In the minus column, they are opaque unless fixed and cleared and difficult to manipulate using genetics. Undiluted, cell-free extracts from Xenopus eggs and early embryos provide access to live imaging and molecular analysis and recapitulate the biology of intact eggs, including scaling relationships (Wilbur and Heald 2013), but it is important to go back to the intact embryo to check validity of key findings where possible. Zebrafish eggs provide a transparent, genetically tractable vertebrate system with very large cells but incomplete cleavage at early stages. Caenorhabditis elegans and Drosophila embryos have excellent imaging and genetics, which are advantages for scaling analysis, especially rate scaling (e.g., Carvalho et al. 2009; Hara and Kimura 2013), but these embryos start smaller, so they provide a lower dynamic range for analyzing size-scaling behavior.