Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Size Scaling of Microtubule Assemblies in Early Xenopus Embryos Timothy J. Mitchison1,2, Keisuke Ishihara1,2, Phuong Nguyen1,2, and Martin Wu¨hr1,2 1Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115 2Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Correspondence: [email protected] 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. ow components and processes within cells embryos (Chang and Ferrell 2013; Tsai et al. Hscale in size and rate with the size of the cell 2014). has become a topic of considerable interest in Size-scaling relationships, which are part of recent years (reviewed in Chan and Marshall the science of allometry, have long informed on 2012; Goehring and Hyman 2012; Levy and whole organism physiology. Explicitly seeking Heald 2012). For molecular machines with pre- them at the subcellular level is a newer endeavor, cise architectures (e.g., ribosomes), size is in- which in our mind holds two kinds of promise. variant, but rates of assembly and function, It can inform on mechanism at the level of in- which depend on regulation and energy, might tegrated cell physiology (e.g., on establishment scale. For assemblies whose dimensions are not of cleavage plane geometry). It can also inform hard wired (e.g., cytoskeleton assemblies and on molecular processes involved in assembly organelles), both size and rate might scale. For growth and dynamics, and perhaps help us dis- pathways involving distributed biochemical cern logic in often frustratingly complex molec- change (e.g., the cell-cycle oscillator), size is ular architectures. It is not obvious, for exam- not well defined, but rate might scale in inter- ple, why 100 protein complexes are required esting ways. Here, we will address only size scal- to build a mitotic spindle in higher eukaryotes ing, and refer the reader to interesting recent (Hutchins et al. 2010), when bacteria can segre- progress on cell-cycle timing in early Xenopus gate plasmids with far fewer (Salje et al. 2010). Editors: Rebecca Heald, Iswar K. Hariharan, and David B. Wake Additional Perspectives on Size Control in Biology: From Organelles to Organisms available at www.cshperspectives.org Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a019182 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019182 1 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press T.J. Mitchison et al. Part of the answer is the need for higher fidelity sions occur with little gene expression and little in the eukaryotic process. Gerhart and Kirsch- change in cell physiology, and it may be reason- ner (1997) also emphasized the need for highly able to assume approximately constant bio- adaptable processes in the evolution of higher chemistry (discussed below), other than period- eukaryotes. At least part of the complexity of ic cell-cycle regulation. After the 12th division, subcellular assemblies might reflect the need cell physiology changes dramatically as part of for adaptable scaling of size, shape, and timing. the midblastulatransition (MBT) (discussed be- Vertebrate embryos derived from large eggs low), which provides a natural cut-off for size- provide a natural experiment in size scaling scaling investigations. An interesting and poten- (Fig. 1). A Xenopus laevis egg, for example, is tially informative complication is that cleaving 1.2 mm in diameter. Following fertilization, it amphibian embryos develop a gradient in blas- cleaves completely 12 times at an approxi- tomere sizes, with larger cells at the vegetal pole mately constant rate of 2 divisions/h(most where yolk is more abundant (evident in Fig. rates in early development are temperature de- 1C,D). Larger blastomeres tend to divide more pendent, and can vary up to about eightfold slowly, which gradually eliminates division syn- over the tolerated range). These divisions gen- chrony (Gerhart 1980). erate a quasispherical array of quasispherical Embryos from different species have pros cells that are, on average, smaller by 212-fold in and cons forexperimental analysis of size scaling volume, or 24-fold in radius. The first 12 divi- during early divisions. Amphibian eggs provide AB E 60 Large-cell regime 50 Meiosis ll m) 40 Mitosis 1 µ Mitosis 2 30 Mitosis 4 Mitosis 5 Small-cell regime 20 Mitosis 7 Stage 8 Spindle size ( Spindle size 10 Stage 9 500 µm 0 0 200 400 600 800 1000 1200 1400 Cell size (µm) C D F DNA Tubulin 30 µm aNuMA 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 byconfocal 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 Wu¨hr et al. 2008; adapted, with permission, from the author; F is an unpub- lished image provided by Jesse Gatlin, University of Wyoming, which is similar to images in Hazel et al. 2013.) 2 Cite this article as Cold Spring Harb Perspect Biol 2015;7:a019182 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Size Scaling of Microtubules in Xenopus Embryos a large dynamic range in cell size, complete di- nuclear import substrates in interphase (Kar- vision, and quasispherical geometryof both cells senti and Vernos 2001; Sillje´ et al. 2006). DNA/ and embryos. In the minus column, they are cytoplasm ratio might affect the degree to which opaque unless fixed and cleared and difficult to such proteins are sequestered inside the nucleus manipulate using genetics. Undiluted, cell-free during the short embryonic cell cycle. This po- extracts from Xenopus eggs and early embryos tential source of size-scaling effects on inter- provide access to live imaging and molecular phase microtubule dynamics has not been inves- analysis and recapitulate the biology of intact tigated. eggs, including scaling relationships (Wilbur Surface-area/volume ratio plays a critical and Heald 2013), but it is important to go back role in size scaling of whole organism physiol- to the intact embryo to check validity of key ogy, and is one reason smaller animals have fast- findings where possible. Zebrafish eggs provide er metabolic rates. It is presumably important a transparent, genetically tractable vertebrate in embryos; for example, it might influence systemwithvery large cells but incomplete cleav- metabolic physiology, or the influence of corti- age at early stages. Caenorhabditis elegans and cal actomyosin on interior cytomechanics, but Drosophila embryos have excellent imaging and this topic has been little investigated. When in- genetics, which are advantages for scaling anal- vestigating nuclear size scaling in Xenopus, Levy ysis, especially rate scaling (e.g., Carvalho et al. and Heald (2010) found that the cytoplasmic 2009; Hara and Kimura 2013), but these em- concentration of the nuclear import factor im- bryos start smaller, so they provide a lower dy- portin-a decreased during cleavage. They pro- namic range for analyzing size-scaling behavior. posed that this was caused by proportionally increased sequestration of importin by the plas- ma membrane. Importin-a is a positive factor SIZE AND RATIO SCALING in nuclear growth, but also a negative factor in As cell size decreases during cleavage, two im- spindle assembly, where it sequesters spindle portant ratios, DNA/cytoplasm and surface- assembly factors (Karsenti and Vernos 2001; area/volume, increase. These three changes are Sillje´ et al. 2006). More work is needed to elu- naturally linked and likely contribute in impor- cidate the biochemistry of importin binding to tant but different waysto size scaling of cell phys- membranes, but this line of research suggests a iology. Initial DNA/cytoplasm ratios can be general mechanism by which the sizes of sub- changed fourfold or more in amphibians using cellular assemblies are controlled by the cell’s haploid or polyploid embryos. This perturba- surface/volume ratio. tion generated important discoveries in cell- size scaling effects at the tissue level (Fankhauser STEADY-STATE VERSUS KINETIC 1945). In a discussion of early embryo scaling DETERMINATION OF ASSEMBLY SIZE effects, the most important impact of DNA/cy- toplasm ratio is to trigger MBTaround the 12th A crucial question in any discussion of assembly division (Newport and Kirschner 1982a). Asthis size is whether size is measured at steady state or ratio approaches somatic values, division rate at some point in a time-varying trajectory.