Research

My current research focuses on cytoplasmic organization in frog eggs, in particular during the period between mitosis and cleavage furrow ingression. During this interval, which coincides with S-phase, a pair of microtubule asters grow out from the two poles of the mitotic spindle. These asters meet at a plane that defines the midplane of the cell, and when they touch the cortex, the plane between them determines where the cleavage furrow initiates. My main experimental approaches are fluorescence microscopy and biochemistry, which I apply to intact eggs and also to an egg extract system that comprises living, undiluted cytoplasm and is able to reconstitute the egg’s spatial organizing mechanisms in a cell free system. The Xenopus egg extract system provides unique access to living cytoplasm and has been used to investigate many aspects of cell biology. I have led technical improvements in this system, in particular the introduction of actin-intact extract in 2011.     I frequently train students and post docs in extract preparation and use

Figure one

Figure 1. Preparation and some uses of Xenopus egg extract, our most important experimental system. Xenopus eggs are collected, packed and crushed and a layer of 1x cytoplasm is collected.  This cytoplasm is arrested in M-phase and will form meiotic spindles when sperm are added.  Addition of calcium releases the arrest and the extract proceeds into interphase forming asters. These microtubule structures recapitulate various aspects of egg biology including cytokinesis signaling when placed on a lipid bilayer. Nguyen et al., 2014, Science (see below).

 

Between 2010 and 2017, I focused on the biochemistry and function of the aster-aster boundary. This boundary recruits two important signaling complexes, Chromosome Passenger Complex (CPC) and Centralspindlin, in both eggs and egg extract. Together, these complexes define the aster boundary and specify the cleavage furrow location. Using poly-spermic fertilization, I discovered that, at least in eggs, only aster boundaries that were close to chromatin at anaphase recruit CPC and Centralspindlin. This finding explains classic observations from the 1910s showing that only asters separated by chromatin can initiate furrows in frog eggs. We proposed that the CPC-positive state spreads from the metaphase plate to the cortex along microtubule as the active state of a bistable, autocatalytic system. In this way, we believe that spread of the CPC-positive state along microtubule bundles at the aster-aster boundary is the spindle-to-cortex signal that emanates from the metaphase plate to triggers furrow initiation, as proposed by Rappaport in the 1960s.

 

Pc 2

Figure 2. Frog egg after poly-spermic fertilization fixed before 1st mitosis and imaged by confocal microscopy. Green shows microtubules, red shows CPC. Note that CPC is recruited to aster-aster boundaries that grow out of metaphase spindles while boundaries formed between different spindles. From Field et al. 2015, Mol Biol Cell (see below).

My current research focuses on regulation and function of F-actin in bulk egg cytoplasm. The whole egg is filled with a network of actin filaments. Most work in the non-muscle actin field has been focused on cortical actomyosin, which defines the mechanics and locomotion of most small cell types in animals. Xenopus eggs are huge cells (1.2 mm diameter), making them an ideal system to study bulk actin, far from the cortex. In 2011, I discovered that the contractile behavior of cytoplasmic actomyosin in regulated by the cell cycle, and is stronger in mitosis. In 2017-19, I observed that F-actin is cleared at CPC-positive aster boundaries, along with keratin networks. This clearing is caused by local activity of the Aurora B kinase subunit of the CPC, and can be observed around beads coated with activated CPC as well as around CPC-positive microtubule bundles. We postulate that this clearing of other cytoskeleton systems is important for implementing furrow ingression during cytokinesis.  How Aurora B kinase activity leads to clearing of F-actin is a current focus of my research. Another focus in the function of F-actin in the bulk cytoplasm. For example, I found that depolymerizing F-actin caused disorganization of the keratin 8-18/19 network that is present through to the egg, and plan to investigate the mechanism.  

fig 3

Figure 3. Aster-aster boundaries in egg extract. The left panel shows actin clearing, the middle panel shows microtubules and CPC. The right panel shows that fluid flow occurs preferentially through aster boundaries where F-actin is cleared. From Field et al 2019 Curr Biol. (see below).

My early research focused on cytokinesis in Drosophila, and I remain interested in how diverse organisms divide. In collaboration with Dr Amy Gladfelter (UNC) and my oldest child, Lorna Mitchison-Field (John Hopkins), I begun collecting, culturing and imaging marine fungi from the waters around the Marine Biological Laboratory in Woods Hole. Live imaging revealed a surprising diversity in division patterns and morphologies that were the topic of a 2019 paper in Current Biology. We are interested in learning more about the mechanisms behind division in these new model organisms.

fig 4

Figure 4 The left panel shows a collection of fungi cultured from the marine environments  around the Marine Biological Laboratory (Buzzards Bay and Vineyard Sound). The right panel is from a movie documenting cell division and growth of one of these fungi, a black yeast,Phaeotheca salicorniae. From Mitchison-Field et al., 2019 Curr Biol (see below).

Selected publications:

Xenopus Eggs

  • Field, CM, Wühr, M, Anderson, GA, Kueh, HY, Strickland, D and Mitchison, TJ. (2011) Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos. J. Cell Sci. 124:2086-2095.
  • Mitchison, TJ, Wühr, M, Nguyen, P, Ishihara K, Groen, A, Field, CM. (2012) Growth, interaction and positioning of microtubule asters in extremely large vertebrate embryo cells. Cytoskeleton 69, 738-750.
  • Nguyen, PA, Groen, AC, Loose, M, Ishihara, K, Wühr, M, Field, CM, Mitchison, TJ. (2014) Spatial organization of cytokinesis signaling reconstituted in a cell-free system. Science 346:244-247.
  • Field, C. M., Groen, A. C., Nguyen, P. A. and Mitchison, T. J. (2015) Spindle to cortex communication in cleaving, polyspermic Xenopus eggs. Mol Biol Cell 26:3628-3640.
  • Mitchison, TJ, Field, CM (2017) Spindle-to-cortex communication in cleaving frog eggs. Cold Spring Harbor Symposia on Quantitative Biology, 82:165-171.
  • Field, CM, Pelletier, JF and Mitchison, TJ. (2019) Disassembly of actin and keratin networks by Aurora B Kinase at the midplane of cleaving Xenopus eggs. Curr Biol. 29:1999-2008.
  • Pelletier JF, Field CM, Fürthauer S, Sonnett M, Mitchison TJ. Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm. Elife. 2020 Dec 7;9. doi: 10.7554/eLife.60047. 
  • Landino J, Leda M, Michaud A, Swider ZT, Prom M, Field CM, Bement WM, Vecchiarelli AG, Goryachev AB, Miller AL. (2021) Rho and F-actin self-organize within an artificial cell cortex. Curr Biol. 2021 Dec 20;31(24):5613-5621.e5. doi:10.1016/j.cub.2021.10.021. 

Marine Fungi

  • Mitchison-Field, LMY, Vargas-Muñiz, JM, Stormo, BM, Vogt,EJD, Van Dierdonck, S, Pelletier, FP, Ehrlich, C, Lew, DJ, Field, CM and Gladfelter, AS. (2019) Unconventional cell division cycles from marine-derived yeasts. Curr Biol 29(20):3439-3456.

 

Xenopus Gastrulation