Research in my laboratory is supported by two highly productive R01s and has focused on two major areas: Cell division is arguably the most dramatic event in the life of a cell. Chromosomes condense, organelles vesiculate, and the microtubule cytoskeleton rearranges into a bipolar spindle that attaches to chromosomes at their kinetochores and segregates a complete set to each daughter cell. Although the morphological changes that occur during mitosis were first observed over a century ago, we still do not understand how these dynamic events are orchestrated. Many factors have been identified that contribute to spindle assembly and function, but the molecular and biophysical mechanisms and interactions that ensure mitotic fidelity remain unclear. Our current projects address outstanding questions including 1) What are the molecular underpinnings and functional consequences of different spindle architectures? Spindle size and organization varies dramatically across cell types and organisms, and factors known to affect these parameters are altered in many cancers, but how specific spindle features are established and their effects on chromosome segregation and cell division are poorly understood. We will leverage morphometric and phylogenetic comparisons together with biochemical and functional assays to investigate the basis and significance of variation in astral microtubule morphology at spindle poles. 2) What activities are sufficient to establish the mechanochemical core of the spindle? Whereas the functions of many individual spindle factors have been studied extensively, reconstituting the spindle from purified components remains a holy grail as the key to a complete understanding of the process. We will extend our bead-based spindle assembly system to define the chromatin-associated activities sufficient for spindle self-organization. 3) What is the role of RNA in kinetochore assembly? Transcription of centromeric sequences appears to be a conserved mechanism required for kinetochore formation, but the fate and mitotic function of nascent transcripts is unclear. We will examine centromeric transcription and RNA processing during mitotic progression using a novel in vitro assay and elucidate its role in spindle assembly. Together, these projects elucidate mitotic mechanisms and advance the field toward a systems-level understanding of the spindle. Absolute and relative size of biological entities varies widely, both within and among species at all levels of organization above the atomic/molecular: the organism, the cells that make up the organism, and the components of the cells. How does scaling occur so that everything fits and functions properly? Correct scaling inside cells is crucial for cell function, architecture, and division, but until recently the contrl systems that a cell uses to regulate the size of its internal structures were virtually unknown. We have established assays to elucidate mechanisms of intracellular scaling between different-sized frog species and during the rapid, reductive cell divisions of early embryogenesis. We are further developing these systems to ask: 1) What scales mitotic chromosome size to cell size? The determinants of mitotic chromosome architecture are poorly understood, and a major challenge in addressing this question is to establish live chromosome labeling methods. We will utilize our new CRISPR-based imaging technique to test the role of candidate factors in chromosome scaling during development. 2) Is there a scaling mechanism that senses the cell surface area-to-volume ratio? Accumulating evidence suggests that cells sense surface area-to-volume as a direct readout for size, and that this information is used to scale subcellular structures. We hypothesize that importin ?, an abundant regulator of spindle and nuclear size that also associates with the plasma membrane and is depleted from the cytoplasm of small cells relative to large cells, acts as a cell size sensor. We will use our size-tunable microfluidi droplet system to test this hypothesis. 3) How is size regulated at the cellular and organism levels? The relative contribution of maternal cytoplasmic factors versus genome content and expression to cell and organism size is unclear. The close phylogenetic relationship between the two Xenopus species used in our lab enables us to generate hybrid frogs of intermediate size and evaluate the role of genome size and content on size relationships. Together, our projects utilizing in vitro and in vivo approaches are identifying cellular and molecular mechanisms underlying biological size control and scaling. The means to address these fundamental cell biological questions is enabled by powerful experimental systems based on cytoplasmic extracts and functional, in vivo assays in vertebrate (Xenopus) embryos. We have established productive collaborations and apply diverse techniques including high-resolution microscopy, biophysical assays, proteomics, RNA sequencing, microfluidics and computational modeling to create new and innovative approaches. Our research will continue to provide novel insight into cell division and size control, processes essential for viability and development, and defective in human diseases including cancer.

Public Health Relevance

During cell division, called mitosis, a critical step for maintaining healthy living cells is segregation of the replicated chromosomes to two daughter cells, which is mediated by a dynamic structure called the mitotic spindle. We do not fully understand how the spindle forms, or how it adjusts its size and shape in different cell types. Investigating this process is important, since errors in spindle function can lead to cancer and other human diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZGM1)
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Gindhart, Joseph G
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University of California Berkeley
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Willsey, Helen Rankin; Walentek, Peter; Exner, Cameron R T et al. (2018) Katanin-like protein Katnal2 is required for ciliogenesis and brain development in Xenopus embryos. Dev Biol 442:276-287
Gibeaux, Romain; Acker, Rachael; Kitaoka, Maiko et al. (2018) Paternal chromosome loss and metabolic crisis contribute to hybrid inviability in Xenopus. Nature 553:337-341
Heald, Rebecca; Gibeaux, Romain (2018) Subcellular scaling: does size matter for cell division? Curr Opin Cell Biol 52:88-95
Gibeaux, Romain; Miller, Kelly; Acker, Rachael et al. (2018) Xenopus Hybrids Provide Insight Into Cell and Organism Size Control. Front Physiol 9:1758
Kitaoka, Maiko; Heald, Rebecca; Gibeaux, Romain (2018) Spindle assembly in egg extracts of the Marsabit clawed frog, Xenopus borealis. Cytoskeleton (Hoboken) 75:244-257
Grenfell, Andrew W; Strzelecka, Magdalena; Heald, Rebecca (2017) Transcription brings the complex(ity) to the centromere. Cell Cycle 16:235-236
Elurbe, Dei M; Paranjpe, Sarita S; Georgiou, Georgios et al. (2017) Regulatory remodeling in the allo-tetraploid frog Xenopus laevis. Genome Biol 18:198
Grenfell, Andrew W; Strzelecka, Magdalena; Crowder, Marina E et al. (2016) A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles. J Cell Biol 213:127-36
Session, Adam M; Uno, Yoshinobu; Kwon, Taejoon et al. (2016) Genome evolution in the allotetraploid frog Xenopus laevis. Nature 538:336-343
Grenfell, Andrew W; Heald, Rebecca; Strzelecka, Magdalena (2016) Correction: Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus. J Cell Biol 214:783

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