research

current research directions

 

cell biology of meiotic drive: genetic cheating in violation of Mendel’s First Law

Every high school biology student learns about the 3:1 phenotype ratio observed by Mendel in his monohybrid crosses of pea plants. Underlying this observation, and at the core of Mendelian genetics, is the concept that gametes are equally likely to carry either allele of a gene. Selfish genetic elements can cheat by violating this principle to increase their representation in the gametes (and therefore in the progeny) by meiotic drive. Examples from a range of eukaryotes show that meiotic drive is a widespread phenomenon that impacts many aspects of reproduction, genetics, and evolution (Werren 2011, Rice 2013, Helleu et al. 2015, Lindholm et al. 2016). These violations of Mendel’s First Law should perhaps not surprise us because the opportunity to cheat is inherent in the haploid-diploid life cycle that underlies sexual reproduction in all eukaryotes, with genetic elements competing for transmission to the offspring through meiosis, the process by which haploids are generated. Female meiosis provides the most straightforward way to cheat because only one egg is produced from two rounds of cell division, so selfish elements compete for transmission to the egg (Sandler and Novitski 1957Pardo-Manuel de Villena and Sapienza, 2001). Our studies focus on the cell biology of female meiotic drive, using a mouse model system in which we can directly observe drive and ask mechanistic questions (Chmátal et al. 2014Iwata-Otsubo et al. 2017, Akera et al. 2017). The area is wide open, as the mechanisms underlying drive are almost completely unknown, and the significance of meiotic drive for genetics and evolutionary and reproductive biology makes the cell biological questions particularly exciting. Furthermore, understanding how selfish centromeres cheat the meiotic segregation machinery will provide new insights into centromere biology and meiotic cell division. Read more or see our papers in this area

Our model system for meiotic drive. A mouse oocyte from a hybrid female produced by crossing strains with different centromeres. Each pair of homologous chromosomes (labeled with histone H2B, red) contains a “weaker” centromere and a “stronger” centromere, which can be distinguished by levels of the centromere protein CENP-B (green). We find that stronger centromeres drive by preferentially segregating to the egg in meiosis I.

 
challenges of the extended meiotic cell cycle in mammalian oocytes and centromere inheritance through the germline

The extended prophase I arrest (lasting months to decades) in mammalian oocytes raises questions about how chromosome cohesion and centromere identity are maintained. Cohesin proteins and CENP-A nucleosomes, which define the centromere, assemble onto chromosomes before the prophase arrest and must persist through the arrest. Using mouse oocytes as our experimental system, we showed that cohesin proteins are gradually lost from chromosomes during prophase I and that the resulting cohesion defects can explain most of the age-related chromosome segregation errors (Duncan et al. 2009, Chiang et al. 20102011). Our findings established a mechanistic basis for the well-known increase in aneuploidy associated with advanced maternal age. In contrast, we showed that CENP-A nucleosomes are stable for the fertile lifespan of the female (>1 yr) in the absence of any new assembly (Smoak et al. 2016). This remarkable stability underlies centromere inheritance through the female germline. Our ongoing studies focus on understanding how this remarkable stability, which underlies centromere inheritance through the female germline, is achieved. Our approach is highly interdisciplinary, in close collaboration with the Black lab, combining structural biology and biophysics with in vivo reproductive biology. Ultimately we hope to connect atomic level structural insights to long-term maintenance of centromere identity and reproductive fitness. See our papers in this area …

CENP-A staining in a mouse oocyte at metaphase I. CENP-A persists at centromeres for >1 year after the gene has been deleted in the oocyte, demonstrating the remarkable stability of centromeric nucleosomes.

See Smoak et al. 2016.

 

optogenetic tools to manipulate living cells

Tools for manipulating protein levels at intracellular structures, with temporal and spatial control, enable the next generation of experiments to dissect dynamic systems in cell biology. We developed an optogenetic system based on a photo-caged chemical dimerizer, in collaboration with the Chenoweth Lab, that allows us to recruit proteins to diverse intracellular structures with light (Ballister et al. 2014, 2015). We are applying these tools to manipulate kinetochores, multi-subunit complexes whose function depends on dynamic changes in localization of kinases, MT-associated proteins, and checkpoint signaling proteins throughout cell division (Zhang et al. 2017). Read more or see our papers in this area

 

previous research areas

 

spatial patterns of Aurora B kinase activity

Aurora B is a key mitotic kinase that redistributes from centromeres to the spindle midzone at anaphase onset, as part of the chromosome passenger complex. To determine how spatial changes in Aurora B signaling regulate cell division, we developed FRET-based biosensors to track changes in kinase activity in living cells with high temporal and spatial resolution. We measured spatial phosphorylation gradients around the spindle midzone in anaphase and around centromeres before anaphase onset, and we established a model for how spatially regulated Aurora B kinase activity can selectively destabilize kinetochore-MT attachment errors (Fuller et al. 2008, Liu et al. 2009, Wang et al. 2011, Zaytsev et al. 2016). Our model is widely cited and is included in a recent textbook (Lodish et al. Molecular Cell Biology, 7th Edition, Figure 19-25).

 

regulation of kinetochore-microtubule interactions

How MT attachments are regulated to ensure accurate chromosome segregation is a long-standing question in cell biology. We addressed this question at multiple levels, using two different systems. In cultured somatic cells, we established a mechanism for phosphatase recruitment to kinetochores to oppose Aurora B (Liu et al. 2010) and showed that Polo-like kinase-1 (Plk1) stabilizes kinetochore MTs in opposition to Aurora B (Liu et al. 2012). Using mouse oocytes as a meiotic model system, we showed that gradually increasing Cdk1 activity acts as a timing mechanism for stabilizing MT attachments after spindle formation (Davydenko et al. 2013). We also demonstrated spatial regulation of attachments by Aurora A at spindle poles (Chmatal et al. 2015), complementary to tension-dependent regulation by Aurora B at centromeres.

bi-orientation Kinetochore microtubule bi-orientation. A metaphase cell stained for microtubules (green) and kinetochores (red). The image is a maximal intensity projection of a confocal z-series. Insets are optical sections showing bi-oriented sister kinetochore pairs.

From Liu et al. 2012.

 

collaborators

Ben Black, Department of Biochemistry and Biophysics

Ekaterina Grishchuk, Department of Physiology

Andrea Liu, Department of Physics and Astronomy

Dave Chenoweth, Department of Chemistry

Roger Greenberg, Department of Cancer Biology

 

research support (past and present)

National Institute of General Medical Sciences (NIGMS)

National Institute of Child Health and Human Development (NICHD)

Searle Scholars Award

Physical Sciences Oncology Center at Penn (PSOC)

Emerson Collective Cancer Research Fund

Nano/Bio Interface Center, University of Pennsylvania

Penn Epigenetics Institute

University Research Foundation, University of Pennsylvania

Abramson Cancer Center, University of Pennsylvania

Institute on Aging, University of Pennsylvania

Penn Genome Frontiers Institute

Translational Biomedical Imaging Center, Institute for Translational Medicine and Therapeutics, University of Pennsylvania

Penn Center for Study of Epigenetics in Reproduction