Publications by authors named "Michael A Lampson"

73 Publications

Parallel pathways for recruiting effector proteins determine centromere drive and suppression.

Cell 2021 Sep 24;184(19):4904-4918.e11. Epub 2021 Aug 24.

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Selfish centromere DNA sequences bias their transmission to the egg in female meiosis. Evolutionary theory suggests that centromere proteins evolve to suppress costs of this "centromere drive." In hybrid mouse models with genetically different maternal and paternal centromeres, selfish centromere DNA exploits a kinetochore pathway to recruit microtubule-destabilizing proteins that act as drive effectors. We show that such functional differences are suppressed by a parallel pathway for effector recruitment by heterochromatin, which is similar between centromeres in this system. Disrupting the kinetochore pathway with a divergent allele of CENP-C reduces functional differences between centromeres, whereas disrupting heterochromatin by CENP-B deletion amplifies the differences. Molecular evolution analyses using Murinae genomes identify adaptive evolution in proteins in both pathways. We propose that centromere proteins have recurrently evolved to minimize the kinetochore pathway, which is exploited by selfish DNA, relative to the heterochromatin pathway that equalizes centromeres, while maintaining essential functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2021.07.037DOI Listing
September 2021

Separase cleaves the kinetochore protein Meikin at the meiosis I/II transition.

Dev Cell 2021 Aug 30;56(15):2192-2206.e8. Epub 2021 Jul 30.

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2021.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8355204PMC
August 2021

Chemical tools for dissecting cell division.

Nat Chem Biol 2021 06 25;17(6):632-640. Epub 2021 May 25.

Department of Biology, University of Pennsylvania, Philadelphia, PA, USA.

Components of the cell division machinery typically function at varying cell cycle stages and intracellular locations. To dissect cellular mechanisms during the rapid division process, small-molecule probes act as complementary approaches to genetic manipulations, with advantages of temporal and in some cases spatial control and applicability to multiple model systems. This Review focuses on recent advances in chemical probes and applications to address select questions in cell division. We discuss uses of both enzyme inhibitors and chemical inducers of dimerization, as well as emerging techniques to promote future investigations. Overall, these concepts may open new research directions for applying chemical probes to advance cell biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-021-00798-3DOI Listing
June 2021

Tension promotes kinetochore-microtubule release by Aurora B kinase.

J Cell Biol 2021 Jun;220(6)

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA.

To ensure accurate chromosome segregation, interactions between kinetochores and microtubules are regulated by a combination of mechanics and biochemistry. Tension provides a signal to discriminate attachment errors from bi-oriented kinetochores with sisters correctly attached to opposite spindle poles. Biochemically, Aurora B kinase phosphorylates kinetochores to destabilize interactions with microtubules. To link mechanics and biochemistry, current models regard tension as an input signal to locally regulate Aurora B activity. Here, we show that the outcome of kinetochore phosphorylation depends on tension. Using optogenetics to manipulate Aurora B at individual kinetochores, we find that kinase activity promotes microtubule release when tension is high. Conversely, when tension is low, Aurora B activity promotes depolymerization of kinetochore-microtubules while maintaining attachment. Thus, phosphorylation converts a catch-bond, in which tension stabilizes attachments, to a slip-bond, which releases microtubules under tension. We propose that tension is a signal inducing distinct error-correction pathways, with release or depolymerization being advantageous for typical errors characterized by high or low tension, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.202007030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082439PMC
June 2021

Mixed knobs in corn cobs.

Genes Dev 2020 09;34(17-18):1110-1112

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Maize heterochromatic knobs cheat female meiosis by forming neocentromeres that bias their segregation into the future egg cell. In this issue of , Swentowsky and colleagues (pp. 1239-1251) show that two types of knobs, those composed of 180-bp and TR1 sequences, recruit their own novel and divergent kinesin-14 family members to form neocentromeres.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.343350.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462059PMC
September 2020

Maternal inheritance of centromeres through the germline.

Curr Top Dev Biol 2020 25;140:35-54. Epub 2020 Apr 25.

Department of Biology, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

The centromere directs chromosome segregation but is not itself genetically encoded. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant CENP-A, independent of the underlying DNA sequence. Therefore, to maintain centromeres and ensure accurate chromosome segregation, CENP-A nucleosomes must be inherited across generations through the germline. In this chapter we discuss three aspects of maternal centromere inheritance. First, we propose mechanisms for maintaining CENP-A nucleosomes through the prolonged prophase arrest in mammalian oocytes. Second, we review mechanisms by which selfish centromeres bias their transmission through female meiosis. Third, we discuss regulation of centromere size through early embryonic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.ctdb.2020.03.004DOI Listing
August 2021

Nuclear body phase separation drives telomere clustering in ALT cancer cells.

Mol Biol Cell 2020 08 24;31(18):2048-2056. Epub 2020 Jun 24.

Penn Center for Genome Integrity, University of Pennsylvania, Philadelphia, PA 19104.

Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liquid condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chemically induced dimerization approach to induce de novo APB condensation in live cells without DNA damage. We show that telomere-binding protein sumoylation nucleates APB condensation via interactions between small ubiquitin-like modifier (SUMO) and SUMO interaction motif (SIM), and that APB coalescence drives telomere clustering. The induced APBs lack DNA repair factors, indicating that APB functions in promoting telomere clustering can be uncoupled from enriching DNA repair factors. Indeed, telomere clustering relies only on liquid properties of the condensate, as an alternative condensation chemistry also induces clustering independent of sumoylation. Our findings introduce a chemical dimerization approach to manipulate phase separation and demonstrate how the material properties and chemical composition of APBs independently contribute to ALT, suggesting a general framework for how chromatin condensates promote cellular functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E19-10-0589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543070PMC
August 2020

Centromere identity and function put to use: construction and transfer of mammalian artificial chromosomes to animal models.

Essays Biochem 2020 09;64(2):185-192

Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

Mammalian artificial chromosomes (MACs) are widely used as gene expression vectors and have various advantages over conventional expression vectors. We review and discuss breakthroughs in MAC construction, initiation of functional centromeres allowing their faithful inheritance, and transfer from cell culture to animal model systems. These advances have contributed to advancements in synthetic biology, biomedical research, and applications in industry and in the clinic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/EBC20190071DOI Listing
September 2020

Photoactivatable trimethoprim-based probes for spatiotemporal control of biological processes.

Methods Enzymol 2020 24;638:273-294. Epub 2020 Apr 24.

Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. By combining the chemical versatility of photoremovable protecting groups with the biological specificity of self-labeling tags, we developed a series of chemi-optogenetic tools that enable protein recruitment with precise spatiotemporal control. To this end, we created a modular platform for chemically inducible proximity (CIP), a technique in which two proteins of interest are brought together by the presence of a small molecule to induce a biological effect. The local proximity of a protein and its substrate has been shown to be sufficient to initiate a desired biological effect, making CIP a valuable technique towards probing cellular processes. The high affinity and specificity of these tags result in rapid initiation of dimerization, allowing biochemical processes to be studied on a biologically relevant timescale. In this chapter, we describe the synthesis and application of chemi-optogenetic probes for spatiotemporal control of protein proximity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.mie.2020.03.015DOI Listing
June 2021

SKP1 drives the prophase I to metaphase I transition during male meiosis.

Sci Adv 2020 03 25;6(13):eaaz2129. Epub 2020 Mar 25.

Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, USA.

The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aaz2129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096161PMC
March 2020

Molecular Strategies of Meiotic Cheating by Selfish Centromeres.

Cell 2019 08 8;178(5):1132-1144.e10. Epub 2019 Aug 8.

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Asymmetric division in female meiosis creates selective pressure favoring selfish centromeres that bias their transmission to the egg. This centromere drive can explain the paradoxical rapid evolution of both centromere DNA and centromere-binding proteins despite conserved centromere function. Here, we define a molecular pathway linking expanded centromeres to histone phosphorylation and recruitment of microtubule destabilizing factors, leading to detachment of selfish centromeres from spindle microtubules that would direct them to the polar body. Exploiting centromere divergence between species, we show that selfish centromeres in two hybrid mouse models use the same molecular pathway but modulate it differently to enrich destabilizing factors. Our results indicate that increasing microtubule destabilizing activity is a general strategy for drive in both models, but centromeres have evolved distinct mechanisms to increase that activity. Furthermore, we show that drive depends on slowing meiotic progression, suggesting that selfish centromeres can be suppressed by regulating meiotic timing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731994PMC
August 2019

Reversible optogenetic control of protein function and localization.

Methods Enzymol 2019 6;624:25-45. Epub 2019 Jun 6.

Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Protein-protein interactions are highly dynamic biological processes that regulate various cellular reactions. They exhibit high specificity and spatiotemporal control in order to efficiently utilize finite resources in a cellular compartment. Photoactivatable chemically inducible dimerization (pCID) has emerged as an attractive technique in the scientific community, leading to the development of systems that can be activated with various wavelengths of light in order to manipulate processes on biologically relevant scales with molecular specificity. These systems can be modified to control various protein functions with unprecedented precision and spatiotemporal resolution. In this chapter, we describe an optogenetic platform that provides reversible control over dimerization of genetically tagged proteins using orthogonal wavelengths of light. We demonstrate photoactivation and photo-reversal of protein localization and transport. Mitosis is manipulated by activating and silencing the spindle assembly checkpoint through recruitment and release of proteins from kinetochores.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.mie.2019.05.002DOI Listing
May 2020

Chromosome Segregation: Poor Supervision in the Early Stage of Life.

Curr Biol 2019 03;29(5):R156-R158

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Abnormal chromosome number, or aneuploidy, is common in early mammalian embryos, although the underlying cell biological basis is still incompletely understood. New research reveals that cells often fail to wait for all chromosomes to properly attach to the spindle machinery before segregation, explaining why early embryonic cell cycles are so error-prone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2019.01.036DOI Listing
March 2019

Reversible Control of Protein Localization in Living Cells Using a Photocaged-Photocleavable Chemical Dimerizer.

J Am Chem Soc 2018 09 14;140(38):11926-11930. Epub 2018 Sep 14.

Many dynamic biological processes are regulated by protein-protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on subcellular levels by controlling protein-protein interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.8b07753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499933PMC
September 2018

Keeping Parents Apart.

Dev Cell 2018 08;46(3):255-256

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Parental genomes are initially separate in the zygote following fertilization. A recent study in Science by Reichmann et al. (2018) reveals that dual spindles assemble around the two pronuclei in mouse embryos to maintain separation of the two parental genomes through the first zygotic division.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2018.07.019DOI Listing
August 2018

Optogenetic Manipulation of Mouse Oocytes.

Methods Mol Biol 2018 ;1818:129-135

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, USA.

Like many biological processes, oocyte development depends on careful orchestration of protein localization. Optogenetic approaches have the potential to manipulate this dynamic system with spatial and temporal precision and molecular specificity. This chapter describes the use of a photocaged chemical inducer of dimerization to control localization of genetically tagged proteins with light. As an example, we recruit a fluorescently tagged protein to one spindle pole in metaphase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8603-3_13DOI Listing
March 2019

Optogenetic control of mitosis with photocaged chemical dimerizers.

Methods Cell Biol 2018 27;144:157-164. Epub 2018 Apr 27.

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Mitosis is a highly dynamic process that depends on coordination of many protein-protein interactions with temporal and spatial precision. A challenge for understanding this complex system is to manipulate it on biologically relevant temporal and spatial scales, with molecular specificity. We describe an optogenetic platform, based on photosensitive chemical inducers of dimerization, which provides control over dimerization of genetically tagged proteins with light. As examples, we drive chromosome transport and activate and silence the spindle assembly checkpoint by recruiting proteins to and releasing them from kinetochores with light.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.mcb.2018.03.006DOI Listing
December 2018

Erratum: Cellular and Molecular Mechanisms of Centromere Drive.

Cold Spring Harb Symp Quant Biol 2018 Feb 26. Epub 2018 Feb 26.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/sqb.2017.82.034736DOI Listing
February 2018

Cellular and Molecular Mechanisms of Centromere Drive.

Cold Spring Harb Symp Quant Biol 2017 12;82:249-257. Epub 2018 Feb 12.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6059.

The asymmetric outcome of female meiosis I, whereby an entire set of chromosomes are discarded into a polar body, presents an opportunity for selfish genetic elements to cheat the process and disproportionately segregate to the egg. Centromeres, the chromosomal loci that connect to spindle microtubules, could potentially act as selfish elements and "drive" in meiosis. We review the current understanding of the genetic and epigenetic contributions to centromere identity and describe recent progress in a powerful model system to study centromere drive in mice. The progress includes mechanistic findings regarding two main requirements for a centromere to exploit the asymmetric outcome of female meiosis. The first is an asymmetry between centromeres of homologous chromosomes, and we found this is accomplished through massive changes in the abundance of the repetitive DNA underlying centromeric chromatin. The second requirement is an asymmetry in the meiotic spindle, which is achieved through signaling from the oocyte cortex that leads to asymmetry in a posttranslational modification of tubulin, tyrosination. Together, these two asymmetries culminate in the biased segregation of expanded centromeres to the egg, and we describe a mechanistic framework to understand this process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/sqb.2017.82.034298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041145PMC
February 2018

Spindle asymmetry drives non-Mendelian chromosome segregation.

Science 2017 11;358(6363):668-672

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA.

Genetic elements compete for transmission through meiosis, when haploid gametes are created from a diploid parent. Selfish elements can enhance their transmission through a process known as meiotic drive. In female meiosis, selfish elements drive by preferentially attaching to the egg side of the spindle. This implies some asymmetry between the two sides of the spindle, but the molecular mechanisms underlying spindle asymmetry are unknown. Here we found that CDC42 signaling from the cell cortex regulated microtubule tyrosination to induce spindle asymmetry and that non-Mendelian segregation depended on this asymmetry. Cortical CDC42 depends on polarization directed by chromosomes, which are positioned near the cortex to allow the asymmetric cell division. Thus, selfish meiotic drivers exploit the asymmetry inherent in female meiosis to bias their transmission.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aan0092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906099PMC
November 2017

Two mechanisms coordinate the recruitment of the chromosomal passenger complex to the plane of cell division.

Mol Biol Cell 2017 Dec 27;28(25):3634-3646. Epub 2017 Sep 27.

Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232

During cytokinesis, the chromosomal passenger complex (CPC) promotes midzone organization, specifies the cleavage plane, and regulates furrow contractility. The localizations of the CPC are coupled to its cytokinetic functions. At the metaphase-to-anaphase transition, the CPC dissociates from centromeres and localizes to midzone microtubules and the equatorial cortex. CPC relocalization to the cell middle is thought to depend on MKlp2-driven, plus end-directed transport. In support of this idea, MKlp2 depletion impairs cytokinesis; however, cytokinesis failure stems from furrow regression rather than failed initiation of furrowing. This suggests that an alternative mechanism(s) may concentrate the CPC at the division plane. We show here that direct actin binding, via the inner centromere protein (INCENP), enhances CPC enrichment at the equatorial cortex, thus acting in tandem with MKlp2. INCENP overexpression rescues furrowing in MKlp2-depleted cells in an INCENP-actin binding-dependent manner. Using live-cell imaging, we also find that MKlp2-dependent targeting of the CPC is biphasic. MKlp2 targets the CPC to the anti-parallel microtubule overlap of the midzone, after which the MKlp2-CPC complex moves in a nondirected manner. Collectively, our work suggests that both actin binding and MKlp2-dependent midzone targeting cooperate to precisely position the CPC during mitotic exit, and that these pathways converge to ensure successful cleavage furrow ingression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E17-06-0399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706991PMC
December 2017

Cell Biology of Cheating-Transmission of Centromeres and Other Selfish Elements Through Asymmetric Meiosis.

Prog Mol Subcell Biol 2017;56:377-396

Department of Biology, University of Pennsylvania, 433 South University Ave, Philadelphia, PA, 19104, USA.

Mendel's First Law of Genetics states that a pair of alleles segregates randomly during meiosis so that one copy of each is represented equally in gametes. Whereas male meiosis produces four equal sperm, in female meiosis only one cell, the egg, survives, and the others degenerate. Meiotic drive is a process in which a selfish DNA element exploits female meiotic asymmetry and segregates preferentially to the egg in violation of Mendel's First Law, thereby increasing its transmission to the offspring and frequency in a population. In principle, the selfish element can consist either of a centromere that increases its transmission via an altered kinetochore connection to the meiotic spindle or a centromere-like element that somehow bypasses the kinetochore altogether in doing so. There are now examples from eukaryotic model systems for both types of meiotic drive. Although meiotic drive has profound evolutionary consequences across many species, relatively little is known about the underlying mechanisms. We discuss examples in various systems and open questions about the underlying cell biology, and propose a mechanism to explain biased segregation in mammalian female meiosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-58592-5_16DOI Listing
May 2019

Optogenetic control of kinetochore function.

Nat Chem Biol 2017 Oct 14;13(10):1096-1101. Epub 2017 Aug 14.

Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Kinetochores act as hubs for multiple activities during cell division, including microtubule interactions and spindle checkpoint signaling. Each kinetochore can act autonomously, and activities change rapidly as proteins are recruited to, or removed from, kinetochores. Understanding this dynamic system requires tools that can manipulate kinetochores on biologically relevant temporal and spatial scales. Optogenetic approaches have the potential to provide temporal and spatial control with molecular specificity. Here we report new chemical inducers of protein dimerization that allow us to both recruit proteins to and release them from kinetochores using light. We use these dimerizers to manipulate checkpoint signaling and molecular motor activity. Our findings demonstrate specialized properties of the CENP-E (kinesin-7) motor for directional chromosome transport to the spindle equator and for maintenance of metaphase alignment. This work establishes a foundation for optogenetic control of kinetochore function, which is broadly applicable to experimental probing of other dynamic cellular processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605432PMC
October 2017

Centromere inheritance through the germline.

Chromosoma 2017 Oct 8;126(5):595-604. Epub 2017 Aug 8.

Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, 19104, USA.

The centromere directs chromosome segregation and genetic inheritance but is not itself heritable in a canonical, DNA-based manner. In most species, centromeres are epigenetically defined by the presence of a histone H3 variant centromere protein A (CENP-A), independent of underlying DNA sequence. Therefore, centromere inheritance depends on maintaining the CENP-A nucleosome mark across generations. Experiments in cycling somatic cells have led to a model in which centromere identity is maintained by a cell cycle-coupled CENP-A chromatin assembly pathway. However, the processes of animal gametogenesis pose unique challenges to centromere inheritance because of the extended cell cycle arrest and the massive genome reorganization in the female and male germline, respectively. Here, we review our current understanding of germline centromere inheritance and highlight outstanding questions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00412-017-0640-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693723PMC
October 2017

Expanded Satellite Repeats Amplify a Discrete CENP-A Nucleosome Assembly Site on Chromosomes that Drive in Female Meiosis.

Curr Biol 2017 Aug 27;27(15):2365-2373.e8. Epub 2017 Jul 27.

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Female meiosis provides an opportunity for selfish genetic elements to violate Mendel's law of segregation by increasing the chance of segregating to the egg [1]. Centromeres and other repetitive sequences can drive in meiosis by cheating the segregation process [2], but the underlying mechanisms are unknown. Here, we show that centromeres with more satellite repeats house more nucleosomes that confer centromere identity, containing the histone H3 variant CENP-A, and bias their segregation to the egg relative to centromeres with fewer repeats. CENP-A nucleosomes predominantly occupy a single site within the repeating unit that becomes limiting for centromere assembly on smaller centromeres. We propose that amplified repetitive sequences act as selfish elements by promoting expansion of CENP-A chromatin and increased transmission through the female germline.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2017.06.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567862PMC
August 2017

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

Dev Cell 2017 04;41(2):143-156.e6

Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2017.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477644PMC
April 2017

Mechanisms to Avoid and Correct Erroneous Kinetochore-Microtubule Attachments.

Biology (Basel) 2017 Jan 5;6(1). Epub 2017 Jan 5.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

In dividing vertebrate cells multiple microtubules must connect to mitotic kinetochores in a highly stereotypical manner, with each sister kinetochore forming microtubule attachments to only one spindle pole. The exact sequence of events by which this goal is achieved varies considerably from cell to cell because of the variable locations of kinetochores and spindle poles, and randomness of initial microtubule attachments. These chance encounters with the kinetochores nonetheless ultimately lead to the desired outcome with high fidelity and in a limited time frame, providing one of the most startling examples of biological self-organization. This chapter discusses mechanisms that contribute to accurate chromosome segregation by helping dividing cells to avoid and resolve improper microtubule attachments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biology6010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371994PMC
January 2017

Cell Cycle Remodeling and Zygotic Gene Activation at the Midblastula Transition.

Adv Exp Med Biol 2017 ;953:441-487

Cell and Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

Following fertilization, vertebrate embryos delay large-scale activation of the zygotic genome from several hours in fish and amphibians to several days in mammals. Externally developing embryos also undergo synchronous and extraordinarily rapid cell divisions that are accelerated by promiscuous licensing of DNA replication origins, absence of gap phases and cell cycle checkpoints, and preloading of the egg with maternal RNAs and proteins needed to drive early development. After a species-specific number of cell divisions, the cell cycle slows and becomes asynchronous, gap phases appear, checkpoint functions are acquired, and large-scale zygotic gene activation begins. These events, along with clearance of maternal RNAs and proteins, define the maternal to zygotic transition and are coordinated at a developmental milestone termed the midblastula transition (MBT). Despite the relative quiescence of the zygotic genome in vertebrate embryos, genes required for clearance of maternal RNAs and for the initial steps in mesoderm induction are robustly transcribed before MBT. The coordination and timing of the MBT depends on a mechanism that senses the ratio of nuclear to cytoplasmic content as well as mechanisms that are independent of the nuclear-cytoplasm ratio. Changes in chromatin architecture anticipate zygotic gene activation, and maternal transcription factors identified as regulators of pluripotency play critical roles in kick-starting the transition from the proliferative, pluripotent state of the early embryo to the more lineage-committed phase of development after the MBT. This chapter describes the regulation of the cell cycle and the activation of zygotic gene expression before and after the MBT in vertebrate embryos.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-46095-6_9DOI Listing
July 2017

In vivo imaging of DNA double-strand break induced telomere mobility during alternative lengthening of telomeres.

Methods 2017 02 1;114:54-59. Epub 2016 Aug 1.

Department of Cancer Biology, Abramson Family Cancer Research Institute, Basser Research Center for BRCA, Perelman School of Medicine, University of Pennsylvania, 421 Curie Blvd, Philadelphia, PA 19104-6160, United States; Department of Pathology, Abramson Family Cancer Research Institute, Basser Research Center for BRCA, Perelman School of Medicine, University of Pennsylvania, 421 Curie Blvd, Philadelphia, PA 19104-6160, United States. Electronic address:

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires mobilization of chromatin for homology searches that allow interaction of the sequence to be repaired and its template DNA. Here we describe a system to rapidly induce DSBs at telomeres and track their movement, as well as a semi-automated workflow for quantitative analysis. We have successfully used this approach to show that DSBs targeted to telomeres in cells utilizing the alternative lengthening of telomeres (ALT) mechanism increase their diffusion and subsequent long-range directional movement to merge with telomeres on other chromosomes. These methods are simple to implement and are compatible with almost any cell line or in vivo microscopy setup. The magnitude of DSB-induced telomere mobility allows the investigator to easily test for factors regulating telomere mobility during ALT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2016.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378164PMC
February 2017

Probing Mitosis by Manipulating the Interactions of Mitotic Regulator Proteins Using Rapamycin-Inducible Dimerization.

Methods Mol Biol 2016 ;1413:325-31

Department of Biology, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Inducible dimerization is a general approach to experimentally manipulate protein-protein interactions with temporal control. This chapter describes the use of rapamycin-inducible dimerization to manipulate mitotic regulatory proteins, for example to control kinetochore localization. A significant feature of this method relative to previously described protocols is the depletion of endogenous FKBP12 protein, which markedly improves dimerization efficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-3542-0_20DOI Listing
December 2017
-->