Biology subjects

Maddox, P. S.

Publications and source records attributed to Maddox, P. S..

5 recordsLinked to original sources

bioRxiv2018

Quiescent cells actively replenish CENP-A nucleosomes to maintain centromere identity and proliferative potential

Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant CENP-A. Prevailing models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres, but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery, but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.

cell biology↗

bioRxiv2018

Nucleo-cytoplasmic trafficking regulates nuclear surface area during nuclear organogenesis

Throughout development, nuclei must be assembled following every cell division to establish a functional organelle from compact, mitotic chromatin. During nuclear organogenesis, chromatin expands to establish a nucleus of a given size seperate from the cytoplasm. Determining how nuclear organogenesis is regulated is particularly significant in the context of certain cancers in which scaling relationships between cell and nuclear sizes are not maintained. Controlling cell size in vitro using a microfluidics approach, we determined that neither nuclear volume nor surface area scale directly with cell size. Looking to explain differential nuclear scaling relationships, we developed a simple mechano-chemical mathematical model. In simulating biological perturbations in silico, our model predicted crucial roles for nucleo-cytoplasmic trafficking in regulating nuclear expansion and in restricting the recruitment of a potential nuclear surface area factor. In mammalian tissue culture, inhibiting nuclear export increased nuclear expansion rates and reduced the amount of nuclear lamin, a candidate surface area factor, being recruited to assembling nuclei, supporting our models predictions. Targeting the principal nuclear export component in the Drosophila syncytial embryo, Embargoed, we show that nuclear expansion rates are also increased in this developmental context, consistent with our model. Using the MS2-reporter system in fly embryos, we demonstrate a role for nuclear export in regulating transcription activation timing and dynamics, suggesting that regulating nuclear assembly is crucial for downstream nuclear function. Taken together, we propose a simple model through which nuclear organogenesis is achieved and demonstrate a role for nuclear export in regulating nuclear assembly.

cell biology↗

bioRxiv2018

Embryo timelapses can be compiled and quantified to understand canonical histone dynamics across multiple cell cycles.

In the last decade, computational analysis of big datasets has facilitated the processing of unprecedented quantities of collected biological data. Thus, automations and big data analyses have been revolutionary in detecting and quantifying subtle phenotypes in cell biological contexts. Analyzing similar quantities of data in larger and more complicated biological systems such as live embryos has been more challenging due to experimental necessities impeding both compilations of data collection and informative analysis. Here we present a streamlined workflow that can quantify cell cycle dynamics in early developing embryos using fluorescently labeled proteins. We benchmark this pipeline using Caenorhabditis elegans (nematode) embryonic development and a fluorescently labeled histone. Using our pipeline, we find that histone proteins are broadly stable in early embryonic development. In sum, we have utilized the large biological and experimental variation associated with quantification of fluorescent proteins in embryonic systems, to quantify nuclear accumulation rate, chromatin incorporation, and turnover/stability of canonical histones during early development.

developmental biology↗

bioRxiv2017

LITE microscopy: a technology for high numerical aperture, low photobleaching fluorescence imaging

Fluorescence microscopy is a powerful approach for studying sub-cellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel LSFM method: Lateral Interference Tilted Excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically-limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

cell biology↗

bioRxiv2017

Crosslinkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis

Cytokinesis and other cell shape changes are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in non-muscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal crosslinking by myosins and non-motor crosslinkers, are thought to promote contractility. Here, we examined how the abundance of motor and non-motor crosslinkers controls the speed of cytokinetic furrowing. We built a minimal model to simulate the contractile dynamics of the C. elegans zygote cytokinetic ring. This model predicted that intermediate levels of non-motor crosslinkers would allow maximal contraction speed, which we found to be the case for the scaffold protein anillin, in vivo. Our model also demonstrated a non-linear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of non-muscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, both motor and non-motor crosslinkers promote cytokinetic ring closure when present at low levels, but act as a brake when present at higher levels. Together, our findings extend the growing appreciation for the roles of crosslinkers, but reveal that they not only drive but also brake cytoskeletal remodeling.

cell biology↗