@article {65240, title = {Aurora B functions at the apical surface after specialized cytokinesis during morphogenesis in C. elegans.}, journal = {Development}, volume = {147}, year = {2020}, month = {2020 Jan}, pages = {1-16}, abstract = {

While cytokinesis has been intensely studied, the way it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant embryonic divisions and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we found several unexpected alterations to cytokinesis including apical midbody migration in polarizing epithelial cells of the gut, pharynx and sensory neurons. Aurora B kinase, which is essential for several aspects of cytokinesis, remains apically localized in each of these tissues after internalization of midbody ring components. Aurora B inactivation disrupts cytokinesis and causes defects in apical structures, even if inactivated post-mitotically. Therefore, cytokinesis is implemented in a specialized way during epithelial polarization and Aurora B has a new role in the formation of the apical surface.

}, issn = {1477-9129}, doi = {10.1242/dev.181099}, author = {Bai, Xiaofei and Melesse, Michael and Sorensen Turpin, Christopher G and Sloan, Dillon and Chen, Chin-Yi and Wang, Wen-Cheng and Lee, Po-Yi and Simmons, James R and Nebenfuehr, Benjamin and Mitchell, Diana and Klebanow, Lindsey R and Mattson, Nicholas and Betzig, Eric and Chen, Bi-Chang and Cheerambathur, Dhanya and Bembenek, Joshua N} } @article {48843, title = {Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes.}, journal = {eLife}, volume = {6}, year = {2017}, month = {2017 Sep 26}, abstract = {

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (\<0.75 {\textmu}m), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

}, issn = {2050-084X}, doi = {10.7554/eLife.26990}, author = {Fritz-Laylin, Lillian K and Riel-Mehan, Megan and Chen, Bi-Chang and Lord, Samuel J and Goddard, Thomas D and Ferrin, Thomas E and Nicholson-Dykstra, Susan M and Higgs, Henry and Johnson, Graham T and Betzig, Eric and Mullins, R Dyche} } @article {48496, title = {Visualizing dynamic microvillar search and stabilization during ligand detection by T cells.}, journal = {Science (New York, N.Y.)}, volume = {356}, year = {2017}, month = {2017 May 12}, abstract = {

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

}, issn = {1095-9203}, doi = {10.1126/science.aal3118}, author = {Cai, En and Marchuk, Kyle and Beemiller, Peter and Beppler, Casey and Rubashkin, Matthew G and Weaver, Valerie M and G{\'e}rard, Audrey and Liu, Tsung-Li and Chen, Bi-Chang and Betzig, Eric and Bartumeus, Frederic and Krummel, Matthew F} } @article {47790, title = {Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy.}, journal = {Molecular Biology of the Cell}, volume = {27}, year = {2016}, month = {2016 Aug 17}, pages = {3418-35}, abstract = {

Membrane remodeling is an essential part for transfer of components to and from the cell surface and membrane-bound organelles, and for changes in cell shape, particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging, mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and non-invasive illumination of the newly developed lattice light sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and showed that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and remained relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and in multicellular assemblies.

}, issn = {1939-4586}, doi = {10.1091/mbc.E16-03-0164}, author = {Aguet, Fran{\c c}ois and Upadhyayula, Srigokul and Gaudin, Rapha{\"e}l and Chou, Yi-ying and Cocucci, Emanuele and He, Kangmin and Chen, Bi-Chang and Mosaliganti, Kishore and Pasham, Mithun and Skillern, Wesley and Legant, Wesley R and Liu, Tsung-Li and Findlay, Greg and Marino, Eric and Danuser, Gaudenz and Megason, Sean and Betzig, Eric and Kirchhausen, Tom} } @article {48118, title = {A plasma membrane template for macropinocytic cups.}, journal = {eLife}, volume = {5}, year = {2016}, month = {2016 Dec 13}, abstract = {

Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery.

}, issn = {2050-084X}, doi = {10.7554/eLife.20085}, author = {Veltman, Douwe M and Williams, Thomas D and Bloomfield, Gareth and Chen, Bi-Chang and Betzig, Eric and Insall, Robert H and Kay, Robert R} } @article {47971, title = {V-1 regulates capping protein activity in vivo.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {113}, year = {2016}, month = {2016 Oct 25}, pages = {E6610-9}, abstract = {

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1{\textquoteright}s ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

}, issn = {1091-6490}, doi = {10.1073/pnas.1605350113}, author = {Jung, Goeh and Alexander, Christopher J and Wu, Xufeng S and Piszczek, Grzegorz and Chen, Bi-Chang and Betzig, Eric and Hammer, John A} } @article {46774, title = {Actin depletion initiates events leading to granule secretion at the immunological synapse.}, journal = {Immunity}, volume = {42}, year = {2015}, month = {2015 May 19}, pages = {864-76}, abstract = {

Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1\ min, cortical actin reduced across the synapse, T\ cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5\ min and reached the synapse after 6\ min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.

}, issn = {1097-4180}, doi = {10.1016/j.immuni.2015.04.013}, author = {Ritter, Alex T and Asano, Yukako and Stinchcombe, Jane C and Dieckmann, N M G and Chen, Bi-Chang and Gawden-Bone, C and van Engelenburg, Schuyler and Legant, Wesley and Gao, Liang and Davidson, Michael W and Betzig, Eric and Lippincott-Schwartz, Jennifer and Griffiths, Gillian M} } @article {46530, title = {Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edge of motile cells and on protrusions of immobile cells.}, journal = {Molecular Biology of the Cell}, volume = {26}, year = {2015}, month = {2015 Apr 7}, pages = {2044-53}, abstract = {

Clathrin/AP2-coated vesicles are the principal endocytic carriers originating at the plasma membrane. In experiments reported here, we have used spinning disk confocal and lattice light sheet microscopy to study the assembly dynamics of coated pits on the dorsal and ventral membranes of migrating U373 glioblastoma cells stably expressing AP2-EGFP and on lateral protrusions from immobile SUM159 breast carcinoma cells, gene edited to express AP2-EGFP. On U373 cells, coated pits initiated on the dorsal membrane at the front of the lamellipodium, as well as at the approximate boundary between the lamellipodium and lamella, and continued to grow as they were swept back toward the cell body; coated pits were absent from the corresponding ventral membrane. We observed a similar dorsal/ventral asymmetry on membrane protrusions from SUM159 cells. Stationary-coated pits formed and budded on the remainder of the dorsal and ventral surfaces of both types of cells. These observations support a previously proposed model that invokes net membrane deposition at the leading edge due to an imbalance between the endocytic and exocytic membrane flow at the front of a migrating cell.

}, issn = {1939-4586}, doi = {10.1091/mbc.E15-01-0055}, author = {Kural, Comert and Akatay, Ahmet Ata and Gaudin, Rapha{\"e}l and Chen, Bi-Chang and Legant, Wesley R and Betzig, Eric and Kirchhausen, Tom} } @article {47020, title = {Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.}, journal = {Science (New York, N.Y.)}, volume = {349}, year = {2015}, month = {2015 Aug 28}, abstract = {

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

}, issn = {1095-9203}, doi = {10.1126/science.aab3500}, author = {Li, Dong and Shao, Lin and Chen, Bi-Chang and Zhang, Xi and Zhang, Mingshu and Moses, Brian and Milkie, Daniel E and Beach, Jordan R and Hammer, John A and Pasham, Mithun and Kirchhausen, Tomas and Baird, Michelle A and Davidson, Michael W and Xu, Pingyong and Betzig, Eric} } @article {47145, title = {Histone H3 threonine phosphorylation regulates asymmetric histone inheritance in the Drosophila male germline.}, journal = {Cell}, volume = {163}, year = {2015}, month = {2015 Nov 5}, pages = {920-33}, abstract = {

A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously, we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here, we show that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.10.002}, author = {Xie, Jing and Wooten, Matthew and Tran, Vuong and Chen, Bi-Chang and Pozmanter, Caitlin and Simbolon, Christine and Betzig, Eric and Chen, Xin} } @article {47183, title = {Three-dimensional tracking of plus-tips by lattice light-sheet microscopy permits the quantification of microtubule growth trajectories within the mitotic apparatus.}, journal = {Journal of Biomedical Optics}, volume = {20}, year = {2015}, month = {2015 Oct 1}, pages = {101206}, issn = {1560-2281}, doi = {10.1117/1.JBO.20.10.101206}, author = {Yamashita, Norio and Morita, Masahiko and Legant, Wesley R and Chen, Bi-Chang and Betzig, Eric and Yokota, Hideo and Mimori-Kiyosue, Yuko} } @article {46922, title = {Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.}, journal = {FASEB Journal}, volume = {29}, year = {2015}, month = {2015 Nov}, pages = {4555-67}, abstract = {

Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation.-Thievessen, I., Fakhri, N., Steinwachs, J., Kraus, V., McIsaac, R. S., Gao, L., Chen, B.-C., Baird, M. A., Davidson, M. W., Betzig, E., Oldenbourg, R., Waterman, C., M., Fabry, B. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

}, issn = {1530-6860}, doi = {10.1096/fj.14-268235}, author = {Thievessen, Ingo and Fakhri, Nikta and Steinwachs, Julian and Kraus, Viola and McIsaac, R Scott and Gao, Liang and Chen, Bi-Chang and Baird, Michelle A and Davidson, Michael W and Betzig, Eric and Oldenbourg, Rudolf and Waterman, Clare M and Fabry, Ben} } @article {45886, title = {3D imaging of Sox2 enhancer clusters in embryonic stem cells.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Dec 24}, abstract = {

Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

}, issn = {2050-084X}, doi = {10.7554/eLife.04236}, author = {Liu, Zhe and Legant, Wesley R and Chen, Bi-Chang and Li, Li and Grimm, Jonathan B and Lavis, Luke D and Betzig, Eric and Tjian, Robert} } @article {6951, title = {3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy.}, journal = {Nature Protocols}, volume = {9}, year = {2014}, month = {2014 May}, pages = {1083-101}, abstract = {

3D live imaging is important for a better understanding of biological processes, but it is challenging with current techniques such as spinning-disk confocal microscopy. Bessel beam plane illumination microscopy allows high-speed 3D live fluorescence imaging of living cellular and multicellular specimens with nearly isotropic spatial resolution, low photobleaching and low photodamage. Unlike conventional fluorescence imaging techniques that usually have a unique operation mode, Bessel plane illumination has several modes that offer different performance with different imaging metrics. To achieve optimal results from this technique, the appropriate operation mode needs to be selected and the experimental setting must be optimized for the specific application and associated sample properties. Here we explain the fundamental working principles of this technique, discuss the pros and cons of each operational mode and show through examples how to optimize experimental parameters. We also describe the procedures needed to construct, align and operate a Bessel beam plane illumination microscope by using our previously reported system as an example, and we list the necessary equipment to build such a microscope. Assuming all components are readily available, it would take a person skilled in optical instrumentation \~{}1 month to assemble and operate a microscope according to this protocol.

}, issn = {1750-2799}, doi = {10.1038/nprot.2014.087}, author = {Gao, Liang and Shao, Lin and Chen, Bi-Chang and Betzig, Eric} } @article {45617, title = {Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.}, journal = {Science}, volume = {346}, year = {2014}, month = {2014 Oct 24}, pages = {1257998}, abstract = {

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

}, issn = {1095-9203}, doi = {10.1126/science.1257998}, author = {Chen, Bi-Chang and Legant, Wesley R and Wang, Kai and Shao, Lin and Milkie, Daniel E and Davidson, Michael W and Janetopoulos, Chris and Wu, Xufeng S and Hammer, John A and Liu, Zhe and English, Brian P and Mimori-Kiyosue, Yuko and Romero, Daniel P and Ritter, Alex T and Lippincott-Schwartz, Jennifer and Fritz-Laylin, Lillian and Mullins, R Dyche and Mitchell, Diana M and Bembenek, Joshua N and Reymann, Anne-Cecile and B{\"o}hme, Ralph and Grill, Stephan W and Wang, Jennifer T and Seydoux, Geraldine and Tulu, U Serdar and Kiehart, Daniel P and Betzig, Eric} } @article {45880, title = {Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans.}, journal = {eLife}, volume = {4}, year = {2014}, month = {2014 Dec 23}, abstract = {

RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2A(PPTR-{\textonehalf}). Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation.

}, issn = {2050-084X}, doi = {10.7554/eLife.04591}, author = {Wang, Jennifer T and Smith, Jarrett and Chen, Bi-Chang and Schmidt, Helen and Rasoloson, Dominique and Paix, Alexandre and Lambrus, Bramwell G and Calidas, Deepika and Betzig, Eric and Seydoux, Geraldine} } @article {6919, title = {Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.}, journal = {Cell}, volume = {156}, year = {2014}, month = {2014 Mar 13}, pages = {1274-85}, abstract = {

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in\ vivo and in\ vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to\ determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2014.01.062}, author = {Chen, Jiji and Zhang, Zhengjian and Li Li and Chen, Bi-Chang and Revyakin, Andrey and Hajj, Bassam and Legant, Wesley and Dahan, Maxime and Lionnet, Timoth{\'e}e and Betzig, Eric and Tjian, Robert and Liu, Zhe} } @article {6291, title = {A localized Wnt signal orients asymmetric stem cell division in vitro.}, journal = {Science}, volume = {339}, year = {2013}, month = {2013 Mar 22}, pages = {1445-8}, abstract = {

Developmental signals such as Wnts are often presented to cells in an oriented manner. To examine the consequences of local Wnt signaling, we immobilized Wnt proteins on beads and introduced them to embryonic stem cells in culture. At the single-cell level, the Wnt-bead induced asymmetric distribution of Wnt-β-catenin signaling components, oriented the plane of mitotic division, and directed asymmetric inheritance of centrosomes. Before cytokinesis was completed, the Wnt-proximal daughter cell expressed high levels of nuclear β-catenin and pluripotency genes, whereas the distal daughter cell acquired hallmarks of differentiation. We suggest that a spatially restricted Wnt signal induces an oriented cell division that generates distinct cell fates at predictable positions relative to the Wnt source.

}, keywords = {Animals, Asymmetric Cell Division, beta Catenin, Cell Differentiation, Cells, Centrosome, Cultured, Cytokinesis, Embryonic Stem Cells, Gene Expression, Homeodomain Proteins, Mice, Mitosis, Octamer Transcription Factor-3, Pluripotent Stem Cells, Recombinant Proteins, Single-Cell Analysis, Transcription Factors, Wnt Proteins, Wnt Signaling Pathway, Wnt3A Protein}, issn = {1095-9203}, doi = {10.1126/science.1231077}, author = {Habib, Shukry J and Chen, Bi-Chang and Tsai, Feng-Chiao and Anastassiadis, Konstantinos and Meyer, Tobias and Betzig, Eric and Nusse, Roel} }