Accepted Manuscript:
Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network
Title: Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network
The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results more »
highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell. « less
Henson, John H.; Samasa, Bakary; Shuster, Charles B.; Wikramanayake, Athula H.(
, PLOS ONE)
Klymkowsky, Michael
(Ed.)
Canonical Wnt/β-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the Frizzled:LRP5/6 receptor complex with β-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica transmission electron microscopy (TEM) to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D structured illumination microscopy (SIM) imaging of isolated egg cortices demonstrated the graded distribution of Dsh in the VCD, whereas higher resolution stimulated emission depletion (STED) imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted withmore »the first cleavage actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for the localized activation of the cWnt pathway at the sea urchin vegetal pole. These observations in sea urchins may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.« less
Lombardo, Andrew T.; Nelson, Shane R.; Kennedy, Guy G.; Trybus, Kathleen M.; Walcott, Sam; Warshaw, David M.(
, Proceedings of the National Academy of Sciences)
The cell’s dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament’s 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D modelmore »that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.
Reiprich, T. H.; Veronica, A.; Pacaud, F.; Ramos-Ceja, M. E.; Ota, N.; Sanders, J.; Kara, M.; Erben, T.; Klein, M.; Erler, J.; et al(
, Astronomy & Astrophysics)
Context. Inferences about dark matter, dark energy, and the missing baryons all depend on the accuracy of our model of large-scale structure evolution. In particular, with cosmological simulations in our model of the Universe, we trace the growth of structure, and visualize the build-up of bigger structures from smaller ones and of gaseous filaments connecting galaxy clusters. Aims. Here we aim to reveal the complexity of the large-scale structure assembly process in great detail and on scales from tens of kiloparsecs up to more than 10 Mpc with new sensitive large-scale observations from the latest generation of instruments. We also aim to compare our findings with expectations from our cosmological model. Methods. We used dedicated SRG/eROSITA performance verification (PV) X-ray, ASKAP/EMU Early Science radio, and DECam optical observations of a ~15 deg 2 region around the nearby interacting galaxy cluster system A3391/95 to study the warm-hot gas in cluster outskirts and filaments, the surrounding large-scale structure and its formation process, the morphological complexity in the inner parts of the clusters, and the (re-)acceleration of plasma. We also used complementary Sunyaev-Zeldovich (SZ) effect data from the Planck survey and custom-made Galactic total (neutral plus molecular) hydrogen column density maps based onmore »the HI4PI and IRAS surveys. We relate the observations to expectations from cosmological hydrodynamic simulations from the Magneticum suite. Results. We trace the irregular morphology of warm and hot gas of the main clusters from their centers out to well beyond their characteristic radii, r 200 . Between the two main cluster systems, we observe an emission bridge on large scale and with good spatial resolution. This bridge includes a known galaxy group but this can only partially explain the emission. Most gas in the bridge appears hot, but thanks to eROSITA’s unique soft response and large field of view, we discover some tantalizing hints for warm, truly primordial filamentary gas connecting the clusters. Several matter clumps physically surrounding the system are detected. For the “Northern Clump,” we provide evidence that it is falling towards A3391 from the X-ray hot gas morphology and radio lobe structure of its central AGN. Moreover, the shapes of these X-ray and radio structures appear to be formed by gas well beyond the virial radius, r 100 , of A3391, thereby providing an indirect way of probing the gas in this elusive environment. Many of the extended sources in the field detected by eROSITA are also known clusters or new clusters in the background, including a known SZ cluster at redshift z = 1. We find roughly an order of magnitude more cluster candidates than the SPT and ACT surveys together in the same area. We discover an emission filament north of the virial radius of A3391 connecting to the Northern Clump. Furthermore, the absorption-corrected eROSITA surface brightness map shows that this emission filament extends south of A3395 and beyond an extended X-ray-emitting object (the “Little Southern Clump”) towards another galaxy cluster, all at the same redshift. The total projected length of this continuous warm-hot emission filament is 15 Mpc, running almost 4 degrees across the entire eROSITA PV observation field. The Northern and Southern Filament are each detected at >4 σ . The Planck SZ map additionally appears to support the presence of both new filaments. Furthermore, the DECam galaxy density map shows galaxy overdensities in the same regions. Overall, the new datasets provide impressive confirmation of the theoretically expected structure formation processes on the individual system level, including the surrounding warm-hot intergalactic medium distribution; the similarities of features found in a similar system in the Magneticum simulation are striking. Our spatially resolved findings show that baryons indeed reside in large-scale warm-hot gas filaments with a clumpy structure.« less
The gene regulatory network (GRN) that underlies echinoderm skeletogenesis is a prominent model of GRN architecture and evolution. KirrelL is an essential downstream effector gene in this network and encodes an Ig-superfamily protein required for the fusion of skeletogenic cells and the formation of the skeleton. In this study, we dissected the transcriptional control region of the kirrelL gene of the purple sea urchin, Strongylocentrotus purpuratus . Using plasmid- and bacterial artificial chromosome-based transgenic reporter assays, we identified key cis -regulatory elements (CREs) and transcription factor inputs that regulate Sp-kirrelL , including direct, positive inputs from two key transcription factors in the skeletogenic GRN, Alx1 and Ets1. We next identified kirrelL cis -regulatory regions from seven other echinoderm species that together represent all classes within the phylum. By introducing these heterologous regulatory regions into developing sea urchin embryos we provide evidence of their remarkable conservation across ~500 million years of evolution. We dissected in detail the kirrelL regulatory region of the sea star, Patiria miniata , and demonstrated that it also receives direct inputs from Alx1 and Ets1. Our findings identify kirrelL as a component of the ancestral echinoderm skeletogenic GRN. They support the view that GRN subcircuits, including specificmore »transcription factor–CRE interactions, can remain stable over vast periods of evolutionary history. Lastly, our analysis of kirrelL establishes direct linkages between a developmental GRN and an effector gene that controls a key morphogenetic cell behavior, cell–cell fusion, providing a paradigm for extending the explanatory power of GRNs.« less
McCall, Patrick M.; MacKintosh, Frederick C.; Kovar, David R.; Gardel, Margaret L.(
, Proceedings of the National Academy of Sciences)
The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how nonequilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actinmore »coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.« less
Garno, Chelsea, Irons, Zoe H., Gamache, Courtney M., McKim, Quenelle, Reyes, Gabriela, Wu, Xufeng, Shuster, Charles B., and Henson, John H.. Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network. Retrieved from https://par.nsf.gov/biblio/10343394. PLOS ONE 16.12 Web. doi:10.1371/journal.pone.0252845.
Garno, Chelsea, Irons, Zoe H., Gamache, Courtney M., McKim, Quenelle, Reyes, Gabriela, Wu, Xufeng, Shuster, Charles B., & Henson, John H.. Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network. PLOS ONE, 16 (12). Retrieved from https://par.nsf.gov/biblio/10343394. https://doi.org/10.1371/journal.pone.0252845
Garno, Chelsea, Irons, Zoe H., Gamache, Courtney M., McKim, Quenelle, Reyes, Gabriela, Wu, Xufeng, Shuster, Charles B., and Henson, John H..
"Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network". PLOS ONE 16 (12). Country unknown/Code not available. https://doi.org/10.1371/journal.pone.0252845.https://par.nsf.gov/biblio/10343394.
@article{osti_10343394,
place = {Country unknown/Code not available},
title = {Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network},
url = {https://par.nsf.gov/biblio/10343394},
DOI = {10.1371/journal.pone.0252845},
abstractNote = {The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament-like network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide the visualization of an apparent septin filament network with the CR structure of an animal cell.},
journal = {PLOS ONE},
volume = {16},
number = {12},
author = {Garno, Chelsea and Irons, Zoe H. and Gamache, Courtney M. and McKim, Quenelle and Reyes, Gabriela and Wu, Xufeng and Shuster, Charles B. and Henson, John H.},
editor = {Prigent, Claude}
}
