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Miriam A Genuth, Christopher D C Allen, Takashi Mikawa, Orion D Weiner
To move directionally, cells can bias the generation of protrusions or select among randomly generated protrusions. Here we use 3D two-photon imaging of chick branchial arch 2 directed neural crest cells to probe how these mechanisms contribute to directed movement, whether a subset or the majority of cells polarize during movement, and how the different classes of protrusions relate to one another. We find that, in contrast to Xenopus, cells throughout the stream are morphologically polarized along the direction of overall stream movement and do not exhibit contact inhibition of locomotion...
March 6, 2018: Developmental Biology
Yang Yang, Ding Xiong, Anne Pipathsouk, Orion D Weiner, Min Wu
Assembly of the endocytic machinery is a constitutively active process that is important for the organization of the plasma membrane, signal transduction, and membrane trafficking. Existing research has focused on the stochastic nature of endocytosis. Here, we report the emergence of the collective dynamics of endocytic proteins as periodic traveling waves on the cell surface. Coordinated clathrin assembly provides the earliest spatial cue for cortical waves and sets the direction of propagation. Surprisingly, the onset of clathrin waves, but not individual endocytic events, requires feedback from downstream factors, including FBP17, Cdc42, and N-WASP...
November 20, 2017: Developmental Cell
Brian R Graziano, Delquin Gong, Karen E Anderson, Anne Pipathsouk, Anna R Goldberg, Orion D Weiner
Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation...
August 7, 2017: Journal of Cell Biology
Anna Reade, Laura B Motta-Mena, Kevin H Gardner, Didier Y Stainier, Orion D Weiner, Stephanie Woo
Here, we describe an optogenetic gene expression system optimized for use in zebrafish. This system overcomes the limitations of current inducible expression systems by enabling robust spatial and temporal regulation of gene expression in living organisms. Because existing optogenetic systems show toxicity in zebrafish, we re-engineered the blue-light-activated EL222 system for minimal toxicity while exhibiting a large range of induction, fine spatial precision and rapid kinetics. We validate several strategies to spatially restrict illumination and thus gene induction with our new TAEL (TA4-EL222) system...
January 15, 2017: Development
Zairan Liu, Orion D Weiner
RhoA controls cleavage furrow formation during cell division, but whether RhoA suffices to orchestrate spatiotemporal dynamics of furrow formation is unknown. In this issue, Wagner and Goltzer (2016. J. Cell Biol show that RhoA activity can induce furrow formation in all cell cortex positions and cell cycle phases.
June 20, 2016: Journal of Cell Biology
Alba Diz-Muñoz, Kevin Thurley, Sana Chintamen, Steven J Altschuler, Lani F Wu, Daniel A Fletcher, Orion D Weiner
For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts...
June 2016: PLoS Biology
Oliver Hoeller, Jared E Toettcher, Huaqing Cai, Yaohui Sun, Chuan-Hsiang Huang, Mariel Freyre, Min Zhao, Peter N Devreotes, Orion D Weiner
For directional movement, eukaryotic cells depend on the proper organization of their actin cytoskeleton. This engine of motility is made up of highly dynamic nonequilibrium actin structures such as flashes, oscillations, and traveling waves. In Dictyostelium, oscillatory actin foci interact with signals such as Ras and phosphatidylinositol 3,4,5-trisphosphate (PIP3) to form protrusions. However, how signaling cues tame actin dynamics to produce a pseudopod and guide cellular motility is a critical open question in eukaryotic chemotaxis...
February 2016: PLoS Biology
Clare E Buckley, Rachel E Moore, Anna Reade, Anna R Goldberg, Orion D Weiner, Jonathan D W Clarke
We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo...
January 11, 2016: Developmental Cell
Miriam A Genuth, Orion D Weiner
For proper spacing or rapid dispersion, some migratory cells are guided by repulsive collisions with their neighbors. A new study reveals that a surprising intercellular coupling of leading edge actin networks forms the basis of mutual repulsion in Drosophila hemocytes.
June 29, 2015: Current Biology: CB
Dan Yu, Michelle A Baird, John R Allen, Elizabeth S Howe, Matthew P Klassen, Anna Reade, Kalpana Makhijani, Yuanquan Song, Songmei Liu, Zehra Murthy, Shao-Qing Zhang, Orion D Weiner, Thomas B Kornberg, Yuh-Nung Jan, Michael W Davidson, Xiaokun Shu
Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.
August 2015: Nature Methods
Anna Payne-Tobin Jost, Orion D Weiner
We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inhibition of Bem1 produces profound defects in cell polarization and cell viability that are not observed in bem1Δ. By disrupting Bem1 activity at specific points in the cell cycle, we demonstrate that Bem1 is essential for the establishment of polarity and bud emergence but is dispensable for the growth of an emerged bud...
October 16, 2015: ACS Synthetic Biology
Sunny S Lou, Alba Diz-Muñoz, Orion D Weiner, Daniel A Fletcher, Julie A Theriot
Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression...
April 27, 2015: Journal of Cell Biology
Julie Wu, Anne Pipathsouk, A Keizer-Gunnink, F Fusetti, W Alkema, Shanshan Liu, Steven Altschuler, Lani Wu, Arjan Kortholt, Orion D Weiner
Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference-mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP3) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex...
May 1, 2015: Molecular Biology of the Cell
Doug Tischer, Orion D Weiner
The light-based control of ion channels has been transformative for the neurosciences, but the optogenetic toolkit does not stop there. An expanding number of proteins and cellular functions have been shown to be controlled by light, and the practical considerations in deciding between reversible optogenetic systems (such as systems that use light-oxygen-voltage domains, phytochrome proteins, cryptochrome proteins and the fluorescent protein Dronpa) are well defined. The field is moving beyond proof of concept to answering real biological questions, such as how cell signalling is regulated in space and time, that were difficult or impossible to address with previous tools...
August 2014: Nature Reviews. Molecular Cell Biology
Brian R Graziano, Orion D Weiner
Many eukaryotic cells regulate their polarity and motility in response to external chemical cues. While we know many of the linear connections that link receptors with downstream actin polymerization events, we have a much murkier understanding of the higher order positive and negative feedback loops that organize these processes in space and time. Importantly, physical forces and actin polymerization events do not simply act downstream of chemotactic inputs but are rather involved in a web of reciprocal interactions with signaling components to generate self-organizing pseudopods and cell polarity...
October 2014: Current Opinion in Cell Biology
Jason S Park, Benjamin Rhau, Aynur Hermann, Krista A McNally, Carmen Zhou, Delquin Gong, Orion D Weiner, Bruce R Conklin, James Onuffer, Wendell A Lim
Directed migration of diverse cell types plays a critical role in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to direct, manipulate, and study cell migration in vitro and in vivo in a specific and facile manner are currently limited. We conceived of a strategy to achieve direct control over cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. Here, we show that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-N-oxide (CNO)...
April 22, 2014: Proceedings of the National Academy of Sciences of the United States of America
Oliver Hoeller, Delquin Gong, Orion D Weiner
Adaptation is the ability of a system to respond and reset itself even in the continuing presence of a stimulus. On one hand, adaptation is a physiological necessity that enables proper neuronal signaling and cell movement. On the other hand, adaptation can be a source of annoyance, as it can make biological systems resistant to experimental perturbations. Here we speculate where adaptation might live in eukaryotic chemotaxis and how it can be encoded in the signaling network. We then discuss tools and strategies that can be used to both understand and outwit adaptation in a wide range of cellular contexts...
March 31, 2014: Developmental Cell
Orion D Weiner
We use many quantitative undergraduate metrics to help select our graduate students, but which of these usefully discriminate successful from underperforming students and which should be ignored? Almost everyone has his or her own pet theory of the most predictive criteria, but I hoped to address this question in a more unbiased manner. I conducted a retrospective analysis of the highest- and lowest-ranked graduate students over the past 20 years in the Tetrad program at the University of California at San Francisco to identify undergraduate metrics that significantly differed between these groups...
February 2014: Molecular Biology of the Cell
Laura B Motta-Mena, Anna Reade, Michael J Mallory, Spencer Glantz, Orion D Weiner, Kristen W Lynch, Kevin H Gardner
Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light...
March 2014: Nature Chemical Biology
Jared E Toettcher, Orion D Weiner, Wendell A Lim
The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk...
December 5, 2013: Cell
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