Julia Dyachok

The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, United States

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Publications (7)46.13 Total impact

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    ABSTRACT: Genetically encoded filamentous actin (F-actin) reporters designed based on fluorescent protein fusions to F-actin binding domains of actin regulatory proteins have emerged as powerful tools to decipher the role of the actin cytoskeleton in plant growth and development. However, these probes could interfere with the function of endogenous actin binding proteins and in turn impact actin organization and plant growth. We therefore surveyed F-actin labeling and compared organ growth in Arabidopsis thaliana lines expressing a variety of F-actin markers. Here we show that the variant of fluorescent protein, type of actin binding domain, and the promoter that drives reporter expression can influence the quality of F-actin labeling particularly in stable plant lines. For example, older red fluorescent protein (RFP)-based probes such as DsRed2 and mOrange induced more aberrant labeling compared to the newer RFP-based, mCherry, GFP, and GFP-derived fluorophores such as YFP and CFP. Moreover, qualitative and quantitative analyses revealed differences in F-actin organization in seedlings expressing Talin- and Lifeact-based reporters in some cell types compared to the fimbrin actin binding domain 2 (ABD2)-based reporters. Finally, the use of the ubiquitin10 (UBQ10) promoter to drive expression of the GFP-ABD2-GFP probe minimized loss of fluorescence and growth defects observed in the 35S-driven version. Taken together, this study shows that care must be taken in the interpretation of data derived from stable expression of certain F-actin reporters and that using alternative promoters such as UBQ10 can overcome some of the pitfalls that accompany the use of in vivo F-actin probes in plants. © 2014 Wiley Periodicals, Inc.
    Cytoskeleton 05/2014; 71(5). DOI:10.1002/cm.21174 · 3.01 Impact Factor
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    Julia Dyachok · Ling Zhu · Fuqi Liao · Ji He · Enamul Huq · Elison B Blancaflor
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    ABSTRACT: The ARP2/3 complex, a highly conserved nucleator of F-actin, and its activator, the SCAR complex, are essential for growth in plants and animals. In this article, we present a pathway through which roots of Arabidopsis thaliana directly perceive light to promote their elongation. The ARP2/3-SCAR complex and the maintenance of longitudinally aligned F-actin arrays are crucial components of this pathway. The involvement of the ARP2/3-SCAR complex in light-regulated root growth is supported by our finding that mutants of the SCAR complex subunit BRK1/HSPC300, or other individual subunits of the ARP2/3-SCAR complex, showed a dramatic inhibition of root elongation in the light, which mirrored reduced growth of wild-type roots in the dark. SCAR1 degradation in dark-grown wild-type roots by constitutive photomorphogenic 1 (COP1) E3 ligase and 26S proteasome accompanied the loss of longitudinal F-actin and reduced root growth. Light perceived by the root photoreceptors, cryptochrome and phytochrome, suppressed COP1-mediated SCAR1 degradation. Taken together, our data provide a biochemical explanation for light-induced promotion of root elongation by the ARP2/3-SCAR complex.
    The Plant Cell 10/2011; 23(10):3610-26. DOI:10.1105/tpc.111.088823 · 9.58 Impact Factor
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    ABSTRACT: During the past decade the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis root cells, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. Parallel to our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use nonembedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.
    Methods in molecular biology (Clifton, N.J.) 01/2009; 586:157-69. DOI:10.1007/978-1-60761-376-3_8 · 1.29 Impact Factor
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    ABSTRACT: The ARP2/3 complex, a highly conserved nucleator of F-actin polymerization, and its activator, the SCAR complex, have been shown to play important roles in leaf epidermal cell morphogenesis in Arabidopsis. However, the intracellular site(s) and function(s) of SCAR and ARP2/3 complex-dependent actin polymerization in plant cells remain unclear. We demonstrate that putative SCAR complex subunits BRK1 and SCAR1 are localized to the plasma membrane at sites of cell growth and wall deposition in expanding cells of leaves and roots. BRK1 localization is SCAR-dependent, providing further evidence of an association between these proteins in vivo. Consistent with plasma membrane localization of SCAR complex subunits, cortical F-actin accumulation in root tip cells is reduced in brk1 mutants. Moreover, mutations disrupting the SCAR or ARP2/3 complex reduce the growth rate of roots and their ability to penetrate semi-solid medium, suggesting reduced rigidity. Cell walls of mutant roots exhibit abnormal structure and composition at intercellular junctions where BRK1 and SCAR1 are enriched in the adjacent plasma membrane. Taken together, our results suggest that SCAR and ARP2/3 complex-dependent actin polymerization promotes processes at the plasma membrane that are important for normal growth and wall assembly.
    Molecular Plant 11/2008; 1(6):990-1006. DOI:10.1093/mp/ssn059 · 6.61 Impact Factor
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    ABSTRACT: The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, is essential for a variety of eukaryotic cellular processes, including epidermal cell morphogenesis in Arabidopsis thaliana. Efficient nucleation of actin filaments by the Arp2/3 complex requires the presence of an activator such as a member of the Scar/WAVE family. In mammalian cells, a multiprotein complex consisting of WAVE, PIR121/Sra-1, Nap1, Abi-2 and HSPC300 mediates responsiveness of WAVE to upstream regulators such as Rac. Essential roles in WAVE complex assembly or function have been demonstrated for PIR121/Sra-1, Nap1 and Abi-2, but the significance of HSPC300 in this complex is unclear. Plant homologs of all mammalian WAVE complex components have been identified, including HSPC300, the mammalian homolog of maize BRICK1 (BRK1). We show that, like mutations disrupting the Arabidopsis homologs of PIR121/Sra-1, Nap1 and Scar/WAVE, mutations in the Arabidopsis BRK1 gene result in trichome and pavement cell morphology defects (and associated alterations in the F-actin cytoskeleton of expanding cells) similar to those caused by mutations disrupting the ARP2/3 complex itself. Analysis of double mutants provides genetic evidence that BRK1 functions in a pathway with the ARP2/3 complex. BRK1 is required for accumulation of SCAR1 protein in vivo, potentially explaining the apparently essential role of BRK1 in ARP2/3 complex function.
    Development 04/2006; 133(6):1091-100. DOI:10.1242/dev.02280 · 6.27 Impact Factor
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    ABSTRACT: The dynamic actin cytoskeleton is important for a myriad of cellular functions, including intracellular transport, cell division, and cell shape. An important regulator of actin polymerization is the actin-related protein2/3 (Arp2/3) complex, which nucleates the polymerization of new actin filaments. In animals, Scar/WAVE family members activate Arp2/3 complex-dependent actin nucleation through interactions with Abi1, Nap1, PIR121, and HSCP300. Mutations in the Arabidopsis thaliana genes encoding homologs of Arp2/3 complex subunits PIR121 and NAP1 all show distorted trichomes as well as additional epidermal cell expansion defects, suggesting that a Scar/WAVE homolog functions in association with PIR121 and NAP1 to activate the Arp2/3 complex in Arabidopsis. In a screen for trichome branching defects, we isolated a mutant that showed irregularities in trichome branch positioning and expansion. We named this gene IRREGULAR TRICHOME BRANCH1 (ITB1). Positional cloning of the ITB1 gene showed that it encodes SCAR2, an Arabidopsis protein related to Scar/WAVE. Here, we show that itb1 mutants display cell expansion defects similar to those reported for the distorted class of trichome mutants, including disruption of actin and microtubule organization. In addition, we show that the scar homology domain (SHD) of ITB1/SCAR2 is necessary and sufficient for in vitro binding to Arabidopsis BRK1, the plant homolog of HSPC300. Overexpression of the SHD in transgenic plants causes a dominant negative phenotype. Our results extend the evidence that the Scar/WAVE pathway of Arp2/3 complex regulation exists in plants and plays an important role in regulating cell expansion.
    The Plant Cell 09/2005; 17(8):2314-26. DOI:10.1105/tpc.104.028670 · 9.58 Impact Factor
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    ABSTRACT: The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, plays a key role in the regulation of actin dynamics eukaryotic cells. In animal cells and yeasts, Wiskott-Aldrich Syndrome protein (WASP)/suppressor of cAMP receptor (Scar)/WASP family verprolin homologous (WAVE) family proteins activate the Arp2/3 complex in response to localized cues. Like other eukaryotes, plants have an Arp2/3 complex, which has recently been shown to play an important role in F-actin organization and cell morphogenesis. However, no activators of the Arp2/3 complex have been identified in plants, which lack obvious homologs of WASP/Scar/WAVE family proteins. Here, we identify a family of Scar/WAVE-related plant Arp2/3 activators. Like Scar/WAVE proteins, four proteins identified in Arabidopsis thaliana (AtSCAR1 to AtSCAR4) and one in maize (ZmSCAR1) have a C-terminal WASP homology 2 (WH2)/acidic (WA)-verprolin homology/cofilin homology/acidic (VCA)-like domain, which we show can activate the bovine Arp2/3 complex. At their N termini, AtSCAR1 to ATSCAR4, along with a fifth protein lacking a VCA/WA-like domain at its C terminus (At4g18600), are related to the N-terminal Scar homology domains of Scar/WAVE family proteins. Analysis of gene expression patterns suggests functional redundancy among members of the AtSCAR family. Full-length AtSCAR1 and ATSCAR3 proteins and their Scar homology domains bind in vitro to AtBRICK 1 (AtBRK1), the Arabidopsis homolog of HSPC300, a WAVE-binding protein recently identified as a component of a complex implicated in the regulation of Scar/WAVE activity. Thus, AtSCAR proteins are likely to function in association with AtBRK1, and perhaps other Arabidopsis homologs of WAVE complex components, to regulate activation of the Arp2,3 complex in vivo.
    Proceedings of the National Academy of Sciences 12/2004; 101(46):16379-84. DOI:10.1073/pnas.0407392101 · 9.81 Impact Factor

Publication Stats

220 Citations
46.13 Total Impact Points


  • 2009–2014
    • The Samuel Roberts Noble Foundation
      • Division of Plant Biology
      Ardmore, Oklahoma, United States
  • 2004–2008
    • University of California, San Diego
      • Section of Cell and Developmental Biology
      San Diego, California, United States