depolymerization
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Actin cytoskeleton is a vital cellular structure primarily known for controlling cell integrity, division and expansion. Here we present a proteomic dissection of Arabidopsis roots treated by actin depolymerizing agent latrunculin B. Pharmacological disintegration of the actin cytoskeleton by latrunculin B caused downregulation of several proteins involved in the actin organization and dynamics. Moreover, this approach helped to identify new protein candidates involved in gene transcription, due to the altered abundance of proteins involved in mRNA nuclear export. Finally, latrunculin B negatively affected the abundance of abscisic acid (ABA) responsive proteins. SIGNIFICANCE: This article substantially contributes to the current knowledge about the importance of actin organization and dynamics in proteome remodelling. We employed gel based and gel free proteomic analyses and identified several new protein candidates and protein networks linking actin dynamics to the gene transcription and to the ABA response in Arabidopsis.
- MeSH
- aktiny chemie metabolismus MeSH
- Arabidopsis chemie MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- kořeny rostlin chemie MeSH
- kyselina abscisová fyziologie MeSH
- mikrofilamenta účinky léků MeSH
- polymerizace účinky léků MeSH
- proteiny huseníčku analýza chemie metabolismus MeSH
- proteom analýza účinky léků MeSH
- proteomika metody MeSH
- thiazolidiny farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The integrity of the actin cytoskeleton is essential for plant immune signalling. Consequently, it is generally assumed that actin disruption reduces plant resistance to pathogen attack. Here, we demonstrate that actin depolymerization induced a dramatic increase in salicylic acid (SA) levels in Arabidopsis thaliana. Transcriptomic analysis showed that the SA pathway was activated due to the action of isochorismate synthase (ICS). The effect was also confirmed in Brassica napus. This raises the question of whether actin depolymerization could, under particular conditions, lead to increased resistance to pathogens. Thus, we explored the effect of pretreatment with actin-depolymerizing drugs on the resistance of Arabidopsis thaliana to the bacterial pathogen Pseudomonas syringae, and on the resistance of an important crop Brassica napus to its natural fungal pathogen Leptosphaeria maculans. In both pathosystems, actin depolymerization activated the SA pathway, leading to increased plant resistance. To our best knowledge, we herein provide the first direct evidence that disruption of the actin cytoskeleton can actually lead to increased plant resistance to pathogens, and that SA is crucial to this process.
- MeSH
- aktiny metabolismus MeSH
- Arabidopsis metabolismus mikrobiologie MeSH
- Ascomycota patogenita MeSH
- Brassica napus metabolismus mikrobiologie MeSH
- intramolekulární transferasy metabolismus MeSH
- kyselina salicylová metabolismus MeSH
- nemoci rostlin mikrobiologie MeSH
- proteiny huseníčku metabolismus MeSH
- Pseudomonas syringae patogenita MeSH
- regulace genové exprese u rostlin fyziologie MeSH
- signální transdukce fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- glukany chemie imunologie terapeutické užití MeSH
- Saccharomyces cerevisiae MeSH
- Publikační typ
- srovnávací studie MeSH
Although actin monomers polymerize into filaments in the cytoplasm, the form of actin in the nucleus remains elusive. We searched for the form and function of β-actin fused to nuclear localization signal and to enhanced yellow fluorescent protein (EN-actin). Our results reveal that EN-actin is either dispersed in the nucleoplasm (homogenous EN-actin) or forms bundled filaments in the nucleus (EN-actin filaments). Formation of such filaments was not connected with increased EN-actin levels. Among numerous actin-binding proteins tested, only cofilin is recruited to the EN-actin filaments. Overexpression of EN-actin causes increase in the nuclear levels of actin-related protein 3 (Arp3). Although Arp3, a member of actin nucleation complex Arp2/3, is responsible for EN-actin filament nucleation and bundling, the way cofilin affects nuclear EN-actin filaments dynamics is not clear. While cells with homogenous EN-actin maintained unaffected mitosis during which EN-actin re-localizes to the plasma membrane, generation of nuclear EN-actin filaments severely decreases cell proliferation and interferes with mitotic progress. The introduction of EN-actin manifests in two mitotic-inborn defects-formation of binucleic cells and generation of micronuclei-suggesting that cells suffer aberrant cytokinesis and/or impaired chromosomal segregation. In interphase, nuclear EN-actin filaments passed through chromatin region, but do not co-localize with either chromatin remodeling complexes or RNA polymerases I and II. Surprisingly presence of EN-actin filaments was connected with increase in the overall transcription levels in the S-phase by yet unknown mechanism. Taken together, EN-actin can form filaments in the nucleus which affect important cellular processes such as transcription and mitosis.
- MeSH
- aktiny metabolismus MeSH
- bakteriální proteiny metabolismus MeSH
- buněčné jádro metabolismus MeSH
- faktory depolymerizující aktin MeSH
- genetická transkripce MeSH
- HEK293 buňky MeSH
- lidé MeSH
- luminescentní proteiny metabolismus MeSH
- mikrofilamenta metabolismus MeSH
- mitóza genetika MeSH
- nádorové buněčné linie MeSH
- protein 3 související s aktinem biosyntéza metabolismus MeSH
- restrukturace chromatinu MeSH
- RNA-polymerasa I genetika MeSH
- RNA-polymerasa II genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
P21-activated kinases (PAKs) are involved in the regulation of multiple processes including cell proliferation, adhesion and migration. However, the current knowledge about their function is mainly based on results obtained in adherent cell types. We investigated the effect of group I PAK inhibition using the compound IPA-3 in a variety of human leukemic cell lines (JURL-MK1, MOLM-7, K562, CML-T1, HL-60, Karpas-299, Jurkat, HEL) as well as in primary blood cells. IPA-3 induced cell death with EC50 ranging from 5 to more than 20 μM. Similar range was found for IPA-3-mediated dephosphorylation of a known PAK downstream effector, cofilin. The cell death was associated with caspase-3 activation, PARP cleavage and apoptotic DNA fragmentation. In parallel, 20 μM IPA-3 treatment induced rapid and marked decrease of the cell adhesivity to fibronectin. Per contra, partial reduction of PAK activity using lower dose IPA-3 or siRNA resulted in a slight increase in the cell adhesivity. The changes in the cell adhesivity were also studied using real-time microimpedance measurement and by interference reflection microscopy. Significant differences in the intracellular IPA-3 level among various cell lines were observed indicating that an active mechanism is involved in IPA-3 transport.
- MeSH
- apoptóza účinky léků MeSH
- buněčná adheze účinky léků MeSH
- disulfidy metabolismus farmakologie MeSH
- faktory depolymerizující aktin chemie metabolismus MeSH
- fibronektiny metabolismus MeSH
- fosforylace účinky léků MeSH
- hematopoéza účinky léků MeSH
- inhibitory proteinkinas metabolismus farmakologie MeSH
- intracelulární prostor účinky léků metabolismus MeSH
- krevní buňky cytologie účinky léků MeSH
- leukemie patologie MeSH
- lidé MeSH
- lymfom patologie MeSH
- malá interferující RNA genetika MeSH
- naftoly metabolismus farmakologie MeSH
- p21 aktivované kinasy antagonisté a inhibitory nedostatek genetika MeSH
- proliferace buněk účinky léků MeSH
- regulace genové exprese účinky léků MeSH
- serin metabolismus MeSH
- umlčování genů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment.
- MeSH
- dospělí MeSH
- faktory depolymerizující aktin biosyntéza genetika MeSH
- invazivní růst nádoru genetika patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfatické metastázy MeSH
- lymfatické uzliny metabolismus patologie MeSH
- molekuly buněčné adheze biosyntéza genetika MeSH
- nádorové biomarkery biosyntéza genetika MeSH
- nádorové proteiny biosyntéza genetika MeSH
- nádory prsu genetika patologie MeSH
- přežití bez známek nemoci MeSH
- prognóza MeSH
- proteiny vázající RNA biosyntéza genetika MeSH
- proteomika MeSH
- receptory pro estrogeny genetika MeSH
- regulace genové exprese u nádorů MeSH
- senioři MeSH
- stathmin biosyntéza genetika MeSH
- thrombospondiny biosyntéza genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
