Determination of the molecular assembly of actin and actin-binding proteins using photoluminescence

Park, Byeongho,Oh, Seunghee,Jo, Seunghan,Kang, Donyoung,Lim, Juhwan,Jung, Youngmo,Lee, Hyungsuk,Jun, Seong Chan Elsevier 2018 Colloids and Surfaces B Vol.169 No.-

<P><B>Abstract</B></P> <P>Actin, the most abundant protein in cells, polymerizes into filaments that play key roles in many cellular dynamics. To understand cell dynamics and functions, it is essential to examine the cytoskeleton structure organized by actin and actin-binding proteins. Here, we pave the way for determining the molecular assembly of the actin cytoskeleton using direct photonic <I>in situ</I> analysis, providing the photoluminescence characteristics of actin as a function of filament length and bundling, without labeling. In experiments for monomeric and filamentous actin reconstituted <I>in vitro</I>, structural forms of actin are identified from the peak positions and intensities of photoluminescence. Actin monomers exhibited small intensity emission peaks at 334 nm, whereas filamentous and bundled actin showed a shifted peak at 323 nm with higher intensity. The peak shift was found to be proportional to the length of the actin filament. With probing structural change of actin in various cells <I>in vivo</I>, our study provides an efficient and precise analytical <I>in situ</I> tool to examine the cytoskeleton structure. It will be beneficial for elucidating the mechanism of various cellular functions such as cell migration, differentiation, cytokinesis and adhesion. Furthermore, our technique can be applied to detect the alterations in the cell cytoskeleton that can occur during many pathological processes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Modification of actin samples with different structures using binding protein. </LI> <LI> Method for monitoring of actin structure from monomer to polymerized protein. </LI> <LI> Polymerization rate of actin under several temperatures and heat energy. </LI> <LI> Label-free detection of protein in both of <I>in vivo</I> and <I>in vitro</I> conditions. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>Actin’s structure and assembly behavior are determined by light absorption and photoluminescence characteristics under both <I>in vitro</I> and <I>in vivo</I> conditions.</P> <P>[DISPLAY OMISSION]</P>

PRP4 kinase induces actin rearrangement and epithelial-mesenchymal transition through modulation of the actin-binding protein cofilin

Islam, Salman Ul,Ahmed, Muhammad Bilal,Lee, Su Jin,Shehzad, Adeeb,Sonn, Jong Kyung,Kwon, Oh-Shin,Lee, Young Sup Elsevier 2018 Experimental cell research Vol.369 No.1

<P><B>Abstract</B></P> <P>Cell actin cytoskeleton is primarily modulated by Rho family proteins. RhoA regulates several downstream targets, including Rho-associated protein kinase (ROCK), LIM-Kinase (LIMK), and cofilin. Pre-mRNA processing factor 4B (PRP4) modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. In this study, we discovered that PRP4 over-expression in HCT116 colon cancer cells induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway. Two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) analysis indicated increased expression of protein phosphatase 1A (PP1A) in PRP4-transfected HCT116 cells. The presence of PRP4 increased the expression of PP1A both at the mRNA and protein levels, which possibly activated cofilin through dephosphorylation and subsequently modulated the cell actin cytoskeleton. Furthermore, we found that PRP4 over-expression did not induce cofilin dephosphorylation in the presence of okadaic acid, a potent phosphatase inhibitor. Moreover, we discovered that PRP4 over-expression in HCT116 cells induced dephosphorylation of migration and invasion inhibitory protein (MIIP), and down-regulation of E-cadherin protein levels, which were further restored by the presence of okadaic acid. These findings indicate a possible molecular mechanism of PRP4-induced actin cytoskeleton remodeling and epithelial-mesenchymal transition, and make PRP4 an important target in colon cancer.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PRP4 is involved in pre-mRNA splicing and cell signalling. </LI> <LI> PRP4 modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. </LI> <LI> PRP4 induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway in HCT116 cells. </LI> <LI> Dephosphorylation of cofilin results in F-actin stabilization, re-distribution of cytoplasmic actin, formation of actin stress fibers, and inhibition of cell motility. </LI> <LI> PRP4 over-expression induces the expressions of PP1A, which directly or indirectly dephosphorylates cofilin, resulting in actin cytoskeleton rearrangement, downregulation of E-cadherin, and EMT induction. Cofilin activation may be associated with EMT properties, and promotes the progression of human colon cancer. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P> <B>Proposed model for PRP4-induced cofilin and MIIP dephosphorylation and epithelial-mesenchymal transition (EMT) induction</B>. PRP4 over-expression results in cofilin and MIIP dephosphorylation, causing actin dynamics to increase, which may lead to EMT. Another proposed pathway for EMT induction by dephosphorylated MIIP is illustrated in the black-dotted panel. MIIP may inhibit the Rac1 signaling pathway through PAK1 (Rac1 downstream target) binding competition, which results in reduced lamellipodia formation and, finally, EMT.</P> <P>[DISPLAY OMISSION]</P>

Role of mechanical flow for actin network organization

Kang, Byungjun,Jo, Seunghan,Baek, Jonghyeok,Nakamura, Fumihiko,Hwang, Wonmuk,Lee, Hyungsuk Elsevier 2019 ACTA BIOMATERIALIA Vol.90 No.-

<P><B>Abstract</B></P> <P>The major cytoskeletal protein actin forms complex networks to provide structural support and perform vital functions in cells. <I>In vitro</I> studies have revealed that the structure of the higher-order actin network is determined primarily by the type of actin binding protein (ABP). By comparison, there are far fewer studies about the role of the mechanical environment for the organization of the actin network. In particular, the duration over which cells reorganize their shape in response to functional demands is relatively short compared to the <I>in vitro</I> protein polymerization time, suggesting that such changes can influence the actin network formation. We hypothesize that mechanical flows in the cytoplasm generated by exogenous and endogenous stimulation play a key role in the spatiotemporal regulation of the actin architecture. To mimic cytoplasmic streaming, we generated a circulating flow using surface acoustic wave in a microfluidic channel and investigated its effect on the formation of networks by actin and ABPs. We found that the mechanical flow affected the orientation and thickness of actin bundles, depending on the type and concentration of ABPs. Our computational model shows that the extent of alignment and thickness of actin bundle are determined by the balance between flow-induced drag forces and the tendency of ABPs to crosslink actin filaments at given angles. These results suggest that local intracellular flows can affect the assembly dynamics and morphology of the actin cytoskeleton.</P> <P><B>Statement of Significance</B></P> <P>Spatiotemporal regulation of actin cytoskeleton structure is essential in many cellular functions. It has been shown that mechanical cues including an applied force and geometric boundary can alter the structural characteristics of actin network. However, even though the cytoplasm accounts for a large portion of the cell volume, the effect of the cytoplasmic streaming flow produced during cell dynamics on actin network organization has not been reported. In this study, we demonstrated that the mechanical flow exerted during actin network organization play an important role in determining the orientation and dimension of actin bundle network. Our result will be beneficial in understanding the mechanism of the actin network reorganization occurred during physiological and pathological processes.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Regulation of Actin Cytoskeleton Dynamics in Cells

Lee, Sung-Haeng,Dominguez, Roberto Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.29 No.4

The dynamic remolding of the actin cytoskeleton is a critical part of most cellular activities, and malfunction of cytoskeletal proteins results in various human diseases. The transition between two forms of actin, monomeric or G-actin and filamentous or F-actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-binding proteins (ABPs). New ABPs are constantly being discovered in the post-genomic era. Most of these proteins are modular, integrating actin binding, protein-protein interaction, membrane-binding, and signaling domains. In response to extracellular signals, often mediated by Rho family GTPases, ABPs control different steps of actin cytoskeleton assembly, including filament nucleation, elongation, severing, capping, and depolymerization. This review summarizes structure-function relationships among ABPs in the regulation of actin cytoskeleton assembly.

Regulation of Actin Cytoskeleton Dynamics in Cells

이성행,Roberto Dominguez 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.4

The dynamic remolding of the actin cytoskeleton is a critical part of most cellular activities, and malfunction of cytoskele-tal proteins results in various human diseases. The transi-tion between two forms of actin, monomeric or G-actin and filamentous or F-actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-binding proteins (ABPs). New ABPs are constantly being discovered in the post-genomic era. Most of these proteins are modular, integrating actin binding, protein-protein inter-action, membrane-binding, and signaling domains. In re-sponse to extracellular signals, often mediated by Rho fam-ily GTPases, ABPs control different steps of actin cytoskele-ton assembly, including filament nucleation, elongation, severing, capping, and depolymerization. This review sum-marizes structure-function relationships among ABPs in the regulation of actin cytoskeleton assembly.

Cytochalasin B Modulates Macrophage-Mediated Inflammatory Responses

( Mi Yeon Kim ),( Jong Hoon Kim ),( Jae Youl Cho ) 한국응용약물학회 2014 Biomolecules & Therapeutics(구 응용약물학회지) Vol.22 No.4

The actin cytoskeleton plays an important role in macrophage-mediated infl ammatory responses by modulating the activation of Src and subsequently inducing nuclear factor (NF)-κB translocation. In spite of its critical functions, few papers have examined how the actin cytoskeleton can be regulated by the activation of toll-like receptor (TLR). Therefore, in this study, we further characterized the biological value of the actin cytoskeleton in the functional activation of macrophages using an actin cytoskeleton disruptor, cytochalasin B (Cyto B), and explored the actin cytoskeleton`s involvement in morphological changes, cellular attachment, and signaling events. Cyto B strongly suppressed the TLR4-mediated mRNA expression of infl ammatory genes such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and inducible nitric oxide (iNOS), without altering cell viability. This compound also strongly suppressed the morphological changes induced by lipopolysaccharide (LPS), a TLR4 ligand. Cyto B also remarkably suppressed NO production under non-adherent conditions but not in an adherent environment. Cyto B did not block the co-localization between surface glycoprotein myeloid differentiation protein-2 (MD2), a LPS signaling glycoprotein, and the actin cytoskeleton under LPS conditions. Interestingly, Cyto B and PP2, a Src inhibitor, enhanced the phagocytic uptake of fl uorescein isothiocyanate (FITC)-dextran. Finally, it was found that Cyto B blocked the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at 1 min and the phosphorylation of heat shock protein 27 (HSP27) at 5 min. Therefore, our data suggest that the actin cytoskeleton may be one of the key components involved in the control of TLR4-mediated infl ammatory responses in macrophages.

p53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest

Hee-Don Chae,So Youn Kim,Sang Eun Park,김정빈,Deug Y Shin 생화학분자생물학회 2012 Experimental and molecular medicine Vol.44 No.3

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin-damaging agents including pectenotoxin-2 (PTX-2)increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced-tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA- PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.

ACTIN2 Functions in Chloroplast Photorelocation Movement in Arabidopsis thaliana

김주영,Jeongsu Ahn,Hanbit Bong,Masamitsu Wada,Sam‑Geun Kong 한국식물학회 2020 Journal of Plant Biology Vol.63 No.5

Chloroplast movement is regulated by dynamic reorganization of the actin cytoskeleton. To gain insight into the function of ACT2 in chloroplast movement, we examined the effect of the act2-3 mutation, in which a T-DNA is inserted at the second exon of the ACT2 gene, and investigated a transgenic Arabidopsis plant expressing the GFP-ACT2 fusion protein. Chloroplast movement in the act2-3 mutant was retarded, especially during the avoidance response, compared with that in the WT. We further verified the physiological response of transgenic Arabidopsis expressing the GFP-ACT2 fusion gene with a 6xGSS linker sequence, which was recently reported to enable the direct visualization of actin filaments in a transient expression system. Although the expression of GFP-ACT2 had highly detrimental effects on the growth and development of transgenic Arabidopsis plants, GFP-ACT2 was incorporated into chloroplast actin (cp-actin) filaments instead of cortical actin filaments. The analyses of the act2 mutant and GFP-ACT2 transgenic plants suggest that ACT2 functions in chloroplast movement as a component of cp-actin filaments and that cp-actin filaments are differently regulated compared to cortical actin filaments.

Disruption of the F-actin Cytoskeleton and Monolayer Barrier Integrity Induced by PAF and the Protective Effect of ITF on Intestinal Epithelium

Ling-fen Xu,Cheng Xu,Zhi-Qin Mao,Xu Teng,Li Ma,Mei Sun 대한약학회 2011 Archives of Pharmacal Research Vol.34 No.2

To explore whether platelet-activating factor (PAF) can disrupt the intestinal epithelial barrier directly and is associated with structural alterations of the F-actin-based cytoskeleton, and to observe the protective effect of intestinal trefoil factor (ITF), we establish an intestinal epithelia barrier model using Caco-2 cells in vitro. Transepithelial electrical resistance and unidirectional flux of lucifer yellow were measured to evaluate barrier permeability; immunofluorescent staining and flow cytometry were applied to observe morphological alterations and to quantify proteins of the F-actin cytoskeleton: the tight junction marker ZO-1 and Claudin-1 were observed using immunofluorescent staining. PAF significantly increased paracellular permeability, at the same time, F-actin and tight junction proteins were disrupted. It was thought that ITF could reverse the high permeability by restoring normal F-actin, ZO-1 and Claudin-1 structures. These results collectively demonstrated that PAF plays an important role in the regulation of mucosal permeability and the effects of PAF are correlated with structural alterations of the F-actin-based cytoskeleton and of tight junctions. ITF can protect intestinal epithelium against PAF-induced disruption by restricting the rearrangement of the F-actin cytoskeleton and of tight junctions.

Regulation of Actin Cytoskeleton by Rap1 Binding to RacGEF1

Hyemin Mun,전택중 한국분자세포생물학회 2012 Molecules and cells Vol.34 No.1

Rap1 is rapidly and transiently activated in response to chemoattractant stimulation and helps establish cell polarity by locally modulating cytoskeletons. Here, we investigated the mechanisms by which Rap1 controls actin cytoskeletal reorganization in Dictyostelium and found that Rap1 interacts with RacGEF1 in vitro and stimulates F-actin polymerization at the sites where Rap1 is activated upon chemoattractant stimulation. Live cell imaging using GFP-coronin, a reporter for F-actin, demonstrates that cells expressing constitutively active Rap1 (Rap1CA) exhibit a high level of F-actin uniformly distributed at the cortex including the posterior and lateral sides of the chemotaxing cell. Examination of the localization of a PH-domain containing PIP3 reporter, PhdA-GFP, and the activation of Akt/Pkb and other Ras proteins in Rap1CA cells reveals that activated Rap1 has no effect on the production of PIP3 or the activation of Akt/Pkb and Ras proteins in response to chemoattractant stimulation. Rac family proteins are crucial regulators in actin cytoskeletal reorganization. In vitro binding assay using truncated RacGEF1 proteins shows that Rap1 interacts with the DH domain of RacGEF1. Taken together, these results suggest that Rap1-mediated F-actin polymerization probably occurs through the Rac signaling pathway by directly binding to RacGEF1.