Cytoplasmic Actin Research Articles

Identification of Major Proteins of a Very Stable High Molecular Mass Multi-Protein Complex of Human Placental Tissue Possessing Nine Different Catalytic Activities

Human placenta is an organ protecting, feeding, and regulating the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. Here we analyzed for the first time human placentas extremely stable multi-protein complex (SPC, ~1000 kDa) by MALDI MS and MS/MS spectrometry using proteins tryptic hydrolyzates after proteins separation by SDS-PAGE and 2-D electrophoresis. The formation of such a very stable complex due to the random association of several various proteins is very unlikely. It was shown that SPCs contain twelve proteins: hemoglobin, alkaline phosphatase, cytoplasmic actin, human serum albumin, chorionic somatomammotropin hormone, heat shock protein beta-1, peroxiredoxin-1, 78 kDa glucose-regulated protein, protein disulfide isomerase A3, serotransferrin, annexin A5, and IgGs. These twelve proteins have in themselves many different and important biological functions, which can be inherent for these proteins in the complex. In addition, the complex demonstrated nine different enzymatic activities: DNase, RNase, ATPase, phosphatase, protease, amylase, catalase, peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent). The efficiency of the catalysis of each of these reactions by SPC preparations from three placentas was comparable. It was shown that hydrolysis of r(pU)23, r(pA)23, and r(pC)23 leads to the formation of 1-22-mer oligonucleotides, while digestion of microRNA mirR137 is a site-specific (3A-4U > 9U-10A > 8U-9U ≥ 15U-16A) resulting in the formation of only four major products. A large number of potentially possible functions of the complex in accordance with the functions of its individual proteins are considered. Progress in the study of placental proteins complexes can promote understanding of their biological functions.

Drosophila and human FHOD family formin proteins nucleate actin filaments

Formins are a conserved group of proteins that nucleate and processively elongate actin filaments. Among them, the formin homology domain-containing protein (FHOD) family of formins contributes to contractility of striated muscle and cell motility in several contexts. However, the mechanisms by which they carry out these functions remain poorly understood. Mammalian FHOD proteins were reported not to accelerate actin assembly in vitro; instead, they were proposed to act as barbed end cappers or filament bundlers. Here, we show that purified Drosophila Fhod and human FHOD1 both accelerate actin assembly by nucleation. The nucleation activity of FHOD1 is restricted to cytoplasmic actin, whereas Drosophila Fhod potently nucleates both cytoplasmic and sarcomeric actin isoforms. Drosophila Fhod binds tightly to barbed ends, where it slows elongation in the absence of profilin and allows, but does not accelerate, elongation in the presence of profilin. Fhod antagonizes capping protein but dissociates from barbed ends relatively quickly. Finally, we determined that Fhod binds the sides of and bundles actin filaments. This work establishes that Fhod shares the capacity of other formins to nucleate and bundle actin filaments but is notably less effective at processively elongating barbed ends than most well studied formins.

Expression of α-smooth muscle actin in benign and malignant salivary gland tumors: An immunohistochemical study.

Myoepithelial cells (ME) are known to contribute in the patterning of salivary gland neoplasms (SGN) and possess cytoplasmic smooth muscle actin (SMA) revealed by alpha SMA (α-SMA). The present study aimed to assess the expression of α-SMA in selected benign and malignant SGN (pleomorphic adenoma [PA], mucoepidermoid carcinoma (MEC), adenoid cystic carcinoma (ACC), and polymorphous low-grade adenocarcinoma (PLGA). The intensity and pattern of expression of α-SMA were studied in 25 cases of SGN's ACC (n = 7), MEC (n = 8), PA (n = 8), and PLGA (n = 2), and correlated with the histological patterns. Maximum expression of α-SMA in the epithelial compartment was seen in ACC, followed by PA, whereas MEC and PLGA showed completely negative staining. The connective tissue expression was mild in ACC and MEC. The myxoid stroma of PA with "melting" pattern was weakly positive for α-SMA. The stroma in PLGA showed complete negativity. In ACC, α-SMA-positive cells were lining the cribriform spaces, small islands, and dispersed within large islands. Small nests showed complete positivity for α-SMA. In ACC, α-SMA expression supports the involvement of ME in epithelial organization explaining the histological patterns seen. In PA, the expression correlates with the predominantly secretory nature of ME. The absence of epithelial positivity in MEC and PLGA suggest that ME has less role to play in their histogenesis. The weak stromal positivity observed in MEC and ACC may be attributed to the positive immunoreactivity of myofibroblasts playing a role in modulating the course of SGN's.

Osteogenic Stimulation of Human Adipose-Derived Mesenchymal Stem Cells Using a Fungal Metabolite That Suppresses the Polycomb Group Protein EZH2.

Strategies for musculoskeletal tissue regeneration apply adult mesenchymal stem/stromal cells (MSCs) that can be sourced from bone marrow‐ and lipo‐aspirates. Adipose tissue‐derived MSCs are more easily harvested in the large quantities required for skeletal tissue‐engineering approaches, but are generally considered to be less osteogenic than bone marrow MSCs. Therefore, we tested a new molecular strategy to improve their osteogenic lineage‐differentiation potential using the fungal metabolite cytochalasin D (CytoD). We show that CytoD, which may function by redistributing the intracellular location of β‐actin (ACTB), is a potent osteogenic stimulant as reflected by significant increases in alkaline phosphatase activity, extracellular matrix mineralization, and osteoblast‐related gene expression (e.g., RUNX2, ALPL, SPARC, and TGFB3). RNA sequencing analyses of MSCs revealed that acute CytoD treatment (24 hours) stimulates a broad program of osteogenic biomarkers and epigenetic regulators. CytoD decreases mRNA and protein levels of the Polycomb chromatin regulator Enhancer of Zeste Homolog 2 (EZH2), which controls heterochromatin formation by mediating trimethylation of histone 3 lysine 27 (H3K27me3). Reduced EZH2 expression decreases cellular H3K27me3 marks indicating a global reduction in heterochromatin. We conclude that CytoD is an effective osteogenic stimulant that mechanistically functions by blocking both cytoplasmic actin polymerization and gene‐suppressive epigenetic mechanisms required for the acquisition of the osteogenic phenotype in adipose tissue‐derived MSCs. This finding supports the use of CytoD in advancing the osteogenic potential of MSCs in skeletal regenerative strategies. Stem Cells Translational Medicine 2018;7:197–209

Profilin 1 plays feedback role in actin-mediated polar body extrusion in mouse oocytes.

Mammalian oocytes undergo several crucial processes during meiosis maturation, including spindle formation and migration and polar body extrusion, which rely on the regulation of actin. As a small actin-binding protein, profilin 1 plays a central role in the regulation of actin assembly. However, the functions of profilin 1 in mammalian oocytes are uncertain. To investigate the function of profilin 1 in oocytes, immunofluorescent staining was first used to examine profilin 1 localisation. The results showed that profilin 1 was localised around the meiotic spindles and was colocalised with cytoplasmic actin. Knockdown (KD) of profilin 1 with specific morpholino microinjection resulted in failure of polar body extrusion. This failure resulted from an increase of actin polymerisation both at membranes and in the cytoplasm. Furthermore, western blot analysis revealed that the expression of Rho-associated kinase (ROCK) and phosphorylation levels of myosin light chain (MLC) were significantly altered after KD of profilin 1. Thus, the results indicate that a feedback mechanism between profilin, actin and ROCK-MLC2 regulates actin assembly during mouse oocyte maturation.

Phosphorylation and degradation of αB-crystallin during enterovirus infection facilitates viral replication and induces viral pathogenesis.

Protein quality control (PQC) plays a key role in maintaining cardiomyocyte function and homeostasis, and malfunction in PQC is implicated in various forms of heart diseases. Molecular chaperones serve as the primary checkpoint for PQC; however, their roles in the pathogenesis of viral myocarditis, an inflammation of the myocardium caused by viral infection, are largely unknown. AlphaB-crystallin (CryAB) is the most abundant chaperone protein in the heart. It interacts with desmin and cytoplasmic actin to prevent protein misfolding and aggregation and to help maintain cytoskeletal integrity and cardiac function. Here we showed that coxsackievirus infection induced desminopathy-like phenotype of the myocardium, as characterized by the accumulation of protein aggregates and the disruption of desmin organization. We further demonstrated that CryAB was phosphorylated during early and downregulated at later stages of infection. Moreover, we showed that phosphorylated CryAB had a shorter half-life and was targeted to the ubiquitin-proteasome system for degradation. Lastly, we found that overexpression of CryAB significantly attenuated viral protein production and progeny release, indicating an anti-viral function for CryAB. Together, our results suggest a mechanism by which coxsackieviral infection induces CryAB degradation and loss-of-function, resulting in desmin aggregation, ultimately contributing to compromised cytoskeletal integrity and viral cardiomyopathy.

FHOD1 regulates cytoplasmic actin-based spindle migration for mouse oocyte asymmetric cell division.

FHOD1 is a member of Diaphanous-related formins (DRFs) which belongs to the Formin family. Previous studies have shown that the DFRs might affect several cellular functions such as morphogenesis, cytokinesis, cell polarity, and embryonic differentiation. However, there is no evidence showing the functions of FHOD1 during oocyte meiosis. This study is aimed at exploring the roles of FHOD1 during the mammalian oocyte maturation. Immunofluorescent staining showed that FHOD1 was restricted to the nucleus in germinal vesicle (GV) stage of the oocytes, after the GV breakdown FHOD1 was primarily located at two poles of the spindle at both metaphases I and II stages. Knockdown of FHOD1 by siRNA injection did not affect polar body extrusion but generated the large polar bodies. In addition, we observed the spindle migration failure in metaphase I oocytes, with a large number of meiotic spindles anchoring in the center of cytoplasm. The expression level of cytoplasmic actin but not cortex actin was significantly reduced, indicating that FHOD1 regulates cytoplasmic actin distribution for the spindle movement. Furthermore, we found that the disruption of ROCK (the Rho-dependent protein kinase) with inhibitor Y-27632 caused the decreased FHOD1 protein expression. Therefore, our data indicate that FHOD1 is regulated by ROCK for cytoplasm actin assembly and spindle migration during mouse oocyte meiosis.

Filamin A is required for spindle migration and asymmetric division in mouse oocytes.

Dynamic changes in the actin network are crucial for the cortical migration of spindles and establishment of polarity, to ensure asymmetric division during meiotic maturation. In this study, filamin A (FLNA) was found to be an essential actin regulator that controlled spindle migration and asymmetric division during oocyte meiosis. FLNA was localized in the cytoplasm and enriched at the cortex and near the chromosomes. Knockdown of FLNA impaired meiotic asymmetric division and spindle migration with a decrease in the amount of cytoplasmic actin mesh and cortical actin levels. Moreover, FLNA knockdown reduced the phosphorylation of cofilin and Rho kinase (ROCK) near the spindle. Similar phenotypes, such as decreased filament actin levels, impaired spindle migration and polar body extrusion, were observed when active cofilin (S3A) was overexpressed or ROCK was inhibited. Notably, we found that FLNA and ROCK interacted directly in mouse oocytes. Taken together, our results show that FLNA plays crucial roles in asymmetric division during meiotic maturation by regulating ROCK-cofilin-mediated actin reorganization.-Wang, H., Guo J., Lin, Z., Namgoong, S., Oh, J. S., Kim, N.-H. Filamin A is required for spindle migration and asymmetric division in mouse oocytes.

Diagnosis of respiratory diseases using the proteomic analysis of exhaled breath condensate

Investigation of exhaled breath condensate (EBC) is a noninvasive diagnostic method in respiratory diseases. The objective of this study was to compare EBC protein spectrum in healthy volunteers and in patients with chronic obstructive pulmonary disease (COPD), pneumonia and lung cancer (NSCLC), as well as to assess a role of proteomic analysis of EBC for diagnosis and differential diagnosis of these diseases. Methods. We examined 18 patients with COPD, 13 patients with community-acquired pneumonia, 26 patients with lung cancer and 24 healthy non-smoking volunteers. EBC was collected using ECoScreen system (VIASYS Healthcare, Germany) and a standardized method. EBC-samples were lyophilized, hydrolyzed and analyzed by HPLC and tandem mass spectrometry. To identify proteins, we used Mascot (Matrix Science, UK) and IPI-human (version 3.82) databases provided by the European Bioinformatics Institute. Results. Proteomic analysis of EBC identified more than 300 different proteins; most of them were types I and II cytoskeletal keratins. Cytokeratin 5, 6, and 14 concentrations in EBC of NSCLC patients were significantly higher than that in healthy volunteers. Dermcidin, immunoglobulin alpha, kininogen, cytoplasmic actin, serum albumin, and Zn-alpha2-glycoprotein were identified in EBC of healthy volunteers and patients with COPD and pneumonia. High concentration of peroxiredoxin in EBC of COPD patients could be due to severe oxidative stress. High levels of acute-phase and hypoxia proteins (annexins A1 and A2, HSP90B, cystatins M and B, collagen and histones fragments) were detected in EBC of pneumonia patients. Also, β- и α-subunit of hemoglobin, nuclear ubiquitin casein (NUCKS), POTEE, high mobility group protein (HMG-I/HMG-Y) and lactoferrin were identified in EBC of NSCLC patients. Conclusion. We found that EBC in healthy nonsmokers and in patients with COPD, pneumonia and NSCLC had characteristic protein spectrum. Most of the identified proteins could be used for diagnosis of these diseases.

ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends

SummaryActin-depolymerizing factor (ADF)/cofilins contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. Here, by monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, we directly quantify the reactions underpinning filament severing and depolymerization from both ends. We find that, in the absence of monomeric actin, soluble ADF/cofilin can associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. We also show that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. Our experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, our results propose that significant barbed-end depolymerization may take place in cells.

Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts.

The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin.

Actin isoforms and neoplastic transformation

The cytoplasmic actins (β and γ) play crucial roles during key cellular processes like adhesion, migration, polarization and cytokinesis. The understanding of their specific underlying mechanisms would be of major relevance not only for fundamental research but also for clinical applications, since modulations of actin isoforms are directly or indirectly correlated with severe pathologies. The major goal of the research was to elucidate the function of the actin isoforms during motile activities, adhesions and cell division and to investigate whether their expression and/or structural organization is related to pathological function. Selective depletion of β- and γ-cytoplasmic actins allowed attributing functional diversities of β- and γ-сytoplasmic actins. β-Сytoplasmic actin plays a preferential role in contractile activities, whereas γ-cytoplasmic actin mainly participates in the formation of a submembranous network necessary for cell shape flexibility and motile activity. The roles of isoforms in regulating the integrity of adherens and tight junctions respectively were demonstrated. Unique roles of β- and γ-cytoplasmic actins in normal cells were shown. Similar results were obtained in cancer cells compared with normal epithelial cells in culture and in human pathological tissue sections of mammary gland, colon, lung and cervix. Malignant cell transformation requires changes in the ability of cells to migrate. The disruption of actin cytoskeleton and intercellular adhesions is an important component of the acquisition of invasive properties in epithelial malignancies.

A change in the expression of membrane-associated proteins and cytoplasmic actin isoforms in the progression of human colon tumors

Тo identify correlations between clinical features, risk of progression and/or recurrence of human colon adenocarcinomas (CAC), and EMT-related tumor markers. Descending colon and sigmoid colon adenocarcinoma samples were examined immunohistochemically. Formalin-fixed paraffin-embedded tissue sections were incubated with antigen-specific antibodies, then secondary antibodies labeled with fluorochromes, and the fluorescence intensity of microscopy images was analyzed. The cells of a tumor compared to those of intact colon tissue showed a weak staining of E-cadherin in the cell-cell contact areas. The reduced membrane staining and nuclear localization of β-catenin were detected in moderately (G2) and poorly (G3) differentiated tumors. There were substantially decreased β-actin levels in almost all tumor samples and increased γ-actin ones, mainly in the samples belonging to stage IV disease. A correlation was found between stage, tumor differentiation grade, risk for relapse or progression of disease, and the impaired expression of different EMT markers: total or partial loss of E-cadherin expression, β-catenin reorganization in cell-cell contacts, and a change in the ratio of cytoplasmic actin isoforms in the late stages of CAC development. We believe that these molecular markers may have a prognostic potential.

New cell motility model observed in parasitic cnidarian Sphaerospora molnari (Myxozoa:Myxosporea) blood stages in fish.

Cellular motility is essential for microscopic parasites, it is used to reach the host, migrate through tissues, or evade host immune reactions. Many cells employ an evolutionary conserved motor protein– actin, to crawl or glide along a substrate. We describe the peculiar movement of Sphaerospora molnari, a myxozoan parasite with proliferating blood stages in its host, common carp. Myxozoa are highly adapted parasitic cnidarians alternately infecting vertebrates and invertebrates. S. molnari blood stages (SMBS) have developed a unique “dancing” behaviour, using the external membrane as a motility effector to rotate and move the cell. SMBS movement is exceptionally fast compared to other myxozoans, non-directional and constant. The movement is based on two cytoplasmic actins that are highly divergent from those of other metazoans. We produced a specific polyclonal actin antibody for the staining and immunolabelling of S. molnari’s microfilaments since we found that neither commercial antibodies nor phalloidin recognised the protein or microfilaments. We show the in situ localization of this actin in the parasite and discuss the importance of this motility for evasion from the cellular host immune response in vitro. This new type of motility holds key insights into the evolution of cellular motility and associated proteins.

Framework to function: mechanosensitive regulators of gene transcription.

Mechanobiology has shifted our understanding of fundamental cellular and physiological functions. Changes to the stiffness of the extracellular matrix, cell rigidity, or shape of the cell environment were considered in the past to be a consequence of aging or pathological processes. We now understand that these factors can actually be causative biological mediators of cell growth to control organ size. Mechanical cues are known to trigger a relatively fast translocation of specific transcriptional co-factors such as MRTFs, YAP and TAZ from the cytoplasm to the cell nucleus to initiate discrete transcriptional programs. The focus of this review is the molecular mechanisms by which biophysical stimuli that induce changes in cytoplasmic actin dynamics are communicated within cells to elicit gene-specific transcription via nuclear localisation or activation of specialized transcription factors, namely MRTFs and the Hippo pathway effectors YAP and TAZ. We propose here that MRTFs, YAP and TAZ closely collaborate as mechano-effectors.

Proteomics Identification of Potential Candidates Involved in Cell Proliferation for Early Stage of Brain Regeneration in the Adult Zebrafish.

The central nervous system (CNS) of the non-mammalian vertebrates has better neuroregenerative capability as compared with the mammalian CNS. Regeneration of habenula was observed 40 days after damage in zebrafish. During the early stage of regeneration, we found a significant increase of apoptotic cells on day-1 post-damage and of proliferative cells on day-3 post-damage. To identify the molecular factor(s) involved in the early stages of neuroregeneration, differentially expressed proteins during sham, 20- and 40-h post-habenula damage were investigated by proteomic approach by using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-ToF) and tandem mass spectrometry. Protein profiles revealed 17 differentially (>1.5-fold) expressed proteins: 10 upregulated, 4 downregulated, 2 proteins were found to be downregulated at the early stage but upregulated at a later stage, and 1 protein was found to be upregulated at 2 different time points. All proteins identified can be summarized under few molecular processes involved in the early stages of neuroregeneration in zebrafish CNS: apoptosis regulation (Wnt inhibitory factor 1 [WIF1]), neuroprotection (metallothionein), cell proliferation (Spred2, ependymin, Lhx1, and Wnts), differentiation (Spred2, Lhx9, and Wnts), and morphogenesis (cytoplasmic actins and draculin). These protein profiling results suggest that drastic molecular changes occur in the neuroregenerative process during this period, which includes cell proliferation, differentiation, and protection.

Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network.

A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization.

Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells.

Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.

Nucleus-associated actin in Amoeba proteus

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

Cycloheximide Inhibits Actin Cytoskeletal Dynamics by Suppressing Signaling via RhoA.

Genome-wide screening of the yeast Saccharomyces cerevisiae knockout collection was used to characterize chemical-genetic interactions of cycloheximide (CHX). The results showed that while the act1Δ mutant was the only deletion mutant in the heterozygous essential gene deletion collection that showed hypersensitivity to sub-inhibitory concentrations of CHX, deletion of nonessential genes that work in concert with either cytoplasmic or nuclear actin in the homozygous deletion collection also highly sensitized yeast to CHX. Fluorescence microscopy analysis revealed that CHX disrupts filamentous actin structures and fluid phase endocytosis in the yeast cell. It also showed that CHX disrupts transforming growth factor-β1 (TGF-β1)-induced actin reorganization and polygonal architecture of microfilaments in mammalian cells. This inhibitory effect is mediated, at least in part, through the actin dynamics signaling pathway via suppression of activation of the small GTPase RhoA. J. Cell. Biochem. 117: 2886-2898, 2016. © 2016 Wiley Periodicals, Inc.