20 research outputs found
Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells
Actins are eukaryotic proteins, which are involved in diverse cellular functions including muscle contraction, cell motility, adhesion and maintenance of cell shape. Cytoplasmic actin isoforms β and γ are ubiquitously expressed and essential for cell functioning. However, their unique contributions are not very well understood. The aim of this study was to determine the effect of β- and γ-actin overexpression on the migration capacity and actin cytoskeleton organization of human colon adenocarcinoma BE cells. In cells overexpressing β- or γ-actin, distinct cytoskeletal actin rearrangements were observed under the laser scanning confocal microscope. Overexpressed actins localized at the submembranous region of the cell body, especially near to the leading edge and on the tips of pseudopodia. The cells transfected with plasmids containing cDNA for β- or γ-actin were characterized by increased migration and invasion capacities. However, the migration velocity was statistically significantly higher only in the case of γ-actin overexpressing cells. In conclusion, the increased level of β- or γ-actin leads to actin cytoskeletal remodeling followed by an increase in migration and invasion capacities of human colon BE cells. These data suggest that expression of both actin isoforms has an impact on cancer cell motility, with the subtle predominance of γ-actin, and may influence invasiveness of human colon cancer
Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells
Integrin-Linked Kinase (ILK) Plays an Important Role in the Laminin-Dependent Development of Dorsal Root Ganglia during Chicken Embryogenesis
Integrin-linked kinase (ILK) is mainly localized in focal adhesions where it interacts and modulates the downstream signaling of integrins affecting cell migration, adhesion, and survival. The interaction of dorsal root ganglia (DRG) cells, being part of the peripheral nervous system (PNS), with the extracellular matrix (ECM) via integrins is crucial for proper PNS development. A few studies have focused on ILK’s role in PNS development, but none of these have focused on chicken. Therefore, we decided to investigate ILK’s role in the development of Gallus gallus domesticus’s DRG. First, using RT-PCR, Western blotting, and in situ hybridization, we show that ILK is expressed in DRG. Next, by immunocytochemistry, we show ILK’s localization both intracellularly and on the cell membrane of DRG neurons and Schwann cell precursors (SCPs). Finally, we describe ILK’s involvement in multiple aspects of DRG development by performing functional experiments in vitro. IgG-mediated interruption of ILK’s action improved DRG neurite outgrowth, modulated their directionality, stimulated SCPs migration, and impacted growth cone morphology in the presence of laminin-1 or laminin-1 mimicking peptide IKVAV. Taken together, our results show that ILK is important for chicken PNS development, probably via its exposure to the ECM
Expression of constructs of WT--cardiac actin and its mutants in different cell lines and primary rat cardiac myocytes
Das Ziel dieser Arbeit war Untersuchung von Mutationen des kardialen -Aktins, die in manchen Fällen der hypertrophen Kardiomyopathie (HCM) gefunden wurden. Mit Konstrukten, die N-terminal einen GFP- (grün fluoreszierte Protein) oder einen Hemagglutinin tag erzeugten, wurden Wildtyp (WT) und die zwei Mutanten - Y166C und M305L - des kardialen -Aktins zunächst fibroblastische NIH3T3, Kardiomyozyten ähnliche HL-1 und humane A431 (epidermoide Tumor-) Zellen transfiziert. Die Ergebnisse dieser Untersuchungen waren widersprüchlich. Die Y166C-Mutante mit GFP konnte intrazellulär nicht polymerisieren, jedoch mit dem HA-tag war diese Mutante fähig zu polymerisieren. Um primäre Cardiomyocyten zu transfizieren wurden zwei rekombinante adenovirale Konstrukte (WT und Y166C) hergestellt. In Herzmuskelzellen konnten WT und die Y166C-Mutante sehr gut polymerisieren und sich in sarkomerische Strukturen einbauen. Jedoch erzeugte die Y166C Mutante kürzere Sarkomere im Vergleich zu WT-kardialem -Aktin
Karakterisasi Pati Talas (Colocasia Esculenta (L.) Schott) Sebagai Alternatif Sumber Pati Industri Di Indonesia
Utilization of taro as the taro flour or taro starch will increase the economic value and the shelf life of products taro. Utilization of taro tubers during this simply as a snack, such as chips. Whereas in other countries namely the United States, Hawaii, Japan, Philippines, Brazil and Columbia have made a variety of taro tuber industrial commodities such as biscuits, bread, pasta and taro. Therefore, this study focused on the manufacture of starch and modified starch taro taro as one alternative source of starch industry in Indonesia. The purpose of this study is to get the characteristics of taro starch and modified starch and starch compared with markets such as corn starch, rice and cassava. In this study analysis, which includes characterization of starch. Characteristics of taro starch were tested consisted of moisture content, starch content, amylose and amylopectin content. The water content of taro starch produced ranged from 5.3 to 13.18 so still meet the quality standards of industrial starch is less than 14%. Starch content of tubers produced by 80% taro, taro flour by 75% while the starch content derived from the modification of taro only 65%. Low levels of starch modification due to the degradation that occurs in the process of modification. The resulting levels of amylose has an average value of 4.41%. Low levels of amylose due to the low starch content. Thus indirectly affect the levels of amylose. While the levels of amylopectin starch modification of taro only 60.88%. This is because the process of modification using acid hydrolysis led to a reduction of amylopectin. Starch is a starch modification process that has undergone physical or chemical treatment in a controlled manner so that changing one or more of its original properties, such as the initial temperature of gelatinization, during gelatinization characteristics, acidification and stirring. While hydrochloric acid hydrolysis of starch which aims to produce a more tenuous structure so that water is more volatile at the time of drying
Are non-muscle actin isoforms functionally equivalent?
Actin is highly conserved and it is the most
widespread protein in eukaryotic cells. One of the most
important features of actin, which allows it to have many
different functions, is its ability to polymerize and
interact with many other proteins. Actins are the major
constituent of the actin cytoskeleton, which is an
important system that is involved in various aspects of
cell function, including cell motility, structure, integrity,
regulation of signal transduction and transcription. Six
mammal actin isoforms are highly conserved and share
common functions. Two of them, β and γ non-muscle
actin isoforms, which differ only by four amino acids
located at the N-terminus of the polypeptide chain, are
required for survival and proper cell functioning. We
also summarized data about actbl2, which is suggested
to be a newly discovered isoactin. Here, we review the
current knowledge about tissue-specific expression of
the non-muscle actin isoforms and possible functional
differences between them. We also discuss molecular
tools, which in recent years have allowed for a better
understanding of the role of these proteins in cell
functioning
Co-localization of exogenous cytoplasmic actin isoform with bleb markers.
<p>Confocal images showing LS174T cells expressing actin isoform β- or γ- encoded by pAcGFP-C1 expression vector were compared to cells transfected with the empty vector pAcGFP-C1. (a) Left panel: AcGFP fluorescence, middle panel: ezrin stained with mouse monoclonal antibodies. Merged images are shown on the right panel. (b) Left panel: AcGFP fluorescence, middle panel: myosin II stained with rabbit polyclonal antibodies. Merged images are shown on the right panel. Enlargements of the boxed, bleb-rich area are shown as insets. Scale bar: 10 μm.</p
Bleb-like protrusions in LS174T colon cancer cells with rounded morphology.
<p>Cells were plated onto coverslips. After fixation with 4% formaldehyde, cells were labeled to visualize filamentous actin (red) and ezrin (green). Merged image is shown on the right picture. Enlargements of the boxed, bleb-rich area are shown as insets. Scale bar: 10 μm</p
Actin organization in LS174T colon cancer cells overexpressing β- and γ-actin isoforms.
<p>(a) Filamentous actin organization in examined cells with increased level of β- or γ-actin. Confocal images showing cells expressing actin isoform β- or γ- encoded by pAcGFP-C1 expression vector were compared to cells transfected with an empty vector pAcGFP-C1. Left panel: AcGFP fluorescence (green). Middle panel: filamentous actin visualized by staining with AlexaFluor 568-conjugated phalloidin (red). Merged images are shown on the right panel. Scale bar: 10 μm. Enlargements of the boxed, bleb-rich area are shown as insets. (b) Subcellular localization of monomeric actin in cells overexpressing β- or γ-actin. β- and γ-actin were encoded by pAcGFP-C1 expression vector. Left panel: AcGFP (green). Middle panel: monomeric actin visualized by staining with DNase I conjugated with Alexa Fluor 594 (red). Merged images are shown on the right panel. Scale bar: 10 μm. (c) Actin polymerization state in colon cancer cells overexpressing β- or γ-actin. Asterisks indicate values statistically different from those obtained for the control cells, transfected with pAcGFP-C1 plasmid. The significance level was set at p ≤ 0.01 in one-way ANOVA followed by Tukey post hoc test. The data were obtained from three independent experiments.</p
Visualization of interactions between β- or γ-actin and myosin II using in situ PLA.
<p>The proximity ligation assay between endogenous β- or γ-actin and myosin II was performed as described in Materials and Methods. Each red spot in the left panel represents a single interaction between endogenous β- or γ-actin and myosin II. The middle panel presents images of LS174T cells in the phase contrast. Merged images (interaction of proteins, phase contrast image, nucleus stained with DAPI) are shown on the right panel. Scale bar: 10 μm.</p