Integration of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G) into Cellular Structures

The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca2+-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.

The Significant Role of the Microfilament System in Tumors

Actin is the structural protein of microfilaments, and it usually exists in two forms: monomer and polymer. Among them, monomer actin is a spherical molecule composed of a polypeptide chain, also known as spherical actin. The function of actin polymers is to produce actin filaments, so it is also called fibroactin. The actin cytoskeleton is considered to be an important subcellular filament system. It interacts with numerous relevant proteins and regulatory cells, regulating basic functions, from cell division and muscle contraction to cell movement and ensuring tissue integrity. The dynamic reorganization of the actin cytoskeleton has immense influence on the progression and metastasis of cancer as well. This paper explores the significance of the microfilament network, the dynamic changes of its structure and function in the presence of a tumor, the formation process around the actin system, and the relevant proteins that may be target molecules for anticancer drugs so as to provide support and reference for interlinked cancer treatment research in the future.

Effect of polyamide nanoparticles on apoptosis of colon cancer cells induced by survivin antisense oligonucleotide

In this study, we aimed to observe the effect of polyamidoamine (PAMAM) liposomes on the apoptosis of human colon cancer cells induced by survivin antisense oligonucleotides (ASODNs). PAMAM liposomes and PAMAM were mixed with survivin ASODNs to obtain antisense gene transfection complexes. In addition, the zeta potentials and encapsulation rates of the complexes were measured. The two gene-containing complexes were transfected into HT-29 colon cancer cells to observe changes in cell morphology, detect the inhibitory effect on tumor cells and changes in apoptosis, and observe changes in the cytoskeleton microfilament system using laser confocal microscopy. Caspase-3 activity in the cells was determined using a kinase activity assay, and p38 mitogen-activated protein kinase (p38 MAPK) activity in the cells was measured using immunoprecipitation analysis. The results showed that the zeta potential of the PAMAM liposome-survivin-ASODN complex was higher than that of the PAMAM-survivin-ASODN complex (P < 0.05). There was no significant difference in the gene encapsulation rates between the two complexes (P > 0.05). PAMAM liposomes may efficiently deliver survivin ASODNs to human colon cancer cells, reduce the expression of survivin protein and at the same time induce G2/M phase arrest in cells, and activate caspase-3 by activating p38 MAPK. Cleavage of caspase-3 destroys the structure of the intracellular skeletal microfilament system, finally resulting in apoptosis of colon cancer cells.

Matrix metalloproteinases: an emerging role in regulation of actin microfilament system

Matrix metalloproteinases (MMPs) are implicated in many physiological and pathological processes, including contraction, migration, differentiation, and proliferation. These processes all involve cell phenotype changes, known to be accompanied by reorganization of actin cytoskeleton. Growing evidence indicates a correlation between MMP activity and the dynamics of actin system, suggesting their mutual regulation. Here, data on the influence of MMPs on the actin microfilament system, on the one hand, and the dependence of MMP expression and activation on the organization of actin structures, on the other hand, are reviewed. The different mechanisms of putative actin-MMP regulation are discussed.

Tissue-specific Tropomyosin Isoform Composition

Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells ( Gunning et al. 1998b ). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.

Uptake Pathway of Polyomavirus via Ganglioside GD1a

ABSTRACT The pathway of entry of polyomavirus (Py) has been investigated with glycolipid-deficient C6 cells and added ganglioside GD1a as a specific virus receptor. Unsupplemented C6 cells show a low basal level of infection but become highly infectable by Py following preincubation with the sialic acid-containing ganglioside GD1a (38). Addition of GD1a has no effect on the overall level of virus binding but mediates the internalization and transit of virus to the endoplasmic reticulum (ER). This pathway of entry is cholesterol and caveola dependent and requires intact microtubules as well as a dynamic state of the microfilament system. In contrast to vesicular transport of other cargo via glycolipids, Py particles do not appear to pass through the Golgi apparatus. Colcemid and brefeldin A block transport of the virus to the ER in GD1a-supplemented cells and lead to accumulation of virus in a caveolin-1-containing environment. Several features distinguish the efficient GD1a-mediated pathway of virus uptake from the less-efficient pathway of basal infection in C6 cells.