Morphological Study of Fibroblasts Treated with Cytochalasin D and Colchicine Using a Confocal Laser Scanning Microscopy

Abstract

Abstract:The role of actin filaments and microtubules in 3D cell morphology was investigated using confocal laser scanning microscopy and image analysis based on a regiongrowing method. Fibroblasts were treated with cytochalasin D or colchicine to disrupt the actin filaments or microtubules, respectively, and the structure and distribution of these cytoskeletal filaments were observed using a confocal laser scanning microscope. From the 3D reconstructed fluorescence images of the cytoskeleton, morphological parameters such as volume, adhesion area, height, and volume ratio of individual cells were determined. The volume ratio was defined as the ratio of the partial volume for every 10% of the height to the total cell volume. The cell volume decreased slightly after the disruption of actin filaments and microtubules, but the change was not significant. The cell adhesion area was significantly decreased after the disruption of actin filaments and microtubules, and was significantly smaller in actin filament-disrupted cells than in microtubule-disrupted cells. Cell height increased significantly after actin filament disruption, whereas it remained almost unchanged after microtubule disruption. Analysis of the volume ratio revealed that the cell shape changed from a cone to a hemisphere after disruption of actin filaments and slightly shifted toward a hemisphere-like shape after microtubule disruption. These results suggest that actin filaments are the major component responsible for the maintenance of global cell shape and that the contribution of microtubules to global cell morphology is much less than that of actin filaments.Key words: cell, cell volume, mechanics, imaging. Cells change their shape by alterations of the structure and distribution of the cytoskeleton in response to changes in the local environment. Cytoskeletons are responsible for the mechanical properties of cells and mechanically and functionally interact with one another [1,2]. In adherent cells, actin fi laments are connected to an extracellular matrix via integrins, transmembrane adhesion receptors, at focal adhesions. Forces acting on the extracellular matrix are transmitted to intracellular actin filaments via integrins, and a rearrangement of the cytoskeleton is induced to balance intracellular and external forces [3]. This dynamic rearrangement of cytoskeletons induces changes not only in the shape but also in the function of cells [4]. Cell shape is closely related to the functions and behaviors of cells, including growth, differentiation, motility, and apoptosis. The cytoskeleton mainly consists of actin fi laments, microtubules, and intermediate fi laments, and each type of cytoskeleton has distinct mechanical properties, dynamics, and biological roles. Therefore, it is very important to understand the contribution of each kind of cytoskeleton to the morphology of cells.The infl uence of the extracellular matrix [5][6][7][8] and the effects of the degree of polymerization of the cytoskeleton [6, 9-12] on cell shape have been examined ...

Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells

“…Consistently with previous reports[25,30], 20-min exposure to 20 μM cytochalasin B sharply decreased the projected area of A549 cells, slightly affected the microtubule network and caused accumulation of actin aggregates in the cortical and central regions of the cytoplasm, indicated by bright red spots. Unlike cytochalasin B, 10 μM vinblastine disrupted the microtubules and increased…”

“…The cells were then washed with PBS, and microtubules were stained with FITC-conjugated anti-mouse-IgG antibody in PBS containing 1% BSA. To visualize microfilaments and the nucleus, the cells were stained with Alexa Fluo488 phalloidin and 4′,6-diamidino-2-phenylindole, respectively, as described in detail elsewhere [25][26][27]. Actin filaments and microtubules were disrupted by 20-30-min treatment with 20 μM cytochalasin B and 10 μM vinblastine, respectively.…”

Effect of Dopamine on Viability of BHK-21 Cells

“…Hence, BHK-21 cells were sensitive to the cytotoxic effect of DA, their sensitivity directly correlated with the content of actin in the globular (not polymerized) aggregate state, which was higher in the cytoplasm of free suspended cells than in the cytoplasm of À attened cells with high content of ¿ lamentous (polymerized) actin [12]. According to the results of model studies, globular actin serves as the target and substrate for DA-induced direct formation of ¿ laments [1,4,5].…”

“…Suspended cells are always round and have virtually no actin, except a thin perimembrane cortical layer. The main pool of cytoplasmic actin is presented by globular (not polymeric) form [12], which we regard as the potential substrate for direct interactions with DA. Adherent rhomboid or polygonal cells have a well-developed cytoskeleton: a strong cortical layer, stress-¿ brils, and cell-cell and focal contacts.…”

The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts

“…In a previous study, rabbit fibroblasts sharply underwent shape alterations after treatment with cytochalasin D (20 µM, 3 h) by disruption of actin filaments. 31 Similarly, the shape of primary human lung fibroblasts cultured on collagen matrix was altered by cytochalasin D (2 µM). Cytochalasin D significantly inhibited the actin polymerization process in lung fibroblasts on collagen without promoting significant cell death (cell viability .88.7%).…”