A quantitative image analysis for the cellular cytoskeleton during in vitro tumor growth

The cellular cytoskeleton is a dynamic subcellular structure that can be a marker of key biological phenomena including cell division, organelle movement, shape changes and locomotion during the avascular tumor phase. Little attention is paid to quantify changes in the cytoskeleton while nuclei and cytoplasmic both are present in subcellular microscopic images. In this paper, we proposed a quantitative image analysis method to analyze subcellular cytoskeletal changes using a texture analysis method preceded by segmentation of nuclei, cytoplasm and ruffling regions (area except nuclei and cytoplasm). To test and validate this model we hypothesized that Mammary Serine Protease Inhibitor (maspin) acts as cytoskeleton regulator that mediates cell-extracellular matrix (ECM) adhesion in tumor. Maspin-a tumor suppressor gene shows multiple tumor suppressive properties such as increasing tumor cell apoptosis and reducing migration, proliferation, invasion, and overall tumor metastasis. The proposed method obtained separated ruffling regions from segmentation steps and then adopted gray–level histograms (GLH) and grey-level co-occurrence matrix (GLCM) texture analysis techniques. In order to verify the reliability, the proposed texture analysis method was used to compare the control and maspin expressing cells grown on different ECM components: plastic, collagen I, fibronectin and laminin. The results show that the texture parameters extracted reflect the different cytoskeletal changes. These changes indicate that maspin acts as a regulator of the cell-ECM enhancement process, while it reduces the cell migration. Overall, this paper not only presents a quantitative image analysis approach to analyze subcellular cytoskeletal architectures but also provides a comprehensive tool for the biologist, pathologist, cancer specialist, and computer scientist to understand cellular and subcellular organization of cells. In long term, this method can be extended to be used in live cell tracking in vivo, image informatics based point-of-care expert system and quantification of various complex architectures in organisms

Effects of Noninhibitory Serpin Maspin on the Actin Cytoskeleton: A Quantitative Image Modeling Approach

Recent developments in quantitative image analysis allow us to interrogate confocal microscopy images to answer biological questions. Clumped and layered cell nuclei and cytoplasm in confocal images challenges the ability to identify subcellular compartments. To date, there is no perfect image analysis method to identify cytoskeletal changes in confocal images. Here, we present a multidisciplinary study where an image analysis model was developed to allow quantitative measurements of changes in the cytoskeleton of cells with different maspin exposure. Maspin, a noninhibitory serpin influences cell migration, adhesion, invasion, proliferation, and apoptosis in ways that are consistent with its identification as a tumor metastasis suppressor. Using different cell types, we tested the hypothesis that reduction in cell migration by maspin would be reflected in the architecture of the actin cytoskeleton. A hybrid marker-controlled watershed segmentation technique was used to segment the nuclei, cytoplasm, and ruffling regions before measuring cytoskeletal changes. This was informed by immunohistochemical staining of cells transfected stably or transiently with maspin proteins, or with added bioactive peptides or protein. Image analysis results showed that the effects of maspin were mirrored by effects on cell architecture, in a way that could be described quantitatively

Binding of Extracellular Maspin to 1 Integrins Inhibits Vascular Smooth Muscle Cell Migration

Maspin is a serpin that has multiple effects on cell behavior, including inhibition of migration. How maspin mediates these diverse effects remains unclear, as it is devoid of protease inhibitory activity. We have previously shown that maspin rapidly inhibits the migration of vascular smooth muscle cells (VSMC), suggesting the involvement of direct interactions with cell surface proteins. Here, using immunofluorescence microscopy, we demonstrate that maspin binds specifically to the surface of VSMC in the dedifferentiated, but not the differentiated, phenotype. Ligand blotting of VSMC lysates revealed the presence of several maspin-binding proteins, with a protein of 150 kDa differentially expressed between the two VSMC phenotypes. Western blotting suggested that this protein was the ß1 integrin subunit, and subsequently both a3ß1 and a5ß1, but not avß3, were shown to associate with maspin by coimmunoprecipitation. Specific binding of these integrins was also observed using maspin-affinity chromatography, using HT1080 cell lysates. Direct binding of maspin to a5ß1 was confirmed using a recombinant a5ß1-Fc fusion protein. Using conformation-dependent anti-ß1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of a5ß1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human a5 integrin, but not those lacking a5 expression. Our observations suggest that maspin engages in specific interactions with a limited number of integrins on VSMC, leading to their inactivation, and that these interactions are responsible for the effects of maspin in the pericellular environment

On the relevance of weaker instruments

We study the asymptotic properties of the standard GMM estimator when additional moment restrictions, weaker than the original ones, are available. We provide conditions under which these additional weaker restrictions improve the efficiency of the GMM estimator. To detect “spurious” identification that may come from invalid moments, we rely on the Hansen J-test that assesses the compatibility between existing restrictions and additional ones. Our simulations reveal that the J-test has good power properties and that its power increases with the weakness of the additional restrictions. Our theoretical characterization of the J-test provides some intuition for why that is

Fractal and image analysis of cytoskeletal changes in tumour cells due to the effects of maspin

Maspin (SERPINB5) is a type II metastasis suppressor that influences multiple cellular functions. To date, maspin has been shown to increase adhesion and apoptosis and to decrease cell migration, proliferation, invasion and metastases in tumour malignancy. At the subcellular level, maspin influences morphological changes in the cell cytoskeleton which regulates complex biological processes including cell migration, cell adhesion and EMT (epithelial to mesenchymal transition). Here non-Euclidian fractal and image analyses have been applied to measure changes in the actin cytoskeleton using confocal microscopy images to confirm the effects of maspin. Results show that maspin contributes to maintaining the regular epithelial like shape, increases cell-cell adhesion and restricts tumour cells from showing the pre-migration and EMT characteristics. Characterization of these changes in the actin cytoskeleton using microscopic image analysis will establish maspin as a potential prognostic marker in future