Two nondimensional parameters for characterizing the nuclear morphology.

Nuclear morphology is an important indicator of cell function. It is regulated by a variety of factors such as the osmotic pressure difference between the nucleoplasm and cytoplasm, cytoskeletal forces, elasticity of the nuclear envelope and chromosomes. Nucleus shape and size are typically quantified using multiple geometrical quantities that are not necessarily independent of one another. This interdependence makes it difficult to decipher the implications of changes in nuclear morphology. We resolved this by analyzing nucleus shapes of populations for multiple cell lines using a mechanics-based model. We deduced two independent nondimensional parameters, namely, flatness index and isometric scale factor. We show that nuclei in a cell population have similar flatness but variable scale factor. Furthermore, nuclei of different cell lines segregate according to flatness. Cellular perturbations using biochemical and biomechanical techniques suggest that the flatness index correlates with actin tension and the scale factor anticorrelates with elastic modulus of nuclear envelope. We argue that nuclear morphology measures such as volume, projected area, height etc., are subsumed by flatness and scale factor, which can unambiguously characterize nuclear morphology.

Anti-tumor effects of Artemisia nilagirica extract on MDA-MB-231 breast cancer cells: deciphering the biochemical and biomechanical properties via TGF-ß upregulation.

PURPOSE: Artemisia nilagirica (AN), which is known to have antimicrobial, antioxidant, antiulcer, and anti-asthmatic properties, has been recently shown to have anti-cancer activity. However, the mechanism responsible for the anti-cancer property and its effect on cellular properties and functions are not known. MATERIAL AND METHODS: We have characterized the biochemical and biomechanical properties of MDA-MB-231 cells treated with the methanolic extract from AN. RESULTS: We show that AN-treatment decreases cell-eccentricity, increases expression of actin and microtubules, and do not affect cell-area. Increased expression of cytoskeletal proteins is known to change the mechanical properties of the cells, which was confirmed using micropipette aspiration and Atomic Force Microscopy. We identified the upregulation of the tumorigenic pathway (TGF-ß) leading to activation of Rho-A as the molecular mechanism responsible for actin upregulation. Since the initial stages of TGF-ß upregulation are known to suppress tumor growth by activating apoptosis, we hypothesized that the mechanism of cell death due to AN-treatment is through TGF-ß activation. We have validated this hypothesis by partially recuing cell death through inhibition of TGF-ß using Alk-5. CONCLUSION: In summary, our study reveals the mechanism of action of Artemisia nilagirica using a synergy between biochemical and biomechanical techniques.

A Nondimensional Model Reveals Alterations in Nuclear Mechanics upon Hepatitis C Virus Replication.

Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of ß-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.

A Scalable Perfusion Culture System with Miniature Peristaltic Pumps for Live-Cell Imaging Assays with Provision for Microfabricated Scaffolds.

We present a perfusion culture system with miniature bioreactors and peristaltic pumps. The bioreactors are designed for perfusion, live-cell imaging studies, easy incorporation of microfabricated scaffolds, and convenience of operation in standard cell culture techniques. By combining with miniature peristaltic pumps-one for each bioreactor to avoid cross-contamination and to maintain desired flow rate in each-we have made a culture system that facilitates perfusion culture inside standard incubators. This scalable system can support multiple parallel perfusion experiments. The major components are fabricated by three-dimensional printing using VeroWhite, which we show to be amenable to ex vivo cell culture. Furthermore, the components of the system can be reused, thus making it economical. We validate the system and illustrate its versatility by culturing primary rat hepatocytes, live imaging the growth of mouse fibroblasts (NIH 3T3) on microfabricated ring-scaffolds inserted into the bioreactor, performing perfusion culture of breast cancer cells (MCF7), and high-magnification imaging of hepatocarcinoma cells (HuH7).