Cells

The purpose of this research is to explore and investigate the biophysical properties of living cells using microfluidics based electrical impedance sensing (EIS) technique. It provides a non-invasive approach to detect label-free biological markers in the regulation of cellular activities even at a molecular level. We specifically focus on the development, testing, and theoretical modeling of electrical impedance spectroscopy for neuroblastoma cells and endothelial cells. First, we demonstrate that the EIS technique can be used to monitor the progressive mitochondrial fission/fusion modification in genetically modified human neuroblastoma cell lines. Our results characterize quantitatively the abnormal mitochondrial dynamics through the variations in cytoplasm conductivity. Secondly, we employ a real time EIS method to determine the biophysical properties of the junctions which join one endothelial cell with one another in a monolayer of endothelial cells. In particular, we examine the role of the protein, c-MYC oncogene, in the barrier function. Our results show that the downregulation of c-MYC oncogene enhances the endothelial barrier dysfunction associated with inflammation. Finally, we measure and find that the electrical admittance (the reciprocal of the impedance) of the monolayer of endothelial cellular networks exhibits an anomalous power law of the form, Y ∝ ωα, over a wide range of frequency, with the value of the exponent, α, depending on the severity of the inflammation. We attribute the power law to the changes of the intercellular electric permeability between neighboring endothelial cells. Thus, the inflammation gives rise to relatively smaller values of α compared to that of the no-inflammation group. Furthermore, we propose a simple percolation model of a large R-C network to confirm the emergent of power law scaling behavior of the complex admittance, suggesting that the endothelial network behaves as a complex microstructural network and its electrical properties may be simulated by a large R-C network.

The kinesin superfamily of microtubule motor proteins is subdivided into families based upon structure and function. KIF9 is the founding member of the Kinesin-9 family, which is a largely uncharacterized group of kinesins. It was originally identified by sequence homology to other kinesins. Subsequent studies have shown that KIF9 interacts with proteins involved in cell shape remodeling, cell migration and proper centrosomal positioning. We have examined KIF9 function in mammalian cells using shRNA-mediated knockdown and GFP-plasmid overexpression. By knocking dow KIF9 expression in these cells, we have seen several effects on normal cell cycle progression. Using various cell cycle markers, we have observed a decrease in the number of cells in late S phase. In addition, there is a marked increase in the number of cells in early mitosis in unexpected time intervals. We propose that KIF9 is required for proper cell progression, via a potentially novel checkpoint mechanism.

The kinesin family of microtubule motors is divided into subfamilies based on structure and function. KIF9, founder of the Kinesin-9 family, has been found to interact with the GTPase Gem. Subsequent studies have shown that KIF9 is vital for flagellar movement and podosome regulation. Previous work has proposed KIF9 is required for microtubule organization as well as proper mitotic entry, progression and completion. In this study, I examined the function of KIF0 in mitotic progression using shRNA-mediated knockdown, and overexpression. In knockdown cells, I saw a significant delay in mitotic progression as well as an increase in multipolarity and multinuclearity, suggesting a failure of cytokinesis. Overexpression of KIF9 produced similar effects on mitotic progression, as well as a marked increase in chromosome distance during anaphase. Taken with previous results, my research indicated that KIF9 is required for normal mitotic progression and completion, possible via regulation of the contractile ring.

Kinesin motors bind to microtubules and function in mitosis and intracellular transport depending on the position of the motor domain within the primary sequence (Hirokawa and Noda 2008). KIF9 has recently been shown to be involved in MTOC positioning and mitotic entry in Dictyostelium (Tikhonenko et al. 2009). To determine if a similar role for KIF9 exists in mammalian cells, we are using siRNA-mediated knockdown of KIF9 in COS-7 cells. Analysis of unsynchronized and cell-cycle synchronized cells treated with siRNA to KIF9 reveal that the transition from G2 to M phase is delayed and that mitotic progression is also affected. Additionally, our data indicates that spindle pole function during anaphase may be abnormal in cells treated with siRNA, suggesting a role for KIF9 during that stage.