PhD Student at Prof. Amitabha Chattopadhya's Laboratory (Shyama Prasad Mukherjee Fellow), CSIR-Centre for Cellular and Molecular Biology
The interplay between membrane cholesterol and the actin cytoskeleton
The function of actin cytoskeleton in cellular motility and trafficking has been widely studied. However, reorganization of actin cytoskeleton upon modulation of membrane cholesterol is addressed only rarely. In the present study, we focus on the role of membrane cholesterol in the regulation of F-actin levels in CHO cells. In order to monitor the changes in the actin cytoskeleton upon cholesterol depletion in a quantitative manner, we used a technique recently developed by us based on high magnification imaging of F-actin in cells followed by image reconstruction. Our results suggest that F-actin content increases in response to membrane cholesterol depletion. Interestingly, depletion of plasma membrane cholesterol has recently been reported to result in reorganization of actin cytoskeleton via a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) dependent manner. As PI(4,5)P2 is known to be enriched in cholesterol-dependent domains, we depleted plasma membrane cholesterol utilizing AY9944 which is a specific metabolic inhibitor of the enzyme required for the final step of cholesterol biosynthesis without disturbing isoprenoid levels . From our results we speculate that reorganization of actin cytoskeleton could be a synergistic effect of Rho GTPases and distribution of plasma membrane PI(4,5)P2. To the best of our knowledge, these results constitute the first comprehensive report quantifying the change in F-actin content upon cholesterol depletion. The dynamic reorganization of the actin cytoskeleton may represent an important determinant in membrane protein signaling in diseases that are due to defects in lipid biosynthesis pathways such as Smith-Lemli-Opitz Syndrome (SLOS) and desmosterolosis.
Abstract: Structural transitions involving shape changes play an important role in cellular physiology. Charged micelles offer a convenient model system in which structural transitions can be suitably induced by increasing the ionic strength of the medium. In this paper, we have explored sphere-to-rod transition in charged micelles of SDS and CTAB by monitoring micellar dipole potential using the dual wavelength ratiometric approach utilizing the potential-sensitive membrane probe di-8-ANEPPS. Our results show that micellar dipole potential is sensitive to sphere-to-rod transition in charged micelles. Micellar dipole potential exhibited increase with increasing ionic strength (salt), irrespective of the nature of micellar charge, implying considerable dipolar reorganization underlying structural transitions. We interpret the increase in dipole potential due to sphere-to-rod transition because of an increase in the population of confined (nonrandom) dipoles induced by micellar organizational change. This is due to the fact that dipole potential arises due to the nonrandom arrangement of micellar dipoles and water molecules at the micelle interface. Our results constitute one of the first reports describing drastic dipolar reorganization due to micellar shape (and size) change. We envision that dipole potential measurements could provide novel insights into micellar processes that are associated with dipolar reorganization.
Pub.: 01 Dec '15, Pinned: 06 Jun '17
Abstract: Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Although dipole potential is generally used in the context of bilayer membranes, the nonrandom arrangement of amphiphiles and water dipoles would also contribute to dipole potential in organized molecular assemblies such as micelles. In this work, we show that the process of micelle formation from monomers for a representative variety of detergents is associated with dipolar rearrangement. We monitor the dipolar reorganization upon micellization as a change in dipole potential, measured by the dual wavelength ratiometric approach utilizing the potential-sensitive membrane probe di-8-ANEPPS. We further utilized this phenomenon to estimate the critical micelle concentration (CMC) of a variety of detergents. CMC determined by this method are in overall agreement with the literature values of CMC for these detergents. To the best of our knowledge, these results constitute the first report showing dipolar reorientation during micellization. We conclude that dipole potential measurements could provide a novel approach to explore micellar organization.
Pub.: 04 Sep '15, Pinned: 06 Jun '17