Roopali Saxena

Cholesterol Biosynthesis and Homeostasis in Regulation of the Cell Cycle

The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer’s disease, that are associated with impaired cholesterol biosynthesis and homeostasis.

Membrane cholesterol stabilizes the human serotonin1A receptor

A number of recently solved crystal structures of G-protein coupled receptors reveal the presence of closely associated cholesterol molecules in the receptor structure. We have previously shown the requirement of membrane cholesterol in the organization, dynamics and function of the serotonin(1A) receptor, a representative G-protein coupled receptor. In this work, we explored the role of membrane cholesterol in the stability of the human serotonin(1A) receptor. Analysis of sensitivity of the receptor to thermal deactivation, pH, and proteolytic digestion in control, cholesterol-depleted and cholesterol-enriched membranes comprehensively demonstrate that membrane cholesterol stabilizes the serotonin(1A) receptor. We conclude that these results could have potential implications in future efforts toward crystallizing the receptor.

Differential effects of cholesterol and desmosterol on the ligand binding function of the hippocampal serotonin1A receptor: Implications in desmosterolosis

Cholesterol is a unique molecule in terms of high level of in-built stringency, fine tuned by natural evolution for its ability to optimize physical properties of higher eukaryotic cell membranes in relation to biological functions. We previously demonstrated the requirement of membrane cholesterol in maintaining the ligand binding activity of the hippocampal serotonin(1A) receptor. In order to test the molecular stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is an immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position in the alkyl side chain. Our results show that replenishment with desmosterol does not restore ligand binding activity of the serotonin(1A) receptor although replenishment with cholesterol led to significant recovery of ligand binding. This is in spite of similar membrane organization (order) in these membranes, as monitored by fluorescence anisotropy measurements. The requirement for restoration of ligand binding activity therefore appears to be more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor in diseases such as desmosterolosis.

Exploring the organization and dynamics of hippocampal membranes utilizing pyrene fluorescence.

The organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors and signal transduction. Previous work from our laboratory has established hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have monitored the organization and dynamics of hippocampal membranes and their modulation by cholesterol using pyrene fluorescence. The apparent dielectric constant experienced by pyrene in hippocampal membranes turns out to be approximately 20+/-3, depending on the experimental condition. Our results show that the polarity of the hippocampal membrane is increased upon cholesterol depletion, as monitored by changes in the ratio of pyrene vibronic peak intensities (I1/I3). This is accompanied by an increase in lateral diffusion, measured as an increase in the pyrene excimer/monomer ratio. These results are relevant in understanding the complex organization and dynamics of hippocampal membranes and could have implications in neuronal diseases characterized by defective cholesterol metabolism.

Membrane organization and dynamics of the serotonin1A receptor in live cells

J. Neurochem. (2011) 116, 726–733.

Location, dynamics and solvent relaxation of a nile red-based phase-sensitive fluorescent membrane probe

Fluorescent membrane probes offer the advantage of high sensitivity, suitable time resolution, and multiplicity of measurable parameters, and provide useful information on model and cell membranes. In this paper, we have explored the location, dynamics, and solvent relaxation characteristics of a novel Nile Red-based phase-sensitive probe (NR12S). Unlike Nile Red, NR12S enjoys unique orientation and location in the membrane, and is localized exclusively in the outer leaflet of the membrane bilayer. By analysis of membrane depth using the parallax approach, we show that the fluorescent group in NR12S is localized at the membrane interface, a region characterized by slow solvent relaxation. Our results show that NR12S exhibits REES (red edge excitation shift), consistent with its interfacial localization. More interestingly, REES of NR12S displays sensitivity to the membrane phase. In addition, fluorescence emission maximum, anisotropy, and lifetime of NR12S are dependent on the membrane phase. We envision that NR12S may prove to be a useful probe in future studies of complex natural membranes.

Identification of a red-emitting fluorescent ligand for in vitro visualization of human serotonin 5-HT 1A receptors

The 5-HT(1A) receptor subtype is the most thoroughly studied serotonin receptor subtype. We report here the design, synthesis and characterization of two new fluorescent ligands for the 5-HT(1A) receptor. The new 1-arylpiperazine-based red-emitting fluorescent compound 6 displayed good binding affinity at the 5-HT(1A) receptor (K(i)=35 nM) and was able to label specifically the human 5-HT(1A) receptor stably expressed in CHO cells visualized using confocal laser scanning microscopy.

Cholesterol-induced changes in hippocampal membranes utilizing a phase-sensitive fluorescence probe.

The function of membrane receptors in the nervous system depends on physicochemical characteristics of neuronal membranes such as membrane order and phase. In this work, we have monitored the changes in hippocampal membrane order and related parameters by cholesterol and protein content utilizing a Nile Red-based phase-sensitive fluorescent membrane probe NR12S. Since alteration of membrane cholesterol is often associated with membrane phase change, the phase-sensitive nature of NR12S fluorescence becomes useful in these experiments. Our results show that fluorescence spectroscopic parameters such as emission maximum, anisotropy, and lifetime of NR12S display characteristic dependence on membrane cholesterol content. Interestingly, cholesterol-dependent red edge excitation shift is displayed by NR12S under these conditions. Hippocampal membranes exhibited reduction in liquid-ordered phase upon cholesterol depletion. These results provide insight into changes in hippocampal membrane order in the overall context of cholesterol and protein modulation.

Cholesterol depletion modulates detergent resistant fraction of human serotonin1A receptors

Abstract Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin1A receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin1A receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (∼ 28%) of the serotonin1A receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin1A receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM1 are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin1A receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin1A receptor in particular, and G-protein coupled receptors in general.

Comparative Analysis of Calcium Spikes upon Activation of Serotonin1A and Purinergic Receptors

Calcium signaling represents one of the most important signaling cascades in cells and regulates diverse processes such as exocytosis, muscle contraction and relaxation, gene expression and cell growth. G protein-coupled receptors (GPCRs) are the most important family of receptors that activate calcium signaling. Since calcium signaling regulates a large number of physiological responses, it is intriguing that how changes in cytosolic calcium levels by a wide range of stimuli lead to signal-specific physiological responses in the cellular interior. In order to address this issue, we have analyzed temporal calcium profiles induced by two GPCRs, the serotonin1A and purinergic receptors. In this work, we have described a set of parameters for the analysis of calcium transients that could provide novel insight into mechanisms responsible for maintaining signal specificity by shaping calcium transients. An interesting feature of calcium signaling that has emerged from our analysis is that the profile of individual transients in a calcium response could play an important role in maintaining downstream signal specificity. In summary, our analysis offers a novel approach to identify differences in calcium response patterns induced by various stimuli.