Browsing by Author "Raghavachari, Sridhar"
Results Per Page
Sort Options
Item Open Access Lateral Diffusion of Receptors at Synapse Influenced by Synapse Geometry and Macromolecular Crowding(2014) Song, YuCells express a variety of proteins on their surface that allows them to sample the world. These proteins are embedded in the plasma membrane, a bilayer of lipids that surrounds the cell. Since the lipid and protein dimensions are in the nanometer range, they are subject to thermal agitation by water molecules and show characteristic diffusive motion. The diffusive movement of these proteins plays a critical role in the cell's ability to react to external signals and regulate its internal environment.
One prominent application of protein diffusion is in the synaptic connection, where is the highly localized concentration of receptors. The receptive dendrite membrane contains many types of receptors that are accumulated to form functional microdomains opposite the presynaptic terminal buttons that release neurotransmitters. Experiments reveal that receptors move from extrasynaptic locations to synaptic locations by lateral diffusion, thereby concentrating receptors at synapses. Two key processes that control synaptic AMPAR numbers are receptor diffusion within the synaptic and extrasynaptic space and interactions between receptors and PSD scaffold proteins. Electron microscopy images suggest that the PSD is highly crowded potentially limiting the ability of receptors to diffuse and interact with scaffold proteins. However, the contribution of macromolecular crowding to receptor retention remains to be tested systematically.
Here, we combine experimental and computational approaches to test the effect of synaptic steric hindrance on receptor mobility and enrichment. We first investigate how the diffusion is influenced by membrane geometry. The membrane itself can have three-dimensional structure, which means that the actual path length of diffusion can be different from a projected path length. Here, we use a position Langevin equation for diffusion, which incorporates curvature and gradient effects of surfaces. Numeric simulation of the equation allows for the prediction of effective diffusion coefficients over corrugated surfaces.
In order to examine the distinct contributions of crowding and receptor-scaffold binding, we developed a computational model for AMPA-receptor diffusion in the synaptic and extrasynaptic space, which contains immobile obstacles, representing scaffolding, receptor and adhesion molecules in the PSD. The spatial distribution of scaffold proteins was determined directly from photo-activated localization microscopy measurements that mapped molecular positions with a resolution of ~20 nm. The AMPAR/scaffold association and dissociation rates were adjusted by computer simulations to fit single-particle tracking and fluorescence recovery after photobleaching measurements. The model predicts the recovery curves are influenced mostly by size changes while variation of kinetic rates did not significantly alter receptor residence time or mobility. We also examined the effect of binding, by adding a single synaptic binding motif to a small transmembrane protein, which slows its diffusion within the synapse. These results suggest that both protein size and binding play important roles in retaining surface-diffusing TM proteins within the excitatory synapse and shed light on the biophysical mechanisms that lead to high density of AMPARs at synapses.
Item Open Access Quantifying Gene Regulatory Networks(2014) Wang, Shangying\abstract
Transcription and translation describe the flow of genetic information from DNA to mRNA to protein. Recent studies show that at a single cell level, these processes are stochastic, which results in the variation of the number of mRNA and proteins even under identical environmental conditions. Because the number of mRNA and protein in each single cell are actually very small, these variations can be crucial for cellular function in diverse contexts, such as development, stress response, immunological and nervous system function. Most studies examine the origin and effects of stochastic gene expression using computer simulations. My goal is to develop a theoretical framework to study activity-dependent gene expression using simplified models that capture essential features.
I have examined the dynamics of stochastic gene regulation in three contexts. First, I examine how fluctuations in promoter accessibility lead to "bursty" transcription, during which genes are turned "on" or "off" stochastically. I describe a mathematical formalism to represent bursty gene expression in a coarse-grained manner as a Markov process and derive a master equation for the time evolution of the probability distribution of the number of mRNA molecules. This allows us to examine how transcript number responds to time varying stimuli. This model forms a basic building block for understanding the signal transmission and noise of the transcription process to time varying inputs as would be sensed by cells in dynamic environments. In addition to synthesis, gene expression is subject to additional modes of regulation. One such mechanism that controls transcript numbers is by microRNAs (miRNAs), which pair with target mRNAs to repress protein production following transcription. Although hundreds of miRNAs have been identified in mammalian genomes, the function of miRNA-based repression in the context of gene regulation networks still remains unclear. I explore the functional roles of feedback regulation by miRNAs and show that protein fluctuations strongly depend on the mode of miRNA-mediated repression. I discuss the functional implications of protein fluctuations arising from miRNA-mediated repression on gene regulatory networks. Finally, I examine the impact of fluctuations on alternative splicing, which is a major source for proteomic complexity in higher eukaryotes. Although the proteins regulating alternative splicing have been extensively studied, little is known about how noise arising from the stochastic nature of alternative splicing contributes to the entire gene expression process. I explore the functional roles and noise properties of alternative splicing, focusing on the case of exon skipping and intron retention. I show that while the overall counts of the mRNAs of the two isoforms are independent and Poisson distributed, diffusion and binding of the splicing factors contributes to the variance in the abundance of the isoforms.
Noise in gene expression may be of particular relevance in the nervous system. Environmental stimuli drive the rapid remodeling of neural circuitry in part by inducing the activation of genes to make proteins that modify neuronal excitability and connectivity, ultimately influencing higher order brain function. Finally, I examine the implications of our studies for activity dependent gene expression in the nervous system.
Item Open Access Quantifying the effects of elastic collisions and non-covalent binding on glutamate receptor trafficking in the post-synaptic density.(PLoS computational biology, 2010) Santamaria, Fidel; Gonzalez, Jossina; Augustine, George J; Raghavachari, SridharOne mechanism of information storage in neurons is believed to be determined by the strength of synaptic contacts. The strength of an excitatory synapse is partially due to the concentration of a particular type of ionotropic glutamate receptor (AMPAR) in the post-synaptic density (PSD). AMPAR concentration in the PSD has to be plastic, to allow the storage of new memories; but it also has to be stable to preserve important information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which AMPAR can enter, leave and remain in the synapse are unclear. We used Monte Carlo simulations to determine the influence of PSD structure and activity in maintaining homeostatic concentrations of AMPARs in the synapse. We found that, the high concentration and excluded volume caused by PSD molecules result in molecular crowding. Diffusion of AMPAR in the PSD under such conditions is anomalous. Anomalous diffusion of AMPAR results in retention of these receptors inside the PSD for periods ranging from minutes to several hours in the absence of strong binding of receptors to PSD molecules. Trapping of receptors in the PSD by crowding effects was very sensitive to the concentration of PSD molecules, showing a switch-like behavior for retention of receptors. Non-covalent binding of AMPAR to anchored PSD molecules allowed the synapse to become well-mixed, resulting in normal diffusion of AMPAR. Binding also allowed the exchange of receptors in and out of the PSD. We propose that molecular crowding is an important biophysical mechanism to maintain homeostatic synaptic concentrations of AMPARs in the PSD without the need of energetically expensive biochemical reactions. In this context, binding of AMPAR with PSD molecules could collaborate with crowding to maintain synaptic homeostasis but could also allow synaptic plasticity by increasing the exchange of these receptors with the surrounding extra-synaptic membrane.