Atomistic Simulations of a Multicomponent Asymmetric Lipid Bilayer

The cell membrane is inherently asymmetric and heterogeneous in its composition, a feature that is crucial for its function. Using atomistic molecular dynamics simulations, the physical properties of a 3-component asymmetric mixed lipid bilayer system comprising an unsaturated POPC (palmitoyloleoylphosphatidylcholine), a saturated PSM (palmitoylsphingomyelin), and cholesterol are investigated. Our simulations explore both the dynamics of coarsening following a quench from the mixed phase and the final phase-segregated regime obtained by equilibrating a fully segregated configuration. Following a quench, the membrane quickly enters a coarsening regime, where the initial stages of liquid ordered, <i>l</i>_o, domain formation are observed. These growing domains are found to be highly enriched in cholesterol and PSM. Consistent with this, the final phase-segregated regime contains large <i>l</i>_o domains at equilibrium, enriched in cholesterol and PSM. Our simulations suggest that the cholesterol molecules may partition into these PSM-dominated regions in the ratio of 3:1 when compared to POPC-dominated regions. PSM molecules exhibit a measurable tilt and long-range tilt correlations within the <i>l</i>_o domain as a consequence of the asymmetry of the bilayer, with implications to local membrane deformation and budding. Tagged particle diffusion for PSM and cholesterol molecules, which reflects spatial variations in the physical environment encountered by the tagged particle, is computed and compared with recent experimental results obtained from high-resolution microscopy

Visualizing Cortical Actin Dynamics Templating Membrane Organization in Blebs and in vitro

<p>Poster at the conference 'Actin Dynamics' in Regensburg, 2012</p

Functional nuclear perturbations alter particle displacement.

<p>Mean square displacements of the beads in the nuclei of cells under pN force at 25C, upon treatment with chemical perturbations of chromatin remodeling, as indicated (). (Insets) Trajectories of typical beads in the nuclei of cells under pN force on treatment with chemical perturbations of chromatin remodeling.</p

Dynamics of Passive and Active Particles in the Cell Nucleus

<div><p>Inspite of being embedded in a dense meshwork of nuclear chromatin, gene loci and large nuclear components are highly dynamic at C. To understand this apparent unfettered movement in an overdense environment, we study the dynamics of a passive micron size bead in live cell nuclei at two different temperatures ( and C) with and without external force. In the absence of a force, the beads are caged over large time scales. On application of a <em>threshold</em> uniaxial force (about 10 pN), the passive beads appear to hop between cages; this large scale movement is absent upon ATP-depletion, inhibition of chromatin remodeling enzymes and RNAi of lamin B1 proteins. Our results suggest that the nucleus behaves like an active solid with a finite yield stress when probed at a micron scale. Spatial analysis of histone fluorescence anisotropy (a measure of local chromatin compaction, defined as the volume fraction of tightly bound chromatin) shows that the bead movement correlates with regions of low chromatin compaction. This suggests that the physical mechanism of the observed yielding is the active opening of free-volume in the nuclear solid via chromatin remodeling. Enriched transcription sites at C also show caging in the absence of the applied force and directed movement beyond a yield stress, in striking contrast with the large scale movement of transcription loci at C in the absence of a force. This suggests that at physiological temperatures, the loci behave as <em>active</em> particles which remodel the nuclear mesh and reduce the local yield stress.</p> </div

Table5_A census of actin-associated proteins in humans.XLSX

Actin filaments help in maintaining the cell structure and coordinating cellular movements and cargo transport within the cell. Actin participates in the interaction with several proteins and also with itself to form the helical filamentous actin (F-actin). Actin-binding proteins (ABPs) and actin-associated proteins (AAPs) coordinate the actin filament assembly and processing, regulate the flux between globular G-actin and F-actin in the cell, and help maintain the cellular structure and integrity. We have used protein–protein interaction data available through multiple sources (STRING, BioGRID, mentha, and a few others), functional annotation, and classical actin-binding domains to identify actin-binding and actin-associated proteins in the human proteome. Here, we report 2482 AAPs and present an analysis of their structural and sequential domains, functions, evolutionary conservation, cellular localization, abundance, and tissue-specific expression patterns. This analysis provides a base for the characterization of proteins involved in actin dynamics and turnover in the cell.</p

Force transduction within the nucleus.

<p>Typical trajectories of TFs in the nuclei of cells at at 25C, (a) in the absence of force and (b) under a pN force, applied on beads. (Inset) Fluorescence image showing H2B-EGFP marked nucleus in green and alexa546-UTP foci in red. Scale bar m. (c) Fluorescence images of the TFs at every s showing directed movement of the compartments in the presence of force. The black line represents the force profile. (d) Type I and Type II trajectories of Alexa546-UTP labelled TCs with pN force applied on m paramagnetic bead for s. TFs in the nucleus show directed movement in the same direction as the bead. Error bars denote standard deviation. (e) Probability distribution of the angle between the instantaneous displacement vector of the TFs and the direction of the pN external force, shows that the TFs move predominantly along the direction of the force. In the absence of a force, the movement of the TFs is isotropic.</p

Force induced displacement of particles in the nucleus.

<p>(a) Time sequence of brightfield images of typical beads in the nuclei of untreated cells in the absence of force and under a super-threshold pN force, and in ATP depleted cells under pN force, respectively. The dotted lines are guides to the eye. Scale bar m. (b) Typical trajectory of a bead in the nucleus of a living cell at 25C under a pN force shows cage-hopping dynamics with a distribution of cage sizes given in Inset. (c) Typical trajectory of a bead in the nucleus of a living cell depleted of ATP at 25C under a pN force shows caging behaviour with hardly any hopping. (Inset) Mean cage size under ATP-depleted and control conditions. While the mean cage sizes are roughly similar, the untreated cell shows cage-hopping dynamics in the presence of a threshold force. (d) Mean square displacement averaged over many beads () versus time in the nuclei of untreated cells and ATP depleted cells under pN force and pN force. (e) Probability distribution of the angle between the instantaneous displacement vector of the bead and the direction of the external force, shows that for untreated cells under a pN force, the beads move predominantly in the direction of the force. In all other cases the bead movement is isotropic.</p

Single particle tracking analysis of particles in the nucleus.

<p>(a) Schematic of the experimental setup. An electromagnet mounted on a fluorescence microscope is used to apply forces on a m paramagnetic bead microinjected into the nucleus. (Inset) Brightfield image of the nucleus showing microinjected magnetic beads. The dotted line represents the edge of the nucleus. Scale bar m. (b) Typical trajectory of a bead in the nucleus of a living cell at C showing caging behaviour. (Inset) Histogram of cage sizes across beads shows a narrow distribution about nm. (c & d) Mean square displacement versus time for beads in the nuclei of untreated cells in the absence of force. beads show Type I behaviour and beads show Type II. (e) Mean effective exponent versus inverse time for Type I and II trajectories together with the combined data. (f) Shear and loss modulus, , (averaged over all beads) as a function of frequency . At low , the response of the nucleus is elastic () and crosses over to a viscous response at higher . Fits at low show that the nucleus behaves as a power-law solid at low frequencies.</p

Table7_A census of actin-associated proteins in humans.XLSX

Actin filaments help in maintaining the cell structure and coordinating cellular movements and cargo transport within the cell. Actin participates in the interaction with several proteins and also with itself to form the helical filamentous actin (F-actin). Actin-binding proteins (ABPs) and actin-associated proteins (AAPs) coordinate the actin filament assembly and processing, regulate the flux between globular G-actin and F-actin in the cell, and help maintain the cellular structure and integrity. We have used protein–protein interaction data available through multiple sources (STRING, BioGRID, mentha, and a few others), functional annotation, and classical actin-binding domains to identify actin-binding and actin-associated proteins in the human proteome. Here, we report 2482 AAPs and present an analysis of their structural and sequential domains, functions, evolutionary conservation, cellular localization, abundance, and tissue-specific expression patterns. This analysis provides a base for the characterization of proteins involved in actin dynamics and turnover in the cell.</p

Table6_A census of actin-associated proteins in humans.XLSX

Actin filaments help in maintaining the cell structure and coordinating cellular movements and cargo transport within the cell. Actin participates in the interaction with several proteins and also with itself to form the helical filamentous actin (F-actin). Actin-binding proteins (ABPs) and actin-associated proteins (AAPs) coordinate the actin filament assembly and processing, regulate the flux between globular G-actin and F-actin in the cell, and help maintain the cellular structure and integrity. We have used protein–protein interaction data available through multiple sources (STRING, BioGRID, mentha, and a few others), functional annotation, and classical actin-binding domains to identify actin-binding and actin-associated proteins in the human proteome. Here, we report 2482 AAPs and present an analysis of their structural and sequential domains, functions, evolutionary conservation, cellular localization, abundance, and tissue-specific expression patterns. This analysis provides a base for the characterization of proteins involved in actin dynamics and turnover in the cell.</p