Combined modeling of cell aggregation and adhesion mediated by receptor–ligand interactions under shear flow

AbstractBlood cell aggregation and adhesion to endothelial cells under shear flow are crucial to many biological processes such as thrombi formation, inflammatory cascade, and tumor metastasis, in which these cellular interactions are mainly mediated by the underlying receptor–ligand bindings. While theoretical modeling of aggregation dynamics and adhesion kinetics of interacting cells have been well studied separately, how to couple these two processes remains unclear. Here we develop a combined model that couples cellular aggregation dynamics and adhesion kinetics under shear flow. The impacts of shear rate (or shear stress) and molecular binding affinity were elucidated. This study provides a unified model where the action of a fluid flow drives cell aggregation and adhesion under the modulations of the mechanical shear flow and receptor–ligand interaction kinetics. It offers an insight into understanding the relevant biological processes and functions

Identification of the Conformational transition pathway in PIP2 Opening Kir Channels

The gating of Kir channels depends critically on phosphatidylinositol 4,5-bisphosphate (PIP2), but the detailed mechanism by which PIP2regulates Kir channels remains obscure. Here, we performed a series of Targeted molecular dynamics simulations on the full-length Kir2.1 channel and, for the first time, were able to achieve the transition from the closed to the open state. Our data show that with the upward motion of the cytoplasmic domain (CTD) the structure of the C-Linker changes from a loop to a helix. The twisting of the C-linker triggers the rotation of the CTD, which induces a small downward movement of the CTD and an upward motion of the slide helix toward the membrane that pulls the inner helix gate open. At the same time, the rotation of the CTD breaks the interaction between the CD- and G-loops thus releasing the G-loop. The G-loop then bounces away from the CD-loop, which leads to the opening of the G-loop gate and the full opening of the pore. We identified a series of interaction networks, between the N-terminus, CD loop, C linker and G loop one by one, which exquisitely regulates the global conformational changes during the opening of Kir channels by PIP2

Identification of the Conformational transition pathway in PIP2 Opening Kir Channels

The gating of Kir channels depends critically on phosphatidylinositol 4,5-bisphosphate (PIP2), but the detailed mechanism by which PIP2 regulates Kir channels remains obscure. Here, we performed a series of Targeted molecular dynamics simulations on the full-length Kir2.1 channel and, for the first time, were able to achieve the transition from the closed to the open state. Our data show that with the upward motion of the cytoplasmic domain (CTD) the structure of the C-Linker changes from a loop to a helix. The twisting of the C-linker triggers the rotation of the CTD, which induces a small downward movement of the CTD and an upward motion of the slide helix toward the membrane that pulls the inner helix gate open. At the same time, the rotation of the CTD breaks the interaction between the CD- and G-loops thus releasing the G-loop. The G-loop then bounces away from the CD-loop, which leads to the opening of the G-loop gate and the full opening of the pore. We identified a series of interaction networks, between the N-terminus, CD loop, C linker and G loop one by one, which exquisitely regulates the global conformational changes during the opening of Kir channels by PIP2

Visualization of Allostery in P-Selectin Lectin Domain Using MD Simulations

Allostery of P-selectin lectin (Lec) domain followed by an epithelial growth factor (EGF)-like domain is essential for its biological functionality, but the underlying pathways have not been well understood. Here the molecular dynamics simulations were performed on the crystallized structures to visualize the dynamic conformational change for state 1 (S1) or state 2 (S2) Lec domain with respective bent (B) or extended (E) EGF orientation. Simulations illustrated that both S1 and S2 conformations were unable to switch from one to another directly. Instead, a novel S1' conformation was observed from S1 when crystallized B-S1 or reconstructed “E-S1” structure was employed, which was superposed well with that of equilibrated S1 Lec domain alone. It was also indicated that the corresponding allosteric pathway from S1 to S1' conformation started with the separation between residues Q30 and K67 and terminated with the release of residue N87 from residue C109. These results provided an insight into understanding the structural transition and the structure-function relationship of P-selectin allostery

Conformational Stability Analyses of Alpha Subunit I Domain of LFA-1 and Mac-1

β2 integrin of lymphocyte function-associated antigen-1 (LFA-1) or macrophage-1 antigen (Mac-1) binds to their common ligand of intercellular adhesion molecule-1 (ICAM-1) and mediates leukocyte-endothelial cell (EC) adhesions in inflammation cascade. Although the two integrins are known to have distinct functions, the corresponding micro-structural bases remain unclear. Here (steered-)molecular dynamics simulations were employed to elucidate the conformational stability of α subunit I domains of LFA-1 and Mac-1 in different affinity states and relevant I domain-ICAM-1 interaction features. Compared with low affinity (LA) Mac-1, the LA LFA-1 I domain was unstable in the presence or absence of ICAM-1 ligand, stemming from diverse orientations of its α7-helix with different motifs of zipper-like hydrophobic junction between α1- and α7-helices. Meanwhile, spontaneous transition of LFA-1 I domain from LA state to intermediate affinity (IA) state was first visualized. All the LA, IA, and high affinity (HA) states of LFA-1 I domain and HA Mac-1 I domain were able to bind to ICAM-1 ligand effectively, while LA Mac-1 I domain was unfavorable for binding ligand presumably due to the specific orientation of S144 side-chain that capped the MIDAS ion. These results furthered our understanding in correlating the structural bases with their functions of LFA-1 and Mac-1 integrins from the viewpoint of I domain conformational stability and of the characteristics of I domain-ICAM-1 interactions

Multi-Patterned Dynamics of Mitochondrial Fission and Fusion in a Living Cell

Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called “kiss and run” model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible, which provided a new insight into mitochondrial dynamics. In addition to kiss and run model of action, our data suggest that, from a global visualization over an entire cell, multiple patterns of action appeared in mitochondrial fusion and fission

Contribution of the CR domain to P-selectin lectin domain allostery by regulating the orientation of the EGF domain.

The allostery of P-selectin has been studied extensively with a focus on the Lec and EGF domains, whereas the contribution of the CR domain remains unclear. Here, molecular dynamics simulations (MDS) combined with homology modeling were preformed to investigate the impact of the CR domain on P-selectin allostery. The results indicated that the CR domain plays a role in the allosteric dynamics of P-selectin in two ways. First, the CR1 domain tends to stabilize the low affinity of P-selectin during the equilibration processes with the transition inhibition from the S1 to S1' state by restraining the extension of the bent EGF orientation, or with the relaxation acceleration of the S2 state by promoting the bending of the extended EGF orientation. Second, the existence of CR domain increases intramolecular extension prior to complex separation, increasing the time available for the allosteric shift during forced dissociation with a prolonged bond duration. These findings further our understanding of the structure-function relationship of P-selectin with the enriched micro-structural bases of the CR domain

Mechanical features of endothelium regulate cell adhesive molecule-induced calcium response in neutrophils

Atherosclerosis is caused by chronic inflammation associated with the adhesion of neutrophils and endothelial cells (ECs) that is mediated by their respective cellular adhesive molecules to stiffened blood vessel walls. However, the stiffness dependence of calcium flux on neutrophils remains unclear yet. Here, the effect of substrate stiffness by ECs on neutrophils' calcium spike was quantified when the individual neutrophils that adhered to the human umbilical vascular endothelial cell (HUVEC) monolayer were pre-placed onto a stiffness-varied polyacrylamide substrate (5 or 34.88 kPa) or glass surface. Our data indicated that E-/P-selectins and intercellular adhesion molecule 1 (ICAM-1) on HUVECs and β2-integrins, P-selectin glycoprotein ligand 1 (PSGL-1), and CD44s on neutrophils were all involved in mediating neutrophil calcium spike in a stiffness-dependent manner, in which the increase in substrate stiffness enhanced the calcium intensity and the oscillation frequency (spike number). Such stiffness-dependent calcium response is associated with the induced selectin related to β2-integrin activation through the Syk/Src signaling pathway, and F-actin/myosin II are also involved in this. Moreover, tension-activated calcium ion channels displayed critical roles in initiating stiffness-dependent calcium spike. These results provide an insight into understanding how the stiffening of vascular walls could regulate the calcium flux of adhered neutrophils, and thus the immune responses in atherosclerosis

Features of P-selectin with different conformations of the <i>R</i>3 loop.

<p>(<i>A</i>) Conformational differences in the <i>R</i>3 loop between <i>S</i>1 (<i>silver</i>), <i>S</i>1’ (<i>cyan</i>) and <i>S</i>2 (<i>blue</i>). The Lec domain is presented in <i>newcartoon</i> format in <i>topview</i>; the other components, except for the <i>R</i>3 loop and calcium ion, are shown in <i>transparent</i> format for clarity. The <i>S</i>1’ conformation is the final snapshot of the 20-<i>ns</i> equilibration of the 1G1Q/P-LEC system of the 1CR set. (<i>B</i>) Differences in receptor-ligand interactions for P-selectin with distinct <i>R</i>3 conformations of <i>S</i>1, <i>S</i>1’ and <i>S</i>2 during the free equilibration process of SGP-3-ligated systems in the 0CR, 1CR and 2CR sets. Non-covalent energy, including van der Waals and electrostatic interactions between P-selectin and the SGP-3 ligand, were quantified, and only the snapshots showing an <i>R</i>3 loop RMSD of less than or equal to 4.0 Å in relation to the reference state <i>S</i>1, <i>S</i>1’ or <i>S</i>2 were counted. The trajectory was counted in statistical analyses of state <i>S</i>1’ if its snapshots fit the rules of both <i>S</i>1 and <i>S</i>1’. The data are presented as the mean ± SD of the different equilibration systems. (<i>C</i>) Differences in dissociation time between the complexes with distinct <i>R</i>3 loop conformations of <i>S</i>1, <i>S</i>1’ and <i>S</i>2 under a constant force of 300 <i>pN</i> pulling on the SGP-3 ligand with EGF end fixation. The data were presented as the mean ± SD of six dissociation runs for each system. The dissociation simulations were based on the final state after a 20 <i>ns</i> equilibration of the 1G1Q/P-LECC-SGP3, 1G1Q/P-LEC-SGP3, and 1G1S/P-LECC-SGP-3 systems for <i>S</i>1, <i>S</i>1’ and <i>S</i>2, respectively.</p

Impact of the CR domain on the constant force dissociation process.

<p>(<i>A</i>) Dissociation time distribution for the EGF end-fixed (<i>black</i>), CR1 end-fixed (<i>white</i>) and CR2 end-fixed (<i>gray</i>) dissociation processes at 300 <i>pN</i> based on 20-<i>ns</i>-equilibrated snapshots of the SGP-3-ligated 1G1Q 2CR complex with the <i>R</i>3 loop in the <i>S</i>1 state (<i>left</i>), the 1G1Q 1CR complex with the <i>R</i>3 loop in the <i>S</i>1’ state (<i>middle</i>) and the 1G1S 2CR complex with the <i>R</i>3 loop in the <i>S</i>2 state (<i>right</i>), respectively. The data are presented as the mean ± SD of six independent runs for each fixation setting. (<i>B</i>, <i>C</i>, <i>D</i>) Corresponding separation-extension profiles of the dissociation processes based on the initial <i>S</i>1 (<i>B</i>), <i>S</i>1’ (<i>C</i>) or <i>S</i>2 (<i>D</i>) system. Six repeated runs for EGF end fixation, CR1 end fixation and CR2 end fixation are shown in the same color (<i>black</i>, <i>red</i> or <i>blue</i>, respectively).</p