Numerical simulation of flow characteristics in a permeable liver sinusoid with leukocytes.

Double-layered channels of sinusoid lumen and Disse space separated by fenestrated liver sinusoidal endothelial cells (LSECs) endow the unique mechanical environment of the liver sinusoid network, which further guarantees its biological function. It is also known that this mechanical environment changes dramatically under liver fibrosis and cirrhosis, including the reduced plasma penetration and metabolite exchange between the two flow channels and the reduced Disse space deformability. The squeezing of leukocytes through narrow sinusoid lumen also affects the mechanical environment of liver sinusoid. To date, the detailed flow-field profile of liver sinusoid is still far from clear due to experimental limitations. It also remains elusive whether and how the varied physical properties of the pathological liver sinusoid regulate the fluid flow characteristics. Here a numerical model based on the immersed boundary method was established, and the effects of Disse space and leukocyte elasticities, endothelium permeability, and sinusoidal stenosis degree on fluid flow as well as leukocyte trafficking were specified upon a mimic liver sinusoid structure. Results showed that endothelium permeability dominantly controlled the plasma penetration velocity across the endothelium, whereas leukocyte squeezing promoted local penetration and significantly regulated wall shear stress on hepatocytes, which was strongly related to the Disse space and leukocyte deformability. Permeability and elasticity cooperatively regulated the process of leukocytes trafficking through the liver sinusoid, especially for stiffer leukocytes. This study will offer new insights into deeper understanding of the elaborate mechanical features of liver sinusoid and corresponding biological function.

E-selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity.

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.

Mapping the morphological identifiers of distinct conformations via the protein translocation current in nanopores.

Conformational changes of proteins play a vital role in implementing their functions and revealing the underlying mechanisms in various biological processes. It is still challenging to monitor protein conformations with temporal fingerprints of current-resistance pulses in the nanopore technique. Here the low-resolution morphologies of different conformations of a typical integrin, αxß2, were estimated via relative blockade currents simulated from all-atom molecular dynamics (MD). Distinct conformational states of αxß2 were directly explained by the volume and shape identifiers. Protein modulation in ionic current was analyzed from the conductivity distribution inside the protein-blocked nanopore. Combining a discrete model with spheroidal approximation, a MD-based approach was developed to theoretically predict the volume and shape of the nanopore for sensing αxß2. This method was also applicable in specifying morphological identifiers of six other proteins, and the theoretical predictions are in good agreement with the experimental measurements. These results potentiated the validity of this method for the conformational identification of proteins in nanopores.

Mechanical Point Loading Induces Cortex Stiffening and Actin Reorganization.

Global cytoskeleton reorganization is well-recognized when cells are exposed to distinct mechanical stimuli, but the localized responses at a specified region of a cell are still unclear. In this work, we mapped the cell-surface mechanical property of single cells in situ before and after static point loading these cells using atomic force microscopy in PeakForce-Quantitative Nano Mechanics mode. Cell-surface stiffness was elevated at a maximum of 1.35-fold at the vicinity of loading site, indicating an enhanced structural protection of the cortex to the cell. Mechanical modeling also elucidated the structural protection from the stiffened cell cortex, in which 9-15% and 10-19% decrease of maximum stress and strain of the nucleus were obtained. Furthermore, the flat-ended atomic force microscopy probes were used to capture cytoskeleton reorganization after point loading quantitatively, revealing that the larger the applied force and the longer the loading time are, the more pronounced cytoskeleton reorganization is. Also, point loading using a microneedle combined with real-time confocal microscopy uncovered the fast dynamics of actin cytoskeleton reorganization for actin-stained live cells after point loading (<10 s). These results furthered the understandings in the transmission of localized mechanical forces into an adherent cell.

Impact of real-time shedding on binding kinetics of membrane-remaining L-selectin to PSGL-1.

L-selectin shedding induced by various cytokines is crucial in activating neutrophils (PMNs) in inflammatory cascade. While the real-time shedding in vivo lasts ~10 min after PMN activation, the impact of time-dependent shedding on binding kinetics of membrane-remaining L-selectins to its ligands is poorly understood at transient or steady state. Here, we developed an in vitro L-selectin shedding dynamics approach, together with competitive assays of cell adhesion, and proposed a theoretical model for quantifying the impact of real-time shedding on the binding kinetics of membrane-remaining L-selectins to P-selectin glycoprotein ligand-1 (PSGL-1). Our data indicated that the extent of L-selectin shedding on PMA activation is higher, but the terminating time is longer for Jurkat cells than those for human PMNs. Meanwhile, fMLF or IL-8 stimulation yields the longer terminating time than that on PMA stimulation but results in a similar shedding extent for PMNs. L-selectin shedding reduces L-selectin-PSGL-1-mediated cell adhesion in three ways: decreasing membrane-anchored L-selectins, increasing soluble L-selectins competitively binding to ligands, and presenting conformational alteration of membrane-remaining L-selectins themselves. Compared with those on intact cells, the binding affinities of membrane-remaining L-selectin-PSGL-1 pairs were all enhanced at initial and lowered at the late shedding phase for both PMN and Jurkat cells even with varied transition time points. The rolling velocities of both PMNs and Jurkat cells were increased following mechanically or biochemically induced shedding of L-selectin under shear flow. These findings help to further our understanding of the function of time-dependent L-selectin shedding during the inflammation cascade.

Salt bridge interactions within the ß2 integrin α7 helix mediate force-induced binding and shear resistance ability.

The functional performance of the αI domain α7 helix in ß2 integrin activation depends on the allostery of the α7 helix, which axially slides down; therefore, it is critical to elucidate what factors regulate the allostery. In this study, we determined that there were two conservative salt bridge interaction pairs that constrain both the upper and bottom ends of the α7 helix. Molecular dynamics (MD) simulations for three ß2 integrin members, lymphocyte function-associated antigen-1 (LFA-1; αL ß2 ), macrophage-1 antigen (Mac-1; αM ß2 ) and αx ß2 , indicated that the magnitude of the salt bridge interaction is related to the stability of the αI domain and the strength of the corresponding force-induced allostery. The disruption of the salt bridge interaction, especially with double mutations in both salt bridges, significantly reduced the force-induced allostery time for all three members. The effects of salt bridge interactions of the αI domain α7 helix on ß2 integrin conformational stability and allostery were experimentally validated using Mac-1 constructs. The results demonstrated that salt bridge mutations did not alter the conformational state of Mac-1, but they did increase the force-induced ligand binding and shear resistance ability, which was consistent with MD simulations. This study offers new insight into the importance of salt bridge interaction constraints of the αI domain α7 helix and external force for ß2 integrin function.

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.