Objective To study how lipid bilayer fluidity modulates the interaction between β1 integrin and CD40L,as well as the formation of CD40L-mediated tumor cell contact interfaces.Methods Supported lipid bilayers(SLB)with different fluidities were prepared through adjusting the 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-l-carboxypentyl)iminodiacetic acid]succinyl nickel salt(DGS-NTA)content.The functionalization of lipid bilayers was achieved by anchoring fluorescently labeled CD40L molecules onto the membrane surface.The contact interface formation of PC9 cells on the functionalized lipid bilayers was observed through confocal fluorescence imaging and fluorescence recovery after photobleaching(FRAP)experiments,and data of two dimensional(2D)reaction kinetics of β1 integrin and CD40L were extracted from Zhu-Golan plots.Results The diffusion coefficient of molecules in lipid bilayer was negatively correlated with DGS-NTA content.High fluidity of lipid bilayer promoted CD40L accumulation at cell contact interface and expanded the cell contact area.The 2D dissociation constants(2D Kd)of β1 integrin-CD40L complexes were approximately 13,31 and 65 molecules/μm2 for the three lipid bilayers with high,moderate and low fluidities,respectively.Conclusions High fluidity of lipid bilayers significantly facilitates diffusion and aggregation of CD40L to the cell contact interface,thus enhancing β1 integrin-CD40L interaction and the stability of cell contact interfaces.

Objective To investigate the conformational states of ADAMTS13 under different pH conditions.Methods Atomic force microscopy(AFM)was applied to pull ADAMTS13 molecules with two distinct pulling systems:the Biotin-Streptavidin system and the 6×His-Anti-His antibody system.The rupture forces and molecular contour lengths were analyzed.Results Under pH 7.4 condition,when ADAMTS13 was pulled by Biotin-Streptavidin system,the mean molecular contour length was(30.93±1.56)nm,exhibiting a bimodal frequency distribution with peak positions at(22.12±0.01)and(49.57±0.05)nm.When ADAMTS13 was pulled using 6×Hist-anti-His antibody system,the mean molecular contour length was(32.77±0.72)nm,also showing a bimodal distribution,with a peak position at(25.73±0.16)and(43.84±0.63)nm,respectively.Under pH 6.0 condition,when ADAMTS13 was pulled by Biotin-Streptavidin system,the mean molecular contour length increased to(47.07±1.6)nm,and the frequency distribution shifted to trimodal,with peak positions at(22±1.25),(55.09±2.62)and(76.69±3.06)nm.The conformation of ADAMTS13 was more extended at pH 6.0 compared with that at pH 7.4.Conclusions ADAMTS13 exists in'closed'and intermediate conformational states at physiological pH 7.4.However,at pH 6.0,ADAMTS13 can adopt'closed',intermediate,and'open'conformational states.This study contributes to a further understanding of the role of ADAMTS13 in normal physiology and thrombotic thrombocytopenic purpura,providing insights for the development of novel recombination ADAMTS13 drugs.

Objective To study how lipid bilayer fluidity modulates the interaction between β1 integrin and CD40L,as well as the formation of CD40L-mediated tumor cell contact interfaces.Methods Supported lipid bilayers(SLB)with different fluidities were prepared through adjusting the 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-l-carboxypentyl)iminodiacetic acid]succinyl nickel salt(DGS-NTA)content.The functionalization of lipid bilayers was achieved by anchoring fluorescently labeled CD40L molecules onto the membrane surface.The contact interface formation of PC9 cells on the functionalized lipid bilayers was observed through confocal fluorescence imaging and fluorescence recovery after photobleaching(FRAP)experiments,and data of two dimensional(2D)reaction kinetics of β1 integrin and CD40L were extracted from Zhu-Golan plots.Results The diffusion coefficient of molecules in lipid bilayer was negatively correlated with DGS-NTA content.High fluidity of lipid bilayer promoted CD40L accumulation at cell contact interface and expanded the cell contact area.The 2D dissociation constants(2D Kd)of β1 integrin-CD40L complexes were approximately 13,31 and 65 molecules/μm2 for the three lipid bilayers with high,moderate and low fluidities,respectively.Conclusions High fluidity of lipid bilayers significantly facilitates diffusion and aggregation of CD40L to the cell contact interface,thus enhancing β1 integrin-CD40L interaction and the stability of cell contact interfaces.

Objective To investigate the conformational states of ADAMTS13 under different pH conditions.Methods Atomic force microscopy(AFM)was applied to pull ADAMTS13 molecules with two distinct pulling systems:the Biotin-Streptavidin system and the 6×His-Anti-His antibody system.The rupture forces and molecular contour lengths were analyzed.Results Under pH 7.4 condition,when ADAMTS13 was pulled by Biotin-Streptavidin system,the mean molecular contour length was(30.93±1.56)nm,exhibiting a bimodal frequency distribution with peak positions at(22.12±0.01)and(49.57±0.05)nm.When ADAMTS13 was pulled using 6×Hist-anti-His antibody system,the mean molecular contour length was(32.77±0.72)nm,also showing a bimodal distribution,with a peak position at(25.73±0.16)and(43.84±0.63)nm,respectively.Under pH 6.0 condition,when ADAMTS13 was pulled by Biotin-Streptavidin system,the mean molecular contour length increased to(47.07±1.6)nm,and the frequency distribution shifted to trimodal,with peak positions at(22±1.25),(55.09±2.62)and(76.69±3.06)nm.The conformation of ADAMTS13 was more extended at pH 6.0 compared with that at pH 7.4.Conclusions ADAMTS13 exists in'closed'and intermediate conformational states at physiological pH 7.4.However,at pH 6.0,ADAMTS13 can adopt'closed',intermediate,and'open'conformational states.This study contributes to a further understanding of the role of ADAMTS13 in normal physiology and thrombotic thrombocytopenic purpura,providing insights for the development of novel recombination ADAMTS13 drugs.

Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.
Humans ; Extracellular Traps ; Tetradecanoylphorbol Acetate/pharmacology* ; Neutrophils ; HL-60 Cells ; Tretinoin/pharmacology*

Neutrophil extracellular traps (NETs) play an important role in the formation of immunothrombosis. However, how vascular endothelial cells mediate the formation of NETs has not been fully understood. We stimulated neutrophils firmly attached on the endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) with lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA) for 4 h, then labeled NETs-DNA with Sytox green dye and the formation of NETs was observed by fluorescent microscopy. The area and fluorescence intensity of NETs-DNA were analyzed to quantify the formation of NETs. The results showed that both PMA and LPS were able to induce firmly adhered neutrophils on ICAM-1 to produce NETs. NETs induced by PMA were independent of neither β2 integrin lymphocyte function-associated antigen-1 (LFA-1) nor macrophage antigen complex-1 (Mac-1). In contrast, LPS-stimulated NETs were mediated by Mac-1 integrin, but not by LFA-1. After inhibition of actin filaments or Talin-1, the formation of NETs irrespective of the stimulus was significantly reduced. This study reveals the mechanism of the direct interaction between neutrophils and endothelial cells to produce NETs under inflammatory conditions, providing a new theoretical basis for the treatment of related diseases and the development of new drugs.
Cytoskeletal Proteins ; Endothelial Cells ; Extracellular Traps ; Integrins ; Intercellular Adhesion Molecule-1 ; Lipopolysaccharides/pharmacology* ; Macrophages ; Neutrophils

P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM), the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellular calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase (Syk). These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectin-induced calcium signaling of neutrophils in flow.
Calcium Signaling ; Female ; Humans ; Male ; Membrane Glycoproteins ; metabolism ; Neutrophils ; metabolism ; P-Selectin ; metabolism ; Stress, Mechanical ; Syk Kinase ; metabolism