Edmondson K E, Denney W S, Diamond S L
Institute for Medicine and Engineering, Department of Bioengineering, University of Pennsylvania, 3320 Smith Walk, Philadelphia, PA 19104, USA.
Biophys J. 2005 Nov;89(5):3603-14. doi: 10.1529/biophysj.105.066134. Epub 2005 Aug 12.
Visualization of flowing neutrophils colliding with adherent 1-mum-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (epsilon), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s(-1), epsilon decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L(tether)(m), increased from 0.35 mum to approximately 2.0 mum over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress off-rate, k(off)(0) = 0.56 s(-1) and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (+/- tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s(-1), pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on epsilon, but increased L(tether)(m) by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-beta-cyclodextrin or formaldehyde fixation had no effect on epsilon, but reduced L(tether)(m) by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.
可视化流动的中性粒细胞与呈现P-选择素的直径1μm的粘附珠碰撞，使得能够同时测量碰撞效率（ε）、膜系留分数（f）、膜系留生长动力学以及PSGL-1/P-选择素结合寿命。对于在25至200 s⁻¹的静脉壁剪切速率下分析的1391次碰撞，ε从0.17降至0.004，而f从0.15增至0.70，并且在此剪切范围内，平均投影膜系留长度L(tether)(m)从0.35μm增加至约2.0μm。在所有测试的剪切速率下，缺乏膜系留的粘附碰撞的平均键寿命短于有系留碰撞所观察到的寿命。对于未能形成膜系留的粘附碰撞，回归的贝尔参数（与单键蒙特卡罗模拟一致）为零应力解离速率k(off)(0)=0.56 s⁻¹和反应性柔顺性r = 0.10 nm，类似于已发表的原子力显微镜（AFM）测量结果。对于所有粘附事件（有或无系留），键寿命分布更类似于通过滚动测定获得的分布，并且用上述贝尔参数和平均1.48个键（n = 1个键（67%），n = 2个键（22%），以及n = 3 - 5个键（11%））的蒙特卡罗模拟能得到最佳拟合。对于100 s⁻¹的碰撞，用肌动蛋白解聚剂拉特罗毒素或细胞松弛素D预处理中性粒细胞对ε没有影响，但使L(tether)(m)分别增加1.74倍或2.65倍，并使平均系留寿命分别延长1.41倍或1.65倍。已知会导致泡状形成的肌动蛋白聚合剂茉莉酮酸酯产生的结果与解聚剂类似。相反，用甲基-β-环糊精进行胆固醇耗竭或甲醛固定对ε没有影响，但分别使L(tether)(m)降低66%或97%，并使平均系留寿命降低30%或42%。中性粒细胞-珠碰撞测定结合了原子力显微镜（小接触区）、凝集测定（离散相互作用）、微量移液器操作（系留可视化）和滚动测定（生理流动加载）的优点。膜系留生长可以通过药理学方法增强或降低，从而对PSGL-1/P-选择素寿命产生影响。
