Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime.
Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime.
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DOI：
10.1529/biophysj.105.066134
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发表时间：
2005-11
影响因子：
3.4
通讯作者：
K. Edmondson;W. S. Denney;S. Diamond
中科院分区：
文献类型：
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作者：
K. Edmondson;W. S. Denney;S. Diamond
流动的中性粒细胞与粘附的1 μ m直径的呈现P-选择素的珠碰撞的可视化允许同时测量碰撞效率（μ m）、膜束缚分数（f）、膜束缚生长动力学和PSGL-1/P-选择素结合寿命。对于1391碰撞分析静脉壁剪切速率从25到200 s（-1），从0.17减少到0.004，而f增加从0.15到0.70，和平均预计膜系绳长度，L（系绳）（m），增加从0.35 μ m到约2.0 μ m在这个剪切范围。在所有的剪切速率测试，粘合剂碰撞缺乏膜系链的平均粘结寿命小于观察到的碰撞与系链。对于未能形成膜系链的粘合剂碰撞，回归的Bell参数（与单键Monte Carlo模拟一致）为零应力解离速率，k（off）（0）= 0.56 s（-1）和反应顺应性，r = 0.10 nm，类似于已发表的原子力显微镜（AFM）测量。对于所有粘附事件（+/-系链），键寿命分布更类似于通过滚动测定获得的那些，并且最好通过Monte Carlo模拟，具有上述Bell参数和平均1.48个键（n = 1个键（67%），n = 2（22%）和n = 3-5（11%））。对于100 s（-1）的碰撞，用肌动蛋白解聚剂、latrunculin或细胞松弛素D预处理中性粒细胞，对细胞分裂没有影响，但L（系链）（m）分别增加了1.74或2.65倍，平均系链寿命分别延长了1.41或1.65倍。已知引起起泡的肌动蛋白聚合剂Jasplakinastatin产生了与解聚剂相似的结果。相反地，用甲基-β-环糊精或甲醛固定的胆固醇消耗对胆固醇没有影响，但L（系链）（m）分别减少了66%或97%，平均系链寿命分别减少了30%或42%。亲和珠碰撞试验结合了原子力显微镜（小接触区）、聚集测定法（离散相互作用）、微量移液管操作（系链可视化）和滚动试验（生理流量加载）的优点。膜系链生长可以增强或减少，从而影响PSGL-1/P-选择素的寿命。
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.