Berk D, Evans E
Department of Pathology, University of British Columbia, Vancouver, Canada.
Biophys J. 1991 Apr;59(4):861-72. doi: 10.1016/S0006-3495(91)82298-6.
An experimental method and analysis are introduced which provide direct quantitation of the strength of adhesive contact for large agglutinin-bonded regions between macroscopically smooth membrane capsules (e.g., red blood cells). The approach yields intrinsic properties for separation of adherent regions independent of mechanical deformation of the membrane capsules during detachment. Conceptually, the micromechanical method involves one rigid test-capsule surface (in the form of a perfect sphere) held fixed by a micropipette and a second deformable capsule maneuvered with another micropipette to force contact with the test capsule. Only the test capsule is bound with agglutinin so that the maximum number of cross-bridges can be formed without steric interference. Following formation of a large adhesion region by mechanical impingement, the deformable capsule is detached from the rigid capsule surface by progressive aspiration into the micropipette. For the particular case modeled here, the deformable capsule is assumed to be a red blood cell which is preswollen by slight osmotic hydration before the test. The caliber of the detachment pipette is chosen so that the capsule will form a smooth cylindrical "piston" inside the pipette as it is aspirated. Because of the high flexibility of the membrane, the capsule naturally seals against the tube wall by pressurization even though it does not adhere to the glass. This arrangement maintains perfect axial symmetry and prevents the membrane from folding or buckling. Hence, it is possible to rigorously analyze the mechanics of deformation of the cell body to obtain the crucial "transducer" relation between pipette suction force and the membrane tension applied directly at the perimeter of the adhesive contact. Further, the geometry of the cell throughout the detachment process is predicted which provides accurate specification of the contact angle theta c between surfaces at the perimeter of the contact. A full analysis of red cell capsules during detachment has been carried out; however, it is shown that the shear rigidity of the red cell membrane can often be neglected so that the red cell can be treated as if it were an under filled lipid bilayer vesicle. From the analysis, the mechanical leverage factor (1-cos theta c) and the membrane tension at the contact perimeter are determined to provide a complete description of the local mechanics of membrane separation as functions of large-scale experimental variables (e.g., suction force, contact diameter, overall cell length). In a companion paper (Evans, E., D. Berk, A. Leung, and N. Mohandas. 1990. Biophys. J. 59:849-860), this approach was applied to the study of separation of large regions of adhesive contact formed between red blood cells by monoclonal antibodies and lectins.
本文介绍了一种实验方法及分析，可直接定量宏观光滑膜囊泡（如红细胞）之间大凝集素结合区域的黏附接触强度。该方法能得出黏附区域分离的内在特性，不受膜囊泡在分离过程中机械变形的影响。从概念上讲，这种微机械方法包括用微量移液器固定一个刚性测试囊泡表面（呈完美球体形式），并用另一个微量移液器操纵第二个可变形囊泡，迫使其与测试囊泡接触。仅测试囊泡与凝集素结合，这样就能在无空间位阻干扰的情况下形成最大数量的交联桥。通过机械碰撞形成大黏附区域后，通过向微量移液器中逐步抽吸，将可变形囊泡从刚性囊泡表面分离。对于此处模拟的特定情况，假设可变形囊泡为红细胞，在测试前通过轻微渗透水合作用使其预肿胀。选择分离移液器的管径，使囊泡在被抽吸时能在移液器内形成光滑的圆柱形“活塞”。由于膜的高柔韧性，囊泡即使不粘附在玻璃上，也会通过加压自然地与管壁密封。这种布置保持了完美的轴对称性，防止膜折叠或弯曲。因此，有可能严格分析细胞体变形的力学，以获得微量移液器吸力与直接施加在黏附接触周边的膜张力之间的关键“换能器”关系。此外，预测了整个分离过程中细胞的几何形状，这为接触周边表面之间的接触角θc提供了准确的规格。已经对红细胞囊泡在分离过程中进行了全面分析；然而，结果表明红细胞膜的剪切刚度通常可以忽略不计，因此红细胞可以被视为一个未充满的脂质双层囊泡。通过分析，确定了机械杠杆因子（1 - cosθc）和接触周边的膜张力，以完整描述膜分离的局部力学作为大规模实验变量（如吸力、接触直径、细胞总长度）的函数。在一篇配套论文（Evans, E., D. Berk, A. Leung, and N. Mohandas. 1990. Biophys. J. 59:849 - 860）中，该方法被应用于研究红细胞之间由单克隆抗体和凝集素形成的大黏附接触区域的分离。
