The mechanism of concanavalin A cap formation in leukocytes.

The process of Concanavalin A (Con A) cap formation on human blood polymorphonuclear leukocytes, monocytes and lymphocytes and rabbit alveolar macrophages has been studied by correlative use of light, fluorescence and electron microscopy. The most important precondition for Con A capping on these cells is the disassembly of cytoplasmic microtubules by colchicine or the glutathione-oxidizing agent, 'diamide'. Incubation of microtubule-depleted leukocytes with fluorescein-conjugated Con A (F-Con A) leads to the aggregation of lectin into a cap which usually occupies a protuberance at one pole of the cell. F-Con A can also be concentrated at a constriction in the cell body. The protuberance is shown to consist of highly plicated membrane subtended by a network of densely packed microfilaments. Additional microfUaments originate from this network and course into individual plications of the protuberance. However, the formation of the protuberance with its organized structure follows the disassembly of microtubules alone and does not require Con A. Thus when cells are treated with colchicine or diamide, then fixed and labelled with F-Con A the typical changes in cell shape that are associated with capping are observed but lectin is distributed homogeneously over the cell surface. Similarly if cells are first capped with low concentrations of unlabelled lectin, then fixed and incubated with F-Con A, fluorescence is again uniformly distributed over the whole membrane. This indicates that membrane Con A receptors have not been concentrated over the protuberance despite the prior aggregation of microfilaments. By contrast, when precapped cells are labelled with F-Con A before fixation, fluorescence is concentrated through the previously established cap. Thus extensive organization of microfilaments and unlabelled lectin does not inhibit the movement of F-Con A-receptor complexes on unfixed cells. Further, the Con A cap is sufficiently fluid to permit mixing of sequentially formed Con A-receptor complexes. Although the aggregation of microfUaments into a protuberance and the concentration of Con A into the membrane of the protuberance are clearly separable events, microfilaments and Con A-receptor complexes are ultimately found in close association in the cap. This association appears to stabilize the localization of both the surface-bound lectin and the submembranous network of microfilaments. Such stabilization could result from physical interactions between microfilaments and Con A within the protuberance. However, we favour an alternative mechanism in which a region of low membrane fluidity that limits further diffusion is established following microtubule disassembly and is preserved by microfilament-membrane interactions.