Colocalization

A method of 'differential redistribution' of membrane components into patches and caps has been broadly used to probe the possible interactions between various plasma membrane components, or between the cell membrane and cytoplasmic structures. If two components A and B were stably associated in the membrane, the clustering and capping of A should always copatch and cocap B. If on the contrary, A and B were never associated, the clustering of one should not affect the distribution of the other. Obviously, since mlg epitopes and major histocompatibility complex (MHC) epitopes were not cocapped, it could be concluded that these were different and independent molecules. Similarly the myosin involvement in the contractile processing and removal of the aggregates from the cell surface was supported by it being found concentrated in the cytoplasmic cortex of the cell just beneath patches of membrane components aggregated by an external ligand.

A number of membrane and cytosolic components were studied in this way and showed 'colocalization'. This led to the discovery of a host of membrane skeletal proteins which may create complex networks of interacting components on and between both faces of the membrane. Similarly, interactions of antigen receptor complexes with proximally recruited CD membrane components in the plasma membrane plane eventually lead to three-dimensional scaffolds of cytosolic protein tyrosine kinases and phosphatases and adaptor proteins as transducer units.

Such differential redistribution studies have a variety of potential pitfalls. Indeed, besides the two aforementioned, simple cases, there were a variety of alternate forms of incomplete and/or unidirectional interactions, three of which are suggested hereafter. 1) Although A and B are molecularly independent, ligand binding might cause conformational changes in A, which then allows binding of A to B, so that capping of A may lead to cocapping of B, whereas the interaction of B with its ligand would not bring A in the cap ('syn-capping'). 2) If there is a large excess of one membrane component over another, a massive aggregation and capping of the major component might entrap the minor one and bring it into the cap, even though the two types of components never showed any affinity for each other during this 'mass effect'. 3) Ligand binding might also trigger a complement-like 'cascade reaction' on the cell surface which may involve several membrane components, both integral proteins and peripheral membrane components in the glycocalix and in the cytosolic membrane skeleton.

Although such colocalization studies may show that a variety of integral and peripheral membrane components do easily interact with each other, they may not always indicate whether there was prior co-localization or whether there has been recruitment. They say little about the natural status of distri bution of such components in a 'resting' cell. To evaluate membrane component interactions, e.g. to determine whether various membrane components constitute homodimers, heteropolymers or large complexes of several different molecules, instantaneous ('pulse') nearest-neighbor-type biochemical studies of the membrane of cells, both resting and in different activated status, are more suitable than patching and capping.

In addition to these restrictions, the possible isomeric forms of some membrane proteins should also be taken into account; the most representative cases are those of proteins which may exist as two widely different membrane forms, either with a classical transmembrane polypeptide anchor ('TM proteins') or with a glycosylphosphatidylinositol anchor (GPI proteins'), and which may show different physico-chemical and/or physiological relationships with other molecular partners in cis on the membrane. Moreover, the hypothesis has been formulated that not only ligand-clustered components but rather whole microdomains of plasma membrane (including unliganded components) might be brought into the cap. This concept puts severe restrictions on the use of capping as a means of selective removal of given membrane components from the plasma membrane.

Finally, it is unclear whether any membrane component may be capped by clustering ligands, and there is also no evidence that all types of membrane component redistributions reported as 'caps' actually proceed through a unique mechanism. Even though the caps were made on the same cell pole and mediated by actin filaments, capping may involve anchorage through integrins for some membrane components, but through cadherins for some others, whereas some 'caps' may actually represent a polar redistribution of microvilli, or a passive recruitment of all clusters into a single one. If whole membrane microdomains rather than individual components show polar redistribution, cocapping may simply reflect the fact that the capped components belong to the same type of domain, whereas independent capping would show they are in different domains, and further conclusions on physical interactions of components with each other are probably speculative.

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