Whereas the Frye-Edidin experiment proved the lateral redistribution of membrane proteins on the micrometer scale , due to technical limitations, it could not resolve whether intermixing of proteins results from the movement of large-scale clusters with constant protein composition or instead, proteins could be exchanged between clusters at the molecular level by dynamic association-dissociation events (dynamically changing composition). In order to address this question, the classical experiment was repeated with some modifications . Intermixing of membrane proteins was studied on homokaryons of human lymphoblasts where, before fusion, the two cells were labeled separately with antibodies (or their Fab fragments) carrying spectrally distinct fluorophores targeting the proteins of interest. The process of intermixing was monitored by simultaneous application of photobleaching FRET and SNOM techniques, which provided a resolution power well beyond that of the original experiment and allowed us to explore the dynamic behavior of both hierarchical levels of protein clusters (see Section 7.2).
As noted in Section 7.3, clustering of MHC glycoproteins is well-characterized and has an important role in antigen presentation [59,60,83,84,102]. Both small-and large-scale clusters of MHC I were found to be dynamic: dissociation and reassociation of small-scale protein complexes and reformation of large-scale associations took place after the fusion of cells that were labeled with fluorescein- and rhodamine-conjugated Fab fragments against MHC I heavy chains. The redistribu tion of micrometer-scale clusters preceded that of the small-scale clusters, corroborating the hierarchical organization of MHC I .
Large-scale homoclusters of MHC II were as dynamic as those of MHC I. Small-and large-scale heteroclusters of MHC I and MHC II also showed dynamic behavior in cell fusion experiments. At the same time, small-scale associations of MHC II were static as revealed by FRET: intermixing of components did not take place between nanometer-scale clusters of MHC II even 80 minutes after fusion . This may be explained by the molecular structure of MHC II which, in its functional dimer form, has two membrane-spanning a-helical transmembrane domains. It can be hypothesized, that hydrophobic interactions between these domains are strong enough to prevent the mixing of small-scale clusters of MHC II .
One may picture this situation as overlapping large-scale clusters of MHC I and MHC II containing dynamic small-scale homoclusters of MHC I and static small-scale clusters of MHC II. Although the small-scale homoclusters of MHC II are not dynamic, they engage in dynamic small-scale association with MHC I .
There was no difference in the behavior of transferrin receptors and the GPI-anchored CD48 protein, representatives of nonraft- and raft-associated proteins, respectively. Therefore, it could be concluded that the lipid microenvironment, on its own, does not determine the dynamic properties of protein associations. Forces resulting in protein clustering, whether they are related to membrane trafficking or to microdomain formation, do not generally inhibit the dynamic exchange of protein components between protein clusters, if the clusters reside in the same type of microdomain .
As a conclusion, one can say that in the composition of both small- and large-scale membrane protein clusters dynamism is the rule, rather than the exception. Whereas preformed large-scale homoclusters of proteins are generally not associated in a static manner, and exchange of components between like clusters is usually observed, tight interactions in the small-scale associations of some proteins could completely block the exchange of these molecules between nanometer-sized clusters .
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