Implication of lipid microdomains in regulation of protein trafficking and epithelial cell morphology

The discovery of the lipid microdomains has provided a new insight to our understanding of cellular membranes and introduced us to a whole new dimension full of answers that can elicit many cellular processes from signalling to protein sorting and infection. The retrieval of these answers requires isolation and biochemical analyses of these microdomains and their components. To date, a variety of non-ionic detergents like Triton X-100, Lubrol WX, and Tween-20 have been successfully used to isolate such microdomains from in biological samples. Growing number of studies employing model proteins and several detergents in different cellular systems have conveyed a great deal of information about the functional characteristics of lipid microdomains. Nevertheless, further information is necessary to produce concrete explanations for the scope of the functions of lipid microdomains in vivo. This study has aimed to elucidate the implications of lipid microdomains in epithelial cells, especially in early protein sorting events and cell differentiation. Employing detergent insolubility as a discrimination method in Madin Darby Canine Kidney (MDCK) cells, it was revealed that GPI-anchor acts as a sorting determinant for a protein’s inclusion into Tween-20 resistant microdomains (Tween-20-DRMs). This interaction between the GPI-anchored protein membrane dipeptidase (MDP) and DRMs was found to be responsible for the rapid and efficient exit of MDP from the endoplasmic reticulum (ER). The removal of the GPI-anchor resulted in the exclusion of MDP from DRMs and an accumulation in the ER. The rate of Golgi to cell surface transport was similar for wild type and anchorless mutant and this has led to the conclusion that ER-exit was the bottleneck in the anterograde transport of MDP and possibly for other DRM-associated proteins. These finding support the theory that proteins are selectively packed in different carriers in the ER and lipid microdomains in the ER membranes contribute to the diversity of these specialized carriers and assure further transport of competent proteins. Such diversity is also present among trans-Golgi network (TGN)-derived vesicles. Detergent insolubility is a determinant for a subgroup of such vesicles that exploit actin based cytoskeletal tracts to reach apical cell membrane. Annexin II is a component of this vesicle species and it has been shown to be essential for the arrival of the contents of these carriers at the apical membrane. Annexin II is a lipid binding protein, which has been shown to interact with lipids from membrane microdomains (cholesterol, PI(4,5)P2). It also can bind F-actin at membrane sites rich in cholesterol and sphingolipids and thereby participate dynamic membrane events such as vesicle fusion and fission. The mechanism how annexin II modulates membrane microdomains and its contribution to processes involving DRMs were investigated in intestinal cell model Caco-2. This was accomplished by stable downregulation of annexin II by RNAi. Caco-2-A4 clone was found to express only 10-15% of normal annexin II levels in wild type cells. Analyses revealed a decrease in expression of several intestinal hydrolases (sucrase isomaltase, angiotensin converting enzyme 2) and microvillus associated protein ezrin as well. Clone Caco-2-A4 also failed to display an increasing pattern of apical hydrolase levels with time. Morphological examination of ultrastructural features of the cells has supported these findings by reduced number and length of microvilli in Caco-2-A4. Additional to the impaired apical transport of brush border enzymes, it was discovered that apical distribution of cytokeratin 19 (K19) was severely disrupted in Caco-2-A4. This could be explained by the appearance of a non-phosphorylated pool of K19, which probably results in depolymerization and subsequent displacement of K19 from the apical cytoskeleton in the clone A4. I suggest that regulation of K19 by annexin II is one of the initial events taking place in cell differentiation. Annexin II is responsible for the apical localization of K19 and this supports the apical cytoskeletal scaffold on which the microvilli are formed.