Membrana del tràfic i l'anexina A6 grup

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Annexin A6 in Membrane Trafficking and Organization

Summary

AnxA6 is a highly dynamic protein involved in a number of cellular events linked to membrane transport such as the cholesterol delivery from late endosomes to the Golgi and the plasma membrane, the cholesterol-dependent recruitment of cPLA2 to the Golgi and its involvement in Golgi vesiculation, and the regulation of SNARE proteins. In this research line we study the molecular and cellular mechanisms that AnxA6 regulates in all these processes.

Description

In recent years, the involvement of annexins in the regulation of membrane traffic has emerged as one of their predominant functions. Annexin A6 (AnxA6) belongs to a family of Ca2+-dependent membrane-binding proteins and is involved in the regulation of endocytic and exocytic pathways. 

 

We previously demonstrated that AnxA6, at the plasma membrane, regulates receptor mediated endocytosis and lysosomal targeting of low-density lipoproteins (LDL) and translocates to cholesterol-enriched late endosomes (LE). Accumulation of LDL-cholesterol is accompanied by the translocation of AnxA6 into pre-lysosomal vesicles, indicating that AnxA6 also participates in the transport of cholesterol across lysosomal membranes.

 

Studies aiming to identify the mechanism how AnxA6 regulates lysosomal targeting and the intracellular transport of cholesterol are relevant for lipid disorders like atherosclerosis and Niemann Pick disease (NPC1).

 

As cholesterol modulates the membrane binding and the cellular location of AnxA6, but also affects the intracellular distribution of caveolin, we investigate the localization and trafficking of caveolin in AnxA6-expressing cells.

 

We have demonstrated that cells expressing high levels of AnxA6 are characterized by an accumulation of caveolin-1 (cav-1) in the Golgi complex. This is associated with a sequestration of cholesterol in the LE and lower levels of cholesterol in the Golgi and the plasma membrane, both likely contributing to retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. Further strengthening these findings, knock down of AnxA6 and the ectopic expression of the Niemann–Pick C1 protein in AnxA6-overexpressing cells restore the cellular distribution of cav-1 and cholesterol, respectively. 

 

Therefore, elevated expression levels of AnxA6 perturb the intracellular distribution of cholesterol, which indirectly inhibits the exit of caveolin from the Golgi complex.

 

On the other hand, the molecular mechanisms regulating the exit of caveolin from the Golgi complex are not fully understood. Cholesterol and sphingolipid availability affects Golgi vesiculation events and involves the activity of cytoplasmic phospholipase A2 (cPLA2).

 

In a recent study we also showed that in Chinese hamster ovary cells overexpressing AnxA6, sequestration of cholesterol in late endosomes, leading to reduced amounts of cholesterol in the Golgi, inhibits cPLA2 activity and its association with the Golgi complex. This correlates with the blockage of caveolin export from the Golgi in cells treated with methyl arachidonyl-fluorophosphonate, a Ca2+-dependent cPLA2 inhibitor. AnxA6-mediated down-regulation of cPLA2 activity was overcome upon the addition of exogenous cholesterol or transfection with small interfering RNA targeting AnxA6. These findings indicate that AnxA6 interferes with caveolin transport through the inhibition of cPLA2.

 

Annexin A6 has been identified in cholesterol-rich lipid rafts/caveolae. Here AnxA6 participates, together with other annexins, in the stabilization of lipid raft membrane domains to regulate the formation and activity of signalling complexes. In our current approach we are studying the effects of Ca2+-induced translocation of AnxA6 into lipid rafts on its lipid and protein composition.

Figure 1
Figure 1. Immunofluorescence staining of Lamp1 (red) and Filipin (blue) of U18666A-treated CHO cells transfected with Rab9-GFP (green)
Figure 2
Figure 2. Immunofluorescence staining of Annexin A6 (red), Filipin (green) and actin cytoskeleton (blue) of U18666A-treated NRK cells
Figure 3
Figure 3. Immunofluorescence staining of Annexin A6 (green), Filipin (red) and actin cytoskeleton (blue) of human skin fibroblats
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