![]() ![]() ![]() These insights were gained, at least in part, through biophysical methods, including fluorescence recovery after photobleaching (FRAP) ( Jacobson et al., 1995 Lippincott-Schwartz et al., 2001). They may be retarded in their lateral diffusion or completely immobilized through interactions with cytoskeletal elements, with extracellular matrix proteins, or by becoming a part of large oligomeric arrays. Since then, it was found that many membrane proteins are not completely free to diffuse in the plane of the lipid bilayer. The fluid mosaic membrane model that was proposed about 30 years ago emphasized the free mobility of membrane proteins in the plane of the membrane ( Singer and Nicolson, 1972). It appears, therefore, that interaction of microtubules with the ER results in the immobilization of translocon complexes that are part of membrane-bound polysomes, and may play a role in the mechanism that segregates the rough and smooth domains of the ER. The most striking increase in the diffusion rate of the translocon complexes was observed in M3/18 cells transfected with a siRNA that effectively knocked down the expression of the endogenous CLIMP-63. Also, the expression of a CLIMP-63 mutant lacking the microtubule-binding domain resulted in a significant increase of the lateral mobility of the translocon complexes. As determined by fluorescence recovery after photobleaching, the breakdown of microtubules caused by drug treatment or by overexpression of the microtubule-severing protein spastin, resulted in an increased lateral mobility of the translocons that are assembled into polysomes. It was, therefore, of interest to investigate whether these microtubules are in fact responsible for the highly restricted lateral mobility of the translocon complexes in M3/18 cells as described before. Microtubules are frequently seen in close proximity to membranes of the endoplasmic reticulum (ER), and the membrane protein CLIMP-63 is thought to mediate specific interaction between these two structures. ![]()
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