The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells.

Abstract

Abstract. Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of MadinDarby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were -5 × l0 -1° cm2/s for the (Na+,K+)ATPase and ~2 × 10 -9 cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.

Exclusion of a Proton ATPase from the Apical Membrane Is Associated with Cell Polarity and Tip Growth inNicotiana tabacumPollen Tubes

“…These proteins were seen to move directionally toward the tip with a mobility of 0.2 to 0.5 mm/min (0.003 to 0.008 mm/s). These values are 100-fold lower than the ones reported for other membrane proteins known to freely diffuse in the plasma membrane, such as in the case of another P-type ATPase, an Na þ ,K þ -ATPase, in low-density MDCK cells (Jesaitis and Yguerabide, 1986) and E-cadherin in epithelial cells before adhesion (Adams et al, 1998). This difference argues for a tethering in the mobility of Nt AHA-GFP since, unlike free diffusion, the movement is directional and therefore active.…”

“…A graphical depiction of this directional movement can be clearly observed in Figure 3B (see Supplemental Movie 2 online). Quantification led to an estimate of an average moving rate for a given fluorescent molecule of 0.33 6 0.14 mmÁmin À1 (n ¼ 8), similar to rates reported for other tethered, nonfreely moving, membrane proteins (Jesaitis and Yguerabide, 1986;Adams et al, 1998). After bleaching, pollen tubes recovered to a growth rate of 0.5 to 1.0 mmÁmin À1 .…”

A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification.

“…Conceivably, some molecules of Na+,K+-ATPase are internalized during endocytosis, as suggested previously (23,31). Some Na+,K+-ATPase activity has also been found to coisolate with highly purified endosome fractions prepared by free flow electrophoresis (24).…”

Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

“…These values are also of a similar magnitude to the present measurements for the shortrange diffusion of the Na+,K+-ATPase, as might be expected, since at high dilution the long-range diffusion in pure lipid bilayers should be relatively unrestricted, which is consistent with complete fluorescence recoveries being obtained after photobleaching. By contrast, in photobleaching experiments on Na+,K+-ATPase from Madin-Darby canine kidney cells, only 50o of the labeled protein exhibited long-range lateral mobility, with a translational diffusion coefficient of0.05 jm2-s-1 (29). Comparison with the present results suggests that there is a considerable difference between the long-range and local translational diffusion of the Na+,K+-ATPase protein in this bipolar cellular system, although it should be noted that the translational diffusion of a lipid probe was also rather slow (Dr 0.2 jum2-s-1) in the same system.…”