Role of kinesin light chain-2 of kinesin-1 in the traffic of Na,K-ATPase-containing vesicles in alveolar epithelial cells

Abstract

Recruitment of the Na,K-ATPase to the plasma membrane of alveolar epithelial cells results in increased active Na(+) transport and fluid clearance in a process that requires an intact microtubule network. However, the microtubule motors involved in this process have not been identified. In the present report, we studied the role of kinesin-1, a plus-end microtubule molecular motor that has been implicated in the movement of organelles in the Na,K-ATPase traffic. We determined by confocal microscopy and biochemical assays that kinesin-1 and the Na,K-ATPase are present in the same membranous cellular compartment. Knockdown of kinesin-1 heavy chain (KHC) or the light chain-2 (KLC2), but not of the light chain-1 (KLC1), decreased the movement of Na,K-ATPase-containing vesicles when compared to sham siRNA-transfected cells (control group). Thus, a specific isoform of kinesin-1 is required for microtubule-dependent recruitment of Na,K-ATPase to the plasma membrane, which is of physiological significance.

Figure 1.

Na,K-ATPase-containing vesicles move using the microtubule network via the molecular motor kinesin-1. A) Live-cell confocal imaging of GFPα1-A549 cells (green) transiently transfected with tubulin-mCherry (red). Movement of GFP-containing vesicles was recorded as multidimensional acquisition stacks. Top panel: localization of Na,K-ATPase-containing vesicles and microtubules in a single cell. Bottom panels: magnification (white square) of 2 stacks showing a single vesicle (white arrow) displacing over the microtubule over time (0 and 30 s). B) Immunofluorescence confocal microscopy images from detergent-permeabilized GFPα1-A549 cells labeled for kinesin-1 using multichannel acquisitions. Red, kinesin-1; green, Na,K-ATPase. Right bottom panel shows an ×4 digital view of the area delineated with a white square. Arrowheads indicate colocalization of kinesin-1 and Na,K-ATPase. C) Total membrane fraction of GFPα1-A549 cells was loaded on top of a sucrose gradient, and 8 fractions were recovered. Distribution of Na,K-ATPase, KHC, KLC1, and KLC2 was analyzed by Western blotting with specific antibodies. Representative Western blot is shown. Scale bars = 10 μm.

Figure 2.

siRNAs against KHC, KLC1, and KLC2 effectively reduce protein abundance. GFPα1-A549 cells were transfected with specific shRNA against KHC (A), a pool of 3 different siRNAs against KLC1 (B), or specific shRNA against KLC2 (C) using lipofectamine LTX. Cell lysates from the transfected cells were obtained at the indicated times. Protein concentration was measured, equal amounts of proteins were resolved by SDS-PAGE, and Western blots were analyzed with specific antibodies for KHC, KLC1, and KLC2. For KHC, actin was used as loading control; KLC2 and KLC1 served as loading control for KLC1 and KLC2, respectively. *P < 0.05; **P < 0.001.

Figure 3.

Knockdown of KHC or KLC2 (but not KLC1) causes redistribution of GFPα1-containing vesicles toward the nucleus. A) GFPα1-A549 cells were transfected with shRNA against KHC and KLC2, or siRNA against KLC1, and at 3 d post-transfection, fluorescence images were taken of 20 cells for each condition. Outer solid line delineates total area of the cell (S1); inner dotted line delineates area of the cell occupied by Na,K-ATPase-containing vesicles (S2). Graph represents ratio of S2 to S1 for each cell. B) MitoTracker-loaded A549 cells were transfected with shRNA against KHC or KLC2, and at 3 d post-transfection, time-lapse fluorescence images were taken of 20 cells for each condition using an inverted fluorescence microscope and MetaMorph software. Trajectories were analyzed using Diatrack 3.0 software. Still fluorescence images were taken of a representative cell for each condition. Red lines show length of each individual trajectory. Graph depicts the average of all trajectories for each condition (relative units). C) A549 cells were transiently transfected with a vector encoding for GFP-Rab7 fusion protein, and 3 d post-transfection, fluorescence images were taken of 20 cells for each condition. Outer line delineates total area of the cell (S1); inner dotted line delineates area of the cell occupied by GFP-Rab7 late endosomes (S2). Graph represents the ratio of S2 to S1 for each cell. Insets: transfections with mCherry shRNA. Scale bars = 10 μm. *P < 0.05.

Figure 4.

Knockdown of KHC or KLC2 causes a reduction in the length of GFPα1-containing vesicle trajectories. A) GFPα1-A549 cells were transfected with shRNA against KHC or KLC2, or siRNA against KLC1; at 3 d post-transfection, time-lapse fluorescence images were taken of 20 cells for each condition, using an inverted fluorescence microscope and MetaMorph software. Trajectories were analyzed using Diatrack 3.0 software. Left panel: still fluorescence images of a representative cell for each condition. Red lines show individual trajectories. Right panel: average of long trajectories (>6 μm) for each condition (relative units). B) Average contour length traveled by vesicles as a function of time, calculated as described in Materials and Methods. Scale bar = 10 μm. *P < 0.05.

Figure 5.

Proposed model for the role of kinesin-1 in the traffic of Na,K-ATPase-containing vesicles. Kinesin-1 (represented as a tetramer of 2 KHC and 2 KLC2) moves Na,K-ATPase-containing vesicles from intracellular pools (minus end) into the plasma membrane (plus end), using microtubules as tracks.