Limits...
CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Reed AA, Loh NY, Terryn S, Lippiat JD, Partridge C, Galvanovskis J, Williams SE, Jouret F, Wu FT, Courtoy PJ, Nesbit MA, Rorsman P, Devuyst O, Ashcroft FM, Thakker RV - Am. J. Physiol. Renal Physiol. (2009)

Bottom Line: Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules.KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin.Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford, United Kingdom.

ABSTRACT
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Show MeSH

Related in: MedlinePlus

Confocal microscopy shows colocalization of CLC-5 and KIF3B. HEK293 cells transfected with fluorescently tagged CLC-5 and KIF3B (CLC-5-RFP and KIF3B-GFP) and costained with anti-α-tubulin or anti-Kinesin-2. A-C: Z-stack dual wavelength confocal microscopy. For each stack, images were obtained in 2 channels over 15 horizontal planes that were 0.7 μm apart. A: HEK293 cells transfected with KIF3B-GFP and CLC-5-RFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized KIF3B and CLC-5 (merge). Confocal Z-stack images and intensity profile line scans demonstrated that KIF3B-GFP and CLC-5-RFP are partially colocalized, as evidenced by the yellow signals or by an overlap of <300 nm, which is the estimated diameter of a proximal tubular endosomal vesicle (19), in the red and green peaks (confocal images 1 to 5). The intensity profile line scans were performed between the white crosses as indicated on confocal images 1 to 5, with the intensity measured in arbitrary units (A.U.). Scale bar top: 10 μm; scale bar confocal images 1 to 5: 2 μm. B: HEK293 cells transfected with CLC-5-RFP and costained with anti-α-tubulin. Red signals: CLC-5; green signals: tubulin; yellow signals: colocalized CLC-5 and tubulin (merge). CLC-5-RFP was present at the plasma membrane and in punctate structures throughout the cytoplasm, where it was localized with tubulin, indicating its association with microtubules. Scale bars: 10 μm. C: HEK293 cells transfected with CLC-5-RFP and costained with anti-Kinesin-2 antibody. Red signals: CLC-5; green signals: Kinesin-2; yellow signals: colocalized CLC-5 and Kinesin-2 (merge). Scale bars: 10 μm. D and E: live cell imaging using dual-wavelength confocal microscopy of HEK293 cells cotransfected with full-length CLC-5-RFP and full-length KIF3B-GFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized CLC-5 and KIF3B. Scale bar: 10 μm. A proportion of CLC-5-RFP-labeled vesicles was transported along KIF3B-labeled cytoskeletal network structures. E: progress of a vesicle (arrowed), over 10 s, is illustrated. The vesicles were estimated to move at an approximate rate of 0.42 μm/s. Scale bar: 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2822520&req=5

Figure 2: Confocal microscopy shows colocalization of CLC-5 and KIF3B. HEK293 cells transfected with fluorescently tagged CLC-5 and KIF3B (CLC-5-RFP and KIF3B-GFP) and costained with anti-α-tubulin or anti-Kinesin-2. A-C: Z-stack dual wavelength confocal microscopy. For each stack, images were obtained in 2 channels over 15 horizontal planes that were 0.7 μm apart. A: HEK293 cells transfected with KIF3B-GFP and CLC-5-RFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized KIF3B and CLC-5 (merge). Confocal Z-stack images and intensity profile line scans demonstrated that KIF3B-GFP and CLC-5-RFP are partially colocalized, as evidenced by the yellow signals or by an overlap of <300 nm, which is the estimated diameter of a proximal tubular endosomal vesicle (19), in the red and green peaks (confocal images 1 to 5). The intensity profile line scans were performed between the white crosses as indicated on confocal images 1 to 5, with the intensity measured in arbitrary units (A.U.). Scale bar top: 10 μm; scale bar confocal images 1 to 5: 2 μm. B: HEK293 cells transfected with CLC-5-RFP and costained with anti-α-tubulin. Red signals: CLC-5; green signals: tubulin; yellow signals: colocalized CLC-5 and tubulin (merge). CLC-5-RFP was present at the plasma membrane and in punctate structures throughout the cytoplasm, where it was localized with tubulin, indicating its association with microtubules. Scale bars: 10 μm. C: HEK293 cells transfected with CLC-5-RFP and costained with anti-Kinesin-2 antibody. Red signals: CLC-5; green signals: Kinesin-2; yellow signals: colocalized CLC-5 and Kinesin-2 (merge). Scale bars: 10 μm. D and E: live cell imaging using dual-wavelength confocal microscopy of HEK293 cells cotransfected with full-length CLC-5-RFP and full-length KIF3B-GFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized CLC-5 and KIF3B. Scale bar: 10 μm. A proportion of CLC-5-RFP-labeled vesicles was transported along KIF3B-labeled cytoskeletal network structures. E: progress of a vesicle (arrowed), over 10 s, is illustrated. The vesicles were estimated to move at an approximate rate of 0.42 μm/s. Scale bar: 5 μm.

Mentions: The basis of the CLC-5 and KIF3B interaction, in mammalian cells, was further investigated by colocalization studies of CLC-5 and KIF3B using transient expression of fluorescently tagged CLC-5 and KIF3B in HEK293 and COS7 (data not shown) cells. This revealed KIF3B to be mostly distributed in a punctate pattern throughout the cytoplasm (Fig. 2A). KIF3B also associated with tubulin (data not shown), and this microtubule-like distribution and colocalization with tubulin are consistent with its presence on microtubules (61). CLC-5 was concentrated at the plasma membrane, and in punctate structures throughout the cytoplasm, where it showed partial colocalization with KIF3B (Fig. 2A). A detailed examination using intensity profile line scan analysis in five cells helped to further demonstrate the colocalization of CLC-5 and KIF3B. Thus, an examination of 138 vesicles revealed that 68 vesicles expressed both KIF3B and CLC-5, 42 vesicles expressed KIF3B only, and 28 vesicles expressed CLC-5 only. Thus, of the 110 vesicles expressing KIF3B, 62% showed colocalization with CLC-5, and of the 96 vesicles expressing CLC-5, 71% showed colocalization with KIF3B. A mean (±SE) of 14 (±1.6) of the 28 (±1.7) vesicles within a cell demonstrated colocalization of CLC-5 with KIF3B, and the total cell area occupied by each of KIF3B- and CLC-5-containing vesicles within these cells was 3.4%. The observed frequency of CLC-5 and KIF3B colocalization was significantly higher at 2.3% (P < 0.001) than that expected to occur at random, which is estimated to be 0.12% (i.e., 3.4% of 3.4%), thereby indicating that the observed colocalization of CLC-5 and KIF3B is ∼20-fold greater than that expected to occur by chance alone. CLC-5 was also demonstrated to be associated with microtubules by costaining with anti-α-tubulin (Fig. 2B) and to colocalize with the Kinesin-2 complex that contains KIF3B (Fig. 2C). In addition, the microtubular location of CLC-5-containing vesicles and KIF3B was further demonstrated by the use of nocodazole, which inhibits polymerization of free tubulin molecules (14). The extent of microtubule depolymerization after nocodazole treatment, and restoration following nocodazole wash-out, was verified by anti-α-tubulin staining. Nocodazole treatment of transfected HEK293 cells resulted in a disruption of microtubule integrity and a dramatic redistribution of CLC-5 and KIF3B (data not shown).


CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Reed AA, Loh NY, Terryn S, Lippiat JD, Partridge C, Galvanovskis J, Williams SE, Jouret F, Wu FT, Courtoy PJ, Nesbit MA, Rorsman P, Devuyst O, Ashcroft FM, Thakker RV - Am. J. Physiol. Renal Physiol. (2009)

Confocal microscopy shows colocalization of CLC-5 and KIF3B. HEK293 cells transfected with fluorescently tagged CLC-5 and KIF3B (CLC-5-RFP and KIF3B-GFP) and costained with anti-α-tubulin or anti-Kinesin-2. A-C: Z-stack dual wavelength confocal microscopy. For each stack, images were obtained in 2 channels over 15 horizontal planes that were 0.7 μm apart. A: HEK293 cells transfected with KIF3B-GFP and CLC-5-RFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized KIF3B and CLC-5 (merge). Confocal Z-stack images and intensity profile line scans demonstrated that KIF3B-GFP and CLC-5-RFP are partially colocalized, as evidenced by the yellow signals or by an overlap of <300 nm, which is the estimated diameter of a proximal tubular endosomal vesicle (19), in the red and green peaks (confocal images 1 to 5). The intensity profile line scans were performed between the white crosses as indicated on confocal images 1 to 5, with the intensity measured in arbitrary units (A.U.). Scale bar top: 10 μm; scale bar confocal images 1 to 5: 2 μm. B: HEK293 cells transfected with CLC-5-RFP and costained with anti-α-tubulin. Red signals: CLC-5; green signals: tubulin; yellow signals: colocalized CLC-5 and tubulin (merge). CLC-5-RFP was present at the plasma membrane and in punctate structures throughout the cytoplasm, where it was localized with tubulin, indicating its association with microtubules. Scale bars: 10 μm. C: HEK293 cells transfected with CLC-5-RFP and costained with anti-Kinesin-2 antibody. Red signals: CLC-5; green signals: Kinesin-2; yellow signals: colocalized CLC-5 and Kinesin-2 (merge). Scale bars: 10 μm. D and E: live cell imaging using dual-wavelength confocal microscopy of HEK293 cells cotransfected with full-length CLC-5-RFP and full-length KIF3B-GFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized CLC-5 and KIF3B. Scale bar: 10 μm. A proportion of CLC-5-RFP-labeled vesicles was transported along KIF3B-labeled cytoskeletal network structures. E: progress of a vesicle (arrowed), over 10 s, is illustrated. The vesicles were estimated to move at an approximate rate of 0.42 μm/s. Scale bar: 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2822520&req=5

Figure 2: Confocal microscopy shows colocalization of CLC-5 and KIF3B. HEK293 cells transfected with fluorescently tagged CLC-5 and KIF3B (CLC-5-RFP and KIF3B-GFP) and costained with anti-α-tubulin or anti-Kinesin-2. A-C: Z-stack dual wavelength confocal microscopy. For each stack, images were obtained in 2 channels over 15 horizontal planes that were 0.7 μm apart. A: HEK293 cells transfected with KIF3B-GFP and CLC-5-RFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized KIF3B and CLC-5 (merge). Confocal Z-stack images and intensity profile line scans demonstrated that KIF3B-GFP and CLC-5-RFP are partially colocalized, as evidenced by the yellow signals or by an overlap of <300 nm, which is the estimated diameter of a proximal tubular endosomal vesicle (19), in the red and green peaks (confocal images 1 to 5). The intensity profile line scans were performed between the white crosses as indicated on confocal images 1 to 5, with the intensity measured in arbitrary units (A.U.). Scale bar top: 10 μm; scale bar confocal images 1 to 5: 2 μm. B: HEK293 cells transfected with CLC-5-RFP and costained with anti-α-tubulin. Red signals: CLC-5; green signals: tubulin; yellow signals: colocalized CLC-5 and tubulin (merge). CLC-5-RFP was present at the plasma membrane and in punctate structures throughout the cytoplasm, where it was localized with tubulin, indicating its association with microtubules. Scale bars: 10 μm. C: HEK293 cells transfected with CLC-5-RFP and costained with anti-Kinesin-2 antibody. Red signals: CLC-5; green signals: Kinesin-2; yellow signals: colocalized CLC-5 and Kinesin-2 (merge). Scale bars: 10 μm. D and E: live cell imaging using dual-wavelength confocal microscopy of HEK293 cells cotransfected with full-length CLC-5-RFP and full-length KIF3B-GFP. Green signals: KIF3B; red signals: CLC-5; yellow signals: colocalized CLC-5 and KIF3B. Scale bar: 10 μm. A proportion of CLC-5-RFP-labeled vesicles was transported along KIF3B-labeled cytoskeletal network structures. E: progress of a vesicle (arrowed), over 10 s, is illustrated. The vesicles were estimated to move at an approximate rate of 0.42 μm/s. Scale bar: 5 μm.
Mentions: The basis of the CLC-5 and KIF3B interaction, in mammalian cells, was further investigated by colocalization studies of CLC-5 and KIF3B using transient expression of fluorescently tagged CLC-5 and KIF3B in HEK293 and COS7 (data not shown) cells. This revealed KIF3B to be mostly distributed in a punctate pattern throughout the cytoplasm (Fig. 2A). KIF3B also associated with tubulin (data not shown), and this microtubule-like distribution and colocalization with tubulin are consistent with its presence on microtubules (61). CLC-5 was concentrated at the plasma membrane, and in punctate structures throughout the cytoplasm, where it showed partial colocalization with KIF3B (Fig. 2A). A detailed examination using intensity profile line scan analysis in five cells helped to further demonstrate the colocalization of CLC-5 and KIF3B. Thus, an examination of 138 vesicles revealed that 68 vesicles expressed both KIF3B and CLC-5, 42 vesicles expressed KIF3B only, and 28 vesicles expressed CLC-5 only. Thus, of the 110 vesicles expressing KIF3B, 62% showed colocalization with CLC-5, and of the 96 vesicles expressing CLC-5, 71% showed colocalization with KIF3B. A mean (±SE) of 14 (±1.6) of the 28 (±1.7) vesicles within a cell demonstrated colocalization of CLC-5 with KIF3B, and the total cell area occupied by each of KIF3B- and CLC-5-containing vesicles within these cells was 3.4%. The observed frequency of CLC-5 and KIF3B colocalization was significantly higher at 2.3% (P < 0.001) than that expected to occur at random, which is estimated to be 0.12% (i.e., 3.4% of 3.4%), thereby indicating that the observed colocalization of CLC-5 and KIF3B is ∼20-fold greater than that expected to occur by chance alone. CLC-5 was also demonstrated to be associated with microtubules by costaining with anti-α-tubulin (Fig. 2B) and to colocalize with the Kinesin-2 complex that contains KIF3B (Fig. 2C). In addition, the microtubular location of CLC-5-containing vesicles and KIF3B was further demonstrated by the use of nocodazole, which inhibits polymerization of free tubulin molecules (14). The extent of microtubule depolymerization after nocodazole treatment, and restoration following nocodazole wash-out, was verified by anti-α-tubulin staining. Nocodazole treatment of transfected HEK293 cells resulted in a disruption of microtubule integrity and a dramatic redistribution of CLC-5 and KIF3B (data not shown).

Bottom Line: Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules.KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin.Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit, Nuffield Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford, United Kingdom.

ABSTRACT
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Show MeSH
Related in: MedlinePlus