Journal: American Journal of Physiology - Renal Physiology

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

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Interactions between CLC-5 and KIF3B subunits. A : CLC-5 consists of 18 helices (A to R) with the NH 2 - and COOH-terminus (C-term) domains being cytoplasmic (membrane boundaries indicated by arrows) ( , ). B : yeast 2-hybrid using the CLC-5 C-term, which contains 2 CBS domains to screen a renal cDNA library, identified KIF3B as a putative interactor. Interaction between large T and p53 provided a positive control. C : 35 S-methionine-labeled CLC-5 C-term was incubated with GST-tagged KIF3B coiled coil (CC)/globular domain (GD). Resin-bound proteins were resolved by SDS-PAGE and detected by autoradiography. Results from 2 independent in vitro translation experiments using 2 different GST-tagged KIF3B clones are shown. The larger band at ∼24.5 kDa represents the 216 amino acid Myc-tagged CLC-5 C-term, whereas the smaller band at ∼18.5 kDa likely represents another translated form of CLC-5 C-term that originated from use of an alternative methionine at codon 583 and hence lacked the NH 2 terminal 32 amino acids. This smaller CLC-5 C-term, which was less efficiently translated, had both CBS domains and hence was pulled down by the GST-tagged KIF3B CC/GD. D : endogenous coimmunoprecipitation using wild-type mouse whole kidney extracts and a KIF3B monoclonal antibody (mAb). KIF3B, KIF3A, and KAP3 mAb were used to confirm endogenous coimmunoprecipitation of these proteins which form a trimeric complex. It is important to note that the interaction between CLC-5 and KIF3B is likely to be transient, as endosomes will be released to continue recycling, and that only a small proportion of the total cellular CLC-5 will be present, at a given time point, on the surface of endosomes that are being trafficked anterogradely; these features are consistent with the relatively low amount of KIF3B that is immunopreciptiated by CLC-5. E : coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-5 and KIF3B domains. Lane 1 : input; lanes 2 , 3 , and 4 : pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively. F : summary of KIF3B domains showing presence (+) or absence (−) of interactions with CLC-5. G : coimmunoprecipitation studies using HEK293 cells demonstrated interactions between CLC-4 and KIF3B, and CLC-7 and KIF3B. Lane 1 : input; lanes 2 , 3 , and 4 : pull-down with no antibody, anti-Myc, or anti-HA antibodies, respectively.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: cDNA Library Assay, Positive Control, Labeling, Incubation, SDS Page, Autoradiography, In Vitro, Clone Assay

Journal: American Journal of Physiology - Renal Physiology

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

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: 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 , 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.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Confocal Microscopy, Transfection, Live Cell Imaging, Labeling

Journal: American Journal of Physiology - Renal Physiology

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

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Electrophysiological, cell surface expression, and albumin uptake studies in nonpolarized HEK293 cells. A-C : representative current recording from a HEK293 cell expressing CLC-5 ( A ), CLC-5 and KIF3B ( B ), and an untransfected HEK293 cell ( C ). D : maximum conductance measured from HEK293 cells cotransfected with CLC-5 and expression plasmids as indicated. Coexpression of KIF3B (FL) significantly increased conductance. E : densitometric analysis of CLC-5 in cell surface fractions. Coexpression of KIF3B (FL) significantly increased the cell surface expression of CLC-5. F : microtubule orientation in nonpolarized HEK293 cells. G : representative Western blot analysis for KIF3B, KIF3A, and tubulin expression in cell lysates obtained from HEK293 cells transfected with control or KIF3B siRNA. KIF3B siRNA suppressed endogenous KIF3B expression, whereas there was no effect on KIF3A and tubulin expression. H : densitometric analysis of KIF3B expression in cell lysates obtained from HEK293 cells transfected with control or KIF3B siRNA. KIF3B siRNA significantly reduced expression of endogenous KIF3B. I : maximum conductance measured from HEK293 cells cotransfected with CLC-5 and KIF3B siRNA or control siRNA plasmids as indicated. KIF3B siRNA significantly decreased conductance compared with coexpression of CLC-5 with control siRNA. J : densitometric analysis of CLC-5 in cell surface fractions. KIF3B siRNA significantly decreased the cell surface expression of CLC-5, compared with coexpression of CLC-5 with control siRNA ( n = 6). K : RT-PCR analysis of cubilin, megalin, KIF3B, calmodulin (positive control), and water blank (negative control) in HEK293 cells and control human kidney (hKid). L : albumin uptake in HEK293 cells transfected with KIF3B constructs. KIF3B (FL) significantly increased albumin uptake, whereas KIF3B siRNA significantly decreased albumin uptake, compared with their respective controls. M : competition assays for albumin uptake. HEK293 cells were transfected with KIF3B constructs, in the absence (no competition) or presence (albumin competition) of excess unlabeled albumin, and the uptake of fluorescently labeled albumin measured ( n = 6); the uptake of fluorescently labeled albumin without competition was taken as the reference and set at 100% and compared with that obtained with competition using unlabeled albumin. Endocytosis of the fluorescently labeled albumin was significantly reduced by competition with unlabeled albumin. Means ± 1 SE and P values calculated by Student's unpaired, 2-tailed t -test are shown. * P < 0.02, ** P < 0.01, and *** P < 0.001.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Construct, Labeling

Journal: American Journal of Physiology - Renal Physiology

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

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Cell surface expression, albumin endocytosis, and transferrin endocytosis studies in polarized opossum kidney (OK) cells. A : microtubule orientation in polarized OK cells [apical membrane (APM) and basolateral membrane (BLM)]. B : confocal microscopy of polarized OK cells stained with ZO1 (green) and EB1 (red). Top : XY image. Bottom : single orthogonal view of ZO1 and EB1. OK cells were polarized in culture as evidenced by the presence of tight junctions (green). In addition, numerous EB1-labeled microtubules are observed below the tight junctions demonstrating that the growing ends of microtubules are predominantly at the basolateral surface of polarized OK cells. Scale bar = 10 μm. C : RT-PCR analysis of KIF3B and calmodulin (positive control) expression in OK and HEK293 cells. A water blank was used as the negative control. OK cells have previously been shown to express CLC-5, megalin, and cubilin ( , ). D : representative Western blot analysis for KIF3B, KIF3A, and tubulin expression in cell lysates obtained from OK cells transfected with control or KIF3B siRNA. KIF3B siRNA suppressed endogenous KIF3B expression, whereas there was no effect on KIF3A and tubulin expression. E : densitometric analysis of KIF3B expression in cell lysates obtained from OK cells transfected with control or KIF3B siRNA. KIF3B siRNA significantly reduced expression of endogenous KIF3B to 55 ± 4.3%, compared with control, i.e., nonsense siRNA ( n = 4). F : confocal microscopy of polarized OK cells transfected with CLC-5-Myc and stained with anti-Myc (red) and anti-ZO1 (green). Top : XY image. Bottom : single orthogonal view of CLC-5-Myc and ZO1. CLC-5-Myc-labeled structures can be observed above the tight junctions (green) demonstrating expression of CLC-5-Myc predominantly at the apical surface of polarized OK cells. Scale bar = 10 μm. G : CLC-5 plasma membrane expression in OK cells (densitometric analysis after surface biotinylation). KIF3B (FL) significantly decreased the cell surface expression of CLC-5, compared with coexpression of CLC-5 with pCMV-HA alone (HA; n = 9). H : albumin uptake in OK cells transfected with KIF3B constructs. KIF3B (FL) significantly decreased albumin uptake ( n = 6), compared with expression of pCMV-HA alone (HA), whereas KIF3B siRNA significantly increased albumin uptake, compared with expression of control siRNA ( n = 12). I : transferrin uptake in OK cells transfected with KIF3B constructs. KIF3B (FL) significantly decreased transferrin uptake ( n = 6), compared with expression of pCMV-HA alone (HA), whereas KIF3B siRNA significantly increased transferrin uptake ( n = 12), compared with expression of control siRNA. J : competition assays for albumin uptake. OK cells were transfected with KIF3B constructs, in the absence (no competition) or presence (competition) of excess unlabeled albumin and transferrin, and the uptake of fluorescently labeled albumin was measured. Endocytosis of fluorescently labeled albumin was significantly reduced by competition with either unlabeled albumin or unlabeled transferrin ( n = 6). K : competition assays for transferrin uptake. OK cells were transfected with KIF3B constructs, in the absence (no competition) or presence (competition) of excess unlabeled albumin and transferrin, and the uptake of fluorescently labeled transferrin was measured. Endocytosis of fluorescently labeled transferrin was significantly reduced by competition with either unlabeled albumin or unlabeled transferrin ( n = 6). Means ± 1 SE and P values calculated by Student's unpaired, 2-tailed t -test are shown; * P < 0.05, ** P < 0.02, $ P < 0.001.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Confocal Microscopy, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Western Blot, Transfection, Construct

Journal: American Journal of Physiology - Renal Physiology

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

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Expression level and subcellular distribution of Kif3b in Clcn5 Y/+ and Clcn5 Y/− mouse kidneys. A : quantitative PCR (qPCR) of mRNA in Clcn5 Y/+ and Clcn5 Y/− kidneys ( n = 4 in each group). Kif3b mRNA levels (adjusted to GAPDH mRNA) are significantly higher in Clcn5 Y/− than control kidneys (155 ± 11%; * P < 0.05). B : representative Western blot for Kif3b in Clcn5 Y/+ and Clcn5 Y/− kidneys ( n = 4 in each group), using 20 μg of protein. C : densitometric analysis normalized to β-actin levels confirms that Kif3b protein is more abundant in Clcn5 Y/− kidneys (153 ± 19% of Clcn5 Y/+ level; * P < 0.05). D : representative Western blot for Kif3b in low-speed (LS), high-speed (HS) membrane, and cytosolic (C) fractions of Clcn5 Y/+ and Clcn5 Y/− kidneys. The samples were loaded at 20 μg total protein per lane, as appropriate for subcellular fractionations . Kif3b is detected in the cytosol of Clcn5 Y/− samples but not in controls. E and F : density distribution after centrifugation of the HS (membrane) fraction, using a Percoll gradient, is presented compared with the initial concentration (C/Ci), so that values >1 reflect organelle enrichment and values <1 reflect organelle depletion. This gradient resolves a low-density peak ( fractions 2–4 ), including early endosomes (Rab5a, V-ATPase), from a high-density peak ( fractions 9–10 ), enriched in lysosomes (cathepsin D). Kif3b partially overlaps with V-ATPase E1 subunit (low-density fractions 2–3 ) and Clc-5 ( fraction 3 ), which supports their association with endosomes ( F ). Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t -test.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Centrifugation, Concentration Assay

Journal: American Journal of Physiology - Renal Physiology

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

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Endogenous Kif3b expression and albumin endocytosis in mouse proximal renal tubular primary cell cultures (mPTCs). A : endogenous expression of Kif3b in mPTC cultures from Clcn5 Y/+ and Clcn5 Y/− mice. The expression of Kif3b was determined by qPCR and was found to be significantly higher in the Clcn5 Y/− mice and consistent with the observations from mouse whole kidney extracts ( Fig. 5 A ). B : expression of human KIF3B (hKIF3B) in transfected mPTCs from Clcn5 Y/+ ( n = 3) and Clcn5 Y/− ( n = 3) mice was assessed by qPCR and this showed an ∼3,000-fold higher expression of the exogenous human KIF3B, compared with the endogenous expression of Kif3b [Kif3b(m)] and Kif3a . C : uptake of FITC-albumin in mPTC cultures from Clcn5 Y/+ and Clcn5 Y/− mice in mock-transfected cells, in the absence or presence of competition by excess unlabeled albumin and transferrin, or after energy depletion by deoxyglucose (DOG) and sodium azide (NaN 3 ), or in KIF3B-transfected cells. In Clcn5 Y/+ mPTCs, overexpression of hKIF3B decreases specific albumin endocytic uptake. In Clcn5 Y/− mPTCs, albumin uptake is fully abrogated (to the level of metabolic inhibition and albumin competition) by hKIF3B overexpression. D : RT-PCR analyses of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in HEK293 cells, OK cells, Clcn5 Y/+ and Clcn5 Y/− whole mouse kidneys, and Clcn5 Y/+ and Clcn5 Y/− mPTCs. GAPDH and a water blank were used as positive and negative controls, respectively. Means ± 1 SE and P values were calculated by the Student's unpaired, 2-tailed t -test.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: Expressing, Transfection, Over Expression, Inhibition, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Physiology - Renal Physiology

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

doi: 10.1152/ajprenal.00038.2009

Figure Lengend Snippet: Schematic representation of a working model for endosomal transport involving CLC-5 and KIF3B interaction in polarized cells. CLC-5 and KIF3B are expressed in polarized renal proximal tubular cells which reabsorb proteins and solutes via the receptor-mediated endocytic pathway that uses megalin and cubilin as the apical plasma membrane (APM) receptors ( , , ). Ligands bind to these receptors at the APM where CLC-5 and interacting proteins Nedd4-2, NHERF2, and cofilin are also present ( , , ). The receptor-ligand complex is internalized in vesicles, containing CLC-5 and CLC-4 , which are anterogradely transported along microtubules via the Kinesin-2 complex . These vesicles fuse with early endosomes where acidification involves the vacuolar H + -ATPase, with counter ions being provided by CLC-5 . In nonpolarized cells, the orientation of the microtubules is reversed ( Fig. 3 F ), and CLC-5 vesicles would be transported to the cell surface.

Article Snippet: The following antibodies were used: affinity-purified rabbit polyclonal antibodies against the NH 2 terminus of human CLC-5 , mouse monoclonal antibodies against KIF3B (BD Bioscience), Rab5a (Santa Cruz Biotechnology), goat polyclonal antibodies against cathepsin D (Santa Cruz Biotechnology), mouse monoclonal antibodies (E11) against the E1 subunit of V-ATPase (Dr. S. Gluck, University of California, San Francisco, CA), and β-actin (Sigma, St. Louis, MO).

Techniques: