Supporting information.

This supporting information includes figures S1 to S6 and their figure legends as well as the legends to the supporting movies S1 to S3. (PDF)</p

Sequence of the single stranded DNA donor (ssDNA donor).

(PDF)</p

Time series of Alexa594-BSA uptake in WT kidney.

(M4V)</p

TIRFM of Clta-eGFP in the cell shown in Fig 5A–5C.

(AVI)</p

<i>In vivo</i> imaging of Alexa594-BSA uptake in Clta-reporter and WT animals.

(A) Calculated ratio of Alexa594-BSA (background corrected) and Clta-eGFP signal at the apical area in PTEC. Alexa594-BSA quickly accumulates in the Clta-eGFP-positive region of S1 proximal tubules, before a steady state of albumin influx and outflux is reached. A one-phase association non-linear fitting determined a half-time for Alexa594-albumin in the Clta-eGFP-positive proximal tubule compartment of 85.53 seconds. Analysis was performed for Clta-reporter animals only, as the defined eGFP signal at the brush border was used for segmentation of the subapical area. Graph shows mean ± SEM. (B) Analysis of Alexa594-BSA uptake in Clta-reporter and WT animals over time was assessed as the ratio of the thickness of Alexa594-albumin-fluorescent region over the total thickness of the respective PTEC for each time point (B inlet, for different time points see S6 Fig in S1 Appendix). No significant difference can be detected at any time point tested (2-way Anova multiple comparison analysis). Dotted line represents basal cell border. TL: lumen. (C) Intravital 2-photon microscopy images of WT and Clta-reporter mouse S1 segment proximal tubules (PTs), before (baseline) and 1 min, 6 min and 15 min post i.v. injection of Alexa594-BSA (red). Compared to WT PTs, Clta-reporter mice identify clathrin by bright eGFP-expression (green) in the brush border (baseline, arrows). Both genotypes reveal autofluorescent lysosomes (yellow) in the apical region of PTECs (baseline, bold arrows). 1 min after injection, Alexa594-BSA is clearly visible in peritubular capillaries (c) and accumulates in the brush border of WT (red) and Clta-reporter (yellow) PTs (arrowheads). Over time, proximal tubular uptake and intracellular trafficking of Alexa594-BSA, leads to increased thickness of red fluorescent area in the apical region of PT cells (arrowheads), until Alexa594-BSA is clearly visible in PT lysosomes at 15 min. TL: lumen; Scale bar: 20 μm.</p

Time series of Alexa594-BSA uptake in Clta-reporter kidney.

(M4V)</p

Localization of the Clta-eGFP signal in kidney sections.

(A) Clta-eGFP reporter kidney section stained for aquaporin-1 (AQP1) showing that the Clta-eGFP signal is localized to proximal tubule. (B) Clta-eGFP reporter kidney sections stained for aquaporin-2 (AQP2) showing that the Clta-eGFP is not detectable in collecting duct of murine kidney. (C) Clta-eGFP reporter co-localizes partly with the brush border resident protein megalin. Scale bar: 25 μm, CD: collecting duct, G: glomerulus, PT: proximal tubule.</p

Clta-eGFP in primary MEFs.

(A) Western blot of three Clta-reporter MEF and two WT MEF cell lines. Clta-eGFP fusion protein is detected using anti-eGFP and anti-Lc antibodies. On each lane 20 μg of total protein were loaded. (B) Co-IP of CHC and Clta-eGFP fusion protein in MEFs. (C) qPCR of Clta and Cltb in MEFs. No significant changes were observed in Clta-reporter and WT MEFs. WT: n = 4; Clta-reporter n = 6 (students t-test). (D) Cy3-Tf uptake in Clta-reporter and WT MEFs: no significant changes in the uptake of Cy3-Tf were detected between WT and Clta-reporter MEFs at 0 min and 30 min (students t-test). WT: n = 6; Clta-reporter n = 9; Scale bar: 50 μm.</p

Tissue expression of Clta-eGFP fusion protein.

(A) Western Blot Analysis of small intestine, brain, kidney and liver lysates show the expression of the Clta-eGFP fusion protein. Additional band in small intestine and kidney arises possibly through tissue specifc splicing or exon-skipping through insertion of eGFP. Partial degradation may not be excluded. On each lane 25 μg of total protein were loaded. *: unspecific cross reactivity of the eGFP antibody. (B) Immunofluorescence of dye separated WT and Clta-reporter cryo sections of kidney and small intestine using confocal microscopy (left, scale bar 30 μm) and 2-PM (right, scale bar 15 μm). The related WT emission spectra in the green spectral range recorded with confocal microscopy, indicating an autofluoresence maxima at 550 nm can be found in S3B Fig in S1 Appendix. (C) Confocal and associated STED images of small intestine of WT and Clta-reporter cryo sections revealed a strong apical occurrence. Scale bar: 5 μm and 1 μm for the cut-outs (1–4). Clta-eGFP appears in clusters of 74 nm in size or smaller. (D) Confocal and associated STED images of kidney of WT and Clta-reporter cryo sections. A strong apical localization of the Clta-eGFP fusion protein was detected. Scale bar: 5 μm and 1 μm for the cutouts (1–2). Darker regions in the STED image are shown by a saturated color table with overglow (OGL) in blue.</p

Injection mixture for pronuclear injection.

Injection mixture for pronuclear injection.</p