Decision letter: Kazrin promotes dynein/dynactin-dependent traffic from early to recycling endosomes

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmental defects, in part derived from altered cell adhesion and migration, as well as failure to undergo epidermal to mesenchymal transition. However, the primary molecular role of kazrin, which might contribute to all these functions, has not been elucidated yet. We previously identified one of its isoforms, kazrin C, as a protein that potently inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knock-out mouse embryonic fibroblasts to investigate its endocytic function. We found that kazrin depletion delays juxtanuclear enrichment of internalized material, indicating a role in endocytic traffic from early to recycling endosomes. Consistently, we found that the C-terminal domain of kazrin C, predicted to be an intrinsically disordered region, directly interacts with several early endosome (EE) components, and that kazrin depletion impairs retrograde motility of these organelles. Further, we noticed that the N-terminus of kazrin C shares homology with dynein/dynactin adaptors and that it directly interacts with the dynactin complex and the dynein light intermediate chain 1. Altogether, the data indicate that one of the primary kazrin functions is to facilitate endocytic recycling by promoting dynein/dynactin-dependent transport of EEs or EE-derived transport intermediates to the recycling endosomes. Editor's evaluation In their paper, Hernandez-Perez et al. perform a detailed and solid analysis of kazrin, a widely expressed protein that appears to be involved in many diverse cellular processes, but whose exact function is unknown. The authors employ mouse embryonic fibroblasts and biochemistry to investigate the function of Kazrin and determine that Kazrin promotes the dynein/dynactin-dependent transport of early endosomes. These valuable findings will be of interest to those in the field of intracellular transport. https://doi.org/10.7554/eLife.83793.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Kazrin is a highly conserved and broadly expressed vertebrate protein, which was first identified as a transcript present in the human brain (Nagase et al., 1999). The human kazrin gene is located on chromosome 1 (1p36.21) and encodes at least seven isoforms (A-F and K), generated by alternative splicing (Groot et al., 2004; Nachat et al., 2009; Wang et al., 2009). From those, kazrin C is the shorter isoform that constitutes the core of all other versions, which bear N or C-terminal extensions. Since its discovery, several laboratories have reported a broad range of roles for the different kazrin isoforms in a myriad of experimental model systems. Thus, in humans, kazrin participates in structuring the skin-cornified envelope and it promotes keratinocyte terminal differentiation (Groot et al., 2004; Sevilla et al., 2008a). In U373MG human astrocytoma cells, kazrin depletion leads to caspase activation and apoptosis (Wang et al., 2009). In Xenopus embryos instead, kazrin depletion causes ectoderm blisters (Sevilla et al., 2008b), as well as craniofacial development defects (Cho et al., 2011), linked to altered cell adhesion (Sevilla et al., 2008b), impaired epidermal to mesenchymal transition (EMT) and defective migration of neural crest cells (Cho et al., 2011). The subcellular localization of kazrin recapitulates its functional diversity. Depending on the isoform and cell type under analysis, kazrin associates with desmosomes (Groot et al., 2004), adherens junction components (Cho et al., 2010; Sevilla et al., 2008a), the nucleus (Groot et al., 2004; Sevilla et al., 2008a), or the microtubule cytoskeleton (Nachat et al., 2009). At the molecular level, the N-terminus of kazrin, predicted to form a coiled-coil, directly interacts with several p120-catenin family members (Sevilla et al., 2008b), as well as with the desmosomal component periplakin (Groot et al., 2004), and it directly or indirectly regulates RhoA activity (Groot et al., 2004; Sevilla et al., 2008a; Cho et al., 2010). How kazrin orchestrates such many cellular functions at the molecular level is far from being understood. We previously identified human kazrin C as a protein that potently inhibits clathrin-mediated endocytosis when overexpressed (Schmelzl and Geli, 2002). In the present work, we generated kazrin knock out (kazKO) Mouse Embryonic Fibroblasts (MEFs) to analyze its role in endocytic traffic in detail. We found that depletion of kazrin caused accumulation of peripheral EEs and delayed transfer of endocytosed transferrin (Tfn) to the pericentriolar juxtanuclear region, where the recycling endosomes (REs) usually concentrate (Granger et al., 2014; Tang and Marshall, 2012). Consequently, cellular functions requiring intact endosomal traffic through the REs, such as cell migration and cytokinetic abscission, were also altered in kazKO cells. Consistent with its role in endocytic traffic, we found that the kazrin C C-terminal portion predicted to be an intrinsically disordered region (IDR), interacted with different components of the EEs, it was required to form foci on these organelles and it was necessary to sustain efficient transport of internalized Tfn to the juxtanuclear region. Further, the N-terminus of kazrin C shared considerable homology with dynein/dynactin activating adaptors, and kazrin directly interacted with the dynactin complex and the dynein light intermediate 1 (LIC1). The data thus suggested that kazrin facilitates the transfer of endocytosed material to the pericentriolar REs by promoting retrograde dynein/dynactin-dependent transport of EEs or EE-derived transport intermediates. Results Kazrin depletion impairs endosomal traffic We originally identified kazrin C as a human brain cDNA, whose overexpression causes accumulation of the transferrin receptor (TfnR) at the plasma membrane (PM) in Cos7 cells (Schmelzl and Geli, 2002), suggesting that kazrin might be involved in clathrin-mediated endocytic uptake from the PM. However, treatment of Cos7 cells with an shRNA directed against kazrin (shKzrn) (Figure 1—figure supplement 1A) did not inhibit endocytic internalization but it rather increased the intracellular signal of Alexa 647-Tfn (A647-Tfn) upon a 2 hr incubation (Figure 1—figure supplement 1B,C), indicating that depletion of kazrin either exacerbated endocytic uptake or inhibited endocytic recycling. The distribution of A647-Tfn labeled endosomes was also altered in the shKzrn treated cells, as compared with that of untreated cells or cells transfected with a control shRNA (shCTR). In wild-type (WT) and shCTR-treated cells, A647-Tfn accumulated in the juxtanuclear region, where the RE is usually located (Granger et al., 2014; Sheff et al., 2002; Shen et al., 2006; Tang and Marshall, 2012; Yamashiro et al., 1984). In contrast, TxR-Tfn labeled endosomes appeared more scattered toward the cell periphery in shKzrn treated cells (Figure 1—figure supplement 1B,C). The accumulation of endocytosed material at the periphery suggested that kazrin plays a post-internalization role in the endocytic pathway, possibly in the transport of material toward the juxtanuclear RE. shRNA transfection in Cos7 cells did not achieve complete kazrin depletion in a reproducible manner and it hampered complementation. To overcome these problems, we generated kazrin knockout MEFs (kazKO MEFs) using the CRISPR-Cas9 technology and we used a lentiviral system to subsequently create two cell lines that expressed GFP or GFP-kazrin C upon doxycycline induction (Figure 1—figure supplement 2A). Immunoblot analysis demonstrated that the expression level of GFP-kazrin C in the absence of doxycycline or upon a short overnight (up to 12 hr) incubation was similar to that of the endogenous kazrin (low expression, 1–4 times the endogenous kazrin expression level) (Figure 1—figure supplement 2B). Under these conditions, the GFP-kazrin C was barely detectable by fluorescence microscopy. This might explain why none of the commercially available or home-made anti-kazrin antibodies detected a specific signal in WT MEFs. Doxycycline incubation for longer periods (up to 24 hr induction) resulted in moderate expression (4–8 times the endogenous kazrin expression levels) (Figure 1—figure supplement 2B), but allowed us to clearly visualize its localization by microscopy (Figure 1—figure supplement 2C). To better discern on the possible effects of kazrin depletion on endocytic uptake or in subsequent trafficking events, WT and kazKO cells were exposed to a short, 10 min incubation pulse with Texas Red-Tfn (TxR-Tfn), fixed, and analyzed. In WT cells, TxRed-Tfn accumulated in a pericentriolar region adjacent to the nucleus, similar to Cos7 cells (Figure 1A and Figure 1—figure supplement 3A). No differences in the amount of internalized TxR-Tfn were observed between WT and kazKO MEF (Figure 1—figure supplement 4), suggesting that kazrin did not play a relevant role in the formation of endocytic vesicles from the PM, but it might rather work downstream in the pathway. In agreement with this view, and similar to the shKzrn Cos7, kazKO MEFs accumulated TxR-Tfn in the cell periphery, as compared with WT cells (Figure 1A and B). Juxtanuclear accumulation of TxR-Tfn was restored in kazKO MEF by low, physiological expression of GFP-kazrin C but not GFP (Figure 1A and B), indicating a direct role of kazrin in the process. No significant difference between the kazKO and the kazKO GFP-expressing cells could be detected in these experiments. Therefore, in order to simplify the experimental design, further assays were normalized to the closest isogenic kazKO background, namely the kazKO cells when compared to the WT, and the kazKO GFP expressing cells when compared to kazKO MEF expressing GFP-kazrin C. Figure 1 with 5 supplements see all Download asset Open asset Kazrin depletion impairs endosomal traffic. (A) Confocal images of wild-type (WT) and kazKO MEF or kazKO MEF expressing low levels (See Materials and methods (M & M)) of GFP or GFP-kazrin C, incubated with Texas Red-Tfn (TxR-Tfn) at 16 °C and chased at 37 °C for 10 min. Scale bar, 10 μm. Cell borders are indicated by dashed lines and nuclei in blue. (B) Scattered plot of the mean ± SD (Standard deviation) TxR-Tfn perinuclear enrichment (See M & M) for the cells described in A, after 10 min incubation at 37 °C. p-values of the two-tailed Mann-Whitney tests are shown. n>58 cells for each sample. Refer also to Figure 1—figure supplement 1 for the effects of kazrin depletion in Cos7 cells, Figure 1—figure supplement 2 for the strategy of kazKO MEF generation, Figure 1—figure supplement 3A for pericentriolar localization of internalized transferrin (Tfn) in WT cells, and Figure 1—figure supplement 4 for the effects of kazrin depletion on TxR-Tfn uptake in MEF. (C) Confocal images of the WT and kazKO MEF, or kazKO MEF expressing low levels of GFP or GFP-kazrin C, fixed and stained with anti-EEA1 and A568-conjugated secondary antibodies. A 17 μm2 magnified insets showing endosomes in the peripheral areas are shown on the right. Scale bar, 10 μm. Cell borders are indicated with dashed lines and nuclei in blue. (D) Scattered plots of the mean ± SD early endosome autoantigen 1 (EEA1) perinuclear enrichment (See M & M) in the cells described in C. The values were normalized to the corresponding kazKO cells (either kazKO or kazKO GFP). p-values of the two-tailed Mann-Whitney tests are shown. n>80 cells for each sample. Refer to Figure 1—figure supplement 3B for pericentriolar localization of EEA1 in WT cells and Figure 1—figure supplement 4 for the effects of kazrin depletion on the RAB11 perinuclear enrichment. (E) Line plot of the mean ± SD TxR-Tfn fluorescence intensity per cell in WT and kazKO MEFs, or kazKO MEFs expressing low levels of GFP and GFP-kazrin C, at the indicated time points after loading early endosomes (EEs) with TxR-Tfn at 16 °C and release at 37 °C to allow recycling (See M & M for further details). Data were normalized to the average intensity at time 0. p-values of the two-tailed Student t-tests are shown. n>16 cells per sample and time point. Figure 1—source data 1 Data for graphs presented in Figure 1B, D and E. https://cdn.elifesciences.org/articles/83793/elife-83793-fig1-data1-v2.zip Download elife-83793-fig1-data1-v2.zip To evaluate if the scattering of TxR-Tfn endosomes was due to a defect in the transfer of material form the EEs to the REs or if it was caused by the dispersal of the REs, we analyzed the distribution of the EE and the RE markers EEA1 (Early endosome autoantigen 1) and RAB11 (Ras-related in brain 11), respectively. We observed that kazKO MEFs accumulated peripheral, often enlarged, EEA1 positive structures, as compared with WT MEF (Figure 1C and D and Figure 1—figure supplement 3B). The juxtanuclear distribution of the REs, was however not significantly affected in the knock-out cells (Figure 1—figure supplement 5). Again, low expression of GFP-kazrin C but not GFP recovered the EEA1 juxtanuclear distribution (Figure 1C and D). The data thus suggested that kazrin promotes the transfer of endocytosed material toward the juxtanuclear region, where the RE is located. Consistent with the role of kazrin in endocytic traffic towards the RE, recycling of TxR-Tfn back to the PM was diminished in kazKO cells (Figure 1E), albeit not completely blocked. A complete block in recycling was not to be expected because, in addition to the RAB11 route, the TfnR can take a RAB4-dependent shortcut to the PM (Sheff et al., 2002). As for the juxtanuclear Tfn enrichment assays, the expression of GFP-kazrin C but not GFP restored the recycling defects installed in the kazKO MEF (Figure 1E). To further confirm the specific role of kazrin in endocytic recycling via the juxtanuclear RE, we analyzed its implication in cellular processes that strongly rely on this pathway, such as cell migration and invasion (Emery and Ramel, 2013; Fan et al., 2004; Jones et al., 2006; Kessler et al., 2012; Mammoto et al., 1999; Powelka et al., 2004; Ramel et al., 2013; Wilson et al., 2018; Yoon et al., 2005). Analysis of the migration of single WT and kazKO cells through Matrigel demonstrated that depletion of kazrin significantly reduced the migration speed, which, similar to endocytic traffic, was recovered upon re-expressing GFP-kazrin C at low levels, but not GFP (Figure 2A and B, Video 1). We also observed an increased persistency in the migration of kazKO cells (Figure 2—figure supplement 1), but it was not recovered with GFP-kazrin C re-expression (Figure 2—figure supplement 1). Increased persistency might be a secondary effect caused by the trafficking block to the RE, which accelerates recycling via the RAB4-dependent shortcut circuit (Perrin et al., 2013; White et al., 2007). The long recycling pathway also plays an important role in the last abscission step during cytokinesis (Fielding et al., 2005; Horgan et al., 2004; Pollard and O’Shaughnessy, 2019; Wilson et al., 2005). Consistent with kazrin playing a role in this process, kazKO cells had a significant delay in cell separation after cytokinesis, which was again restored by GFP-kazrin C expression (Figure 2C and D and Video 2). Figure 2 with 1 supplement see all Download asset Open asset Kazrin depletion impairs cell migration and division. (A) Paths described by individually migrating wild-type (WT) and kazKO MEF or kazKO MEFs expressing GFP or GFP-kazrin C at low levels (See M & M). The cells were embedded in Matrigel and tracked for 9 hr with a 10 min time lapse. All tracks start at the (0,0) coordinate in the graph. See also Video 1 for examples of individual migrating cells. (B) Scattered plot of the mean ± SD speed of cells described in (A). The data wre normalized to the mean of the corresponding KO cells (either kazKO or kazKO expressing GFP). p-values of the two-tailed Mann-Whitney tests are shown. n>100 cells per condition. See also Figure 2—figure supplement 1 for the effects of kazrin depletion on directionality. (C) Time-lapse epifluorescence images of WT and kazKO MEFs or kazKO MEFs expressing GFP or GFP-kazrin C at low levels, as they divide. Cells were recorded every 10 min. See also Video 2 for examples of individual dividing cells. Windows are 55 x 74 μm2 for WT MEF, 38 x 50 μm2 for kazKO MEF and 60 x 80 μm2 for GFP and GFP-kazrin C expressing MEF. (D) Mean time ± SD between substrate attachment and complete cell separation of the cells described in C. The data were normalized to the mean of the corresponding KO (kazKO or kazKO expressing GFP). p-values of the two-tailed Mann-Whitney tests are shown. n>68 dividing cells per condition. Figure 2—source data 1 Data for graphs presented in Figure 2B and D. https://cdn.elifesciences.org/articles/83793/elife-83793-fig2-data1-v2.zip Download elife-83793-fig2-data1-v2.zip Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Videos of individually migrating wild-type (WT) and kazKO MEF, and kazKO MEF expressing low levels of GFP and GFP-kazrin C. The cells were embedded in Matrigel and imaged with an epifluorescence microscope. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Videos of dividing wild-type (WT) and kazKO MEF, and kazKO MEF expressing low levels of GFP and GFP-kazrin C, from the moment the mother cell attached to the substrate until the daughter cells were completely separated. Scale bar = 10 μm. The cells were embedded in Matrigel and imaged with an epifluorescence microscope. Kazrin is recruited to EEs and directly interacts with components of the endosomal machinery through its C-terminal predicted IDR Next, we investigated whether endogenous kazrin was present in EEs. For that purpose, we initially used subcellular fractionation and immunoblot because the endogenous protein was not detectable by fluorescence microscopy, nor was GFP-kazrin C expressed at physiological levels. As shown in Figure 3A, endogenous kazrin neatly co-fractionated in the lightest fractions with EE markers such as the tethering factor EEA1 and EHD (Eps15 homology domain) proteins, most likely corresponding to EHD1 and EHD3. On the contrary, it only partially co-fractionated with a transitional early-to-late endosome marker (Vacuolar Protein Sorting 35 ortholog, VPS35) and did not with markers of recycling endosomes (RAB11) or the Golgi apparatus (Golgi Matrix protein 130, GM130) (Figure 3A). Moderately overexpressed GFP-kazrin C also co-fractionated with EEs, although it appeared slightly more spread towards the RE and Golgi fractions in the gradient (Figure 3A). Endogenous kazrin localization at EEs was confirmed by subcellular fractionation experiments in mIMCD3 cells (Figure 3—figure supplement 1). Figure 3 with 2 supplements see all Download asset Open asset Kazrin is an endosomal protein. (A) Left, immunoblots of Optiprep density gradient fractionations of membrane lysates of wild-type (WT) and kazKO MEF or kazKO MEF moderately expressing (See M & M) GFP or GFP-kazrin C. The membranes were probed with antibodies against the kazrin C N-terminus, EEA1, and EHD1 (EE markers), VPS35 (RAB5/RAB7 transition endosomal marker), RAB11 (RE/Golgi marker), GM130 (cis-Golgi marker) and BIP (Binding immunoglobulin protein) (ER marker). The antibody against EHD1 is likely to recognize other Eps15 homology domain (EHD) proteins. Band intensity plots per fraction for kazrin or the indicated intracellular membrane markers are shown on the right. The signal intensities of each fraction were normalized to the maximum for each antibody. All gradients were loaded with the same amount of total protein. Refer also to Figure 3—figure supplement 1 for co-fractionation of kazrin and early endosome autoantigen 1 (EEA1) in the lightest gradient fractions in IMCD3 cells. (B) Immunoblots of anti-GFP-agarose precipitates from lysates of kazKO MEF moderately expressing GFP or GFP-kazrin C, probed with antibodies against the indicated proteins. 10 µg of total protein were loaded as input. (C) Immunoblots of pull-downs from glutathione-Sepharose beads coated with GST, or GST fused to full-length EHD1 or EHD3, the clathrin heavy chain terminal domain (CHC-TD) or the γ-adaptin ear domain, incubated with purified 6xHis-kazrin C. The membranes were probed with an anti-kazrin antibody (ab74114, from Abcam) and stained with Ponceau red to visualize the GST fusion constructs. Refer also to Figure 3—figure supplement 2 for evidence indicating co-immunoprecipitation of endogenous kazrin with γ-adaptin and clathrin. (D) Immunoblots of pull-downs from glutathione-Sepharose beads coated with GST, or GST fused to the N- (amino acids 1–174) or C- (amino acids 161–327) terminal portions of kazrin C, incubated with non-denaturing extracts from MEFs. 10 µg of total protein were loaded as input. Ponceau red staining of the same membrane (lower panels) is shown to visualize the protein extract or the GST fusion constructs. (E) Prediction of IDRs in kazrin C. The graph shows the probability of each residue of being part of an intrinsically disordered region (IDR), according to the IUPred2A software. Residues in the shaded area have a consistent probability over 0.5 to form part of an IDR. (F) Immunoblots of a lipid-binding assay performed with either the purified GST-kazrin C C-terminal portion (amino acids 161–327) (GST-kaz-Ct) or an equivalent construct in which the poly-K region has been mutated to poly-A. The membranes used in this assay contain a concentration gradient of the indicated phosphoinositides. Membranes were probed with an anti-GST antibody. (G) Immunoblot of a liposome pelleting assay probed with an anti-GST antibody. GST or GST-kazrin C were incubated in the presence (+) or absence (−) of liposomes containing 5% phosphatidylinositol 3-phosphate (PI3P). Liposomes were recovered at 100.000 g for 1.5 hr. One equivalent of the input (T), one equivalent of the supernatant (S), and ten equivalents of the pellet (P) were loaded per sample. Figure 3—source data 1 Un-cropped blots for Figure 3A, B, C, D and G. https://cdn.elifesciences.org/articles/83793/elife-83793-fig3-data1-v2.zip Download elife-83793-fig3-data1-v2.zip To confirm the interaction of kazrin C with endosomes, we immunoprecipitated GFP-kazrin C from native cellular extracts and probed the immunoprecipitates for a number of proteins involved in endosomal trafficking. We detected specific interactions of GFP-kazrin C and γ-adaptin, a component of the Golgi and endosomal clathrin adaptor AP-1 (Adaptor Protein 1), as well as clathrin and EHD proteins (Figure 3B). No interaction with the retromer subunit VPS35, the tethering factor EEA1, or the clathrin adaptors GGA2 (Golgi-localized Gamma-ear-containing ADP-ribosylation factor-binding 2) or AP-2 (Adaptor Protein 2) could be detected in immunoprecipitation assays (Figure 3B), indicating that kazrin C binds the machinery implicated in endosomal traffic from EEs to or through REs (Caplan et al., 2002; George et al., 2007; Grant et al., 2001; Grant and Caplan, 2008; Jović et al., 2007; Lin et al., 2001; Naslavsky et al., 2006; Perrin et al., 2013; Rapaport et al., 2006). Pull-down assays with purified components demonstrated that kazrin C can directly interact with EHD1 and EHD3, the clathrin heavy chain terminal domain, and the γ-adaptin ear (Figure 3C). Pull-down assays from cell extracts showed that the EHD proteins and the AP-1 complex bound to the C-terminus of kazrin C, predicted to be an IDR, but not to the N-terminus (Figure 3D and E). Most kazrin interacting partners were previously defined to bind its N-terminal region predicted to form a coiled-coil (Groot et al., 2004; Sevilla et al., 2008b). The interaction of endogenous kazrin with γ-adaptin could also be confirmed in co-immunoprecipitation experiments from MEFs, using a polyclonal antibody against the C-terminus of kazrin C (Figure 3—figure supplement 2). In lipid overlay and liposome pelleting assays, we also found that purified kazrin C interacted with PhosphatidylInositol 3-Phosphate (PI3P) (Figure 3F and G), a lipid particularly enriched on EEs (Gillooly et al., 2000; Wang et al., 2019). The interaction required the poly-Lys stretch in the C-terminus of kazrin C (Figure 3F), previously proposed to constitute a nuclear localization signal (Groot et al., 2004). The data suggested that the predicted kazrin C IDR had multiple binding sites for EE components, and therefore, it might be required for its EE recruitment and its function in endosomal traffic. To investigate the role of the C-terminal region of kazrin C in its recruitment to endosomes and its function in endocytic traffic, we generated kazKO cells expressing a GFP-kazrin C construct lacking the C-terminal predicted IDR (lacking amino acids 161–327) (kazKO GFP-kazrin C-Nt), using the lentivirus system (Figure 4—figure supplement 1). We then analyzed its subcellular localization and its capacity to complement the kazKO endocytic defects, as compared with full-length GFP-kazrin C or GFP. As shown in Figure 4A and B, moderately expressed GFP-kazrin C significantly associated with the microsomal fraction containing the EEs. In contrast, GFP and GFP-kazrin C-Nt were mostly cytosolic, indicating that the C-terminal predicted IDR, which binds PI3P, γ-adaptin, and EHD proteins, might be required to bring kazrin to cellular membranes. Next, we proceeded to image cells expressing moderate levels of GFP-kazrin C and GFP-kazrin C-Nt, upon loading of EEs with TxR-Tfn at 16 °C. The previously reported localizations of kazrin C in the nucleus and at cell-cell contacts were evident in these cells (Figure 4—figure supplement 2; Groot et al., 2004). At the PM, GFP-kazrin C neatly co-localized with the adherens junction components N-cadherin, β-catenin, and p120-catenin, but not with desmoglein, a desmosomal cadherin (Figure 4—figure supplement 2). In addition to the previously reported localizations, GFP-kazrin C formed small foci, which associated with the surface of the TxR-Tfn labeled endosomes (Figure 4C and D; Figure 4—figure supplement 3 and Video 3). Co-localization of GFP-kazrin C foci with EHD-labeled structures could also be observed in the cell periphery (Figure 4—figure supplement 4 and Video 4). GFP-kazrin C-Nt and GFP staining at similar expression levels appeared mostly cytosolic, with nearly no visible (for GFP) or scarce (for GFP-kazrin C-Nt) foci per cell (Figure 4C to E). The few GFP-kazrin C-Nt foci observable appeared less associated with TxR-Tfn loaded endosomes, as compared to GFP-kazrin C (Figure 4C and D; Figure 4—figure supplement 3 and Videos 3 and 5). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Four 3D reconstructions of Z stacks of kazKO MEF expressing moderate amounts of GFP-kazrin C loaded with Texas Red-Tfn (TxR-Tfn) at 16℃ to accumulate endocytic cargo in early endosome s (EEs). Cells were shifted to 37℃ and fixed after 10 min. The windows are 5 × 5 µm2. Figure 4 with 4 supplements see all Download asset Open asset The predicted intrinsically disordered region (IDR) of kazrin C is required for its endocytic function. (A) Immunoblots of subcellular fractionations from kazKO cells expressing moderate amounts (See M & M) of GFP, GFP-kazrin C or a GFP-kazrin C construct lacking the C-terminal predicted IDR (GFP-kazrin C-Nt). Cells were lysed in a non-denaturing buffer and centrifuged at 186,000 g for 1 hr to separate membranes (Mic) from the cytosol (Cyt). 15 µg of the total extract (Tot), and 1 and 5 equivalents of the cytosolic and membrane fractions were loaded per lane, respectively. (B) Scattered plot of the mean ± SD percentage of the GFP-signal associated with the microsomal fraction (Mic) in kazKO MEF expressing moderate amounts of GFP, GFP-kazrin C or GFP-kazrin C-Nt. Student´s t-tests p-values are shown. n=5 independent experiments for each sample. See M & M for experimental details. (C) MIP of confocal images of kazKO MEF expressing moderate amounts of GFP, GFP-kazrin C, or GFP-kazrin-Nt, loaded with 20 µg/ml of Texas Red-Tfn (TxR-Tfn) at 16 °C to accumulate endocytic cargo on early Endosomes endosomes (EEs). The images from the GFP and TxR channels and the merge from 5 × 5 µm2 fields are shown on the right. (D) Frames showing consecutive 60o turn snapshots of the 5 × 5 µm2 3D reconstruction animations of the insets shown in C for GFP-kazrin C and GFP-kazrin-C-Nt, showing the association of GFP-kazrin C foci, but not GFP-kazrin C-Nt, with TxR-Tfn-loaded EEs. (E) Scattered plot of the mean ± SD of the number of condensates per cell, visible with the GFP filter channel in the kazKO cells described in (B). p-values of the two-tailed Mann-Whitney test are shown. n=29 cells for each sample. Refer also to Video 3 for four 3D reconstruction animations of TxR-Tfn loaded endosomes associated with GFP-kazrin C, and Video 5 for GFP-kazrin C-Nt; Figure 4—figure supplement 2 for co-localization of GFP