AIF1L regulates actomyosin contractility and filopodial extensions in human podocytes

<div><p>Podocytes are highly-specialized epithelial cells essentially required for the generation and the maintenance of the kidney filtration barrier. This elementary function is directly based on an elaborated cytoskeletal apparatus establishing a complex network of primary and secondary processes. Here, we identify the actin-bundling protein allograft-inflammatory-inhibitor 1 like (AIF1L) as a selectively expressed podocyte protein <i>in vivo</i>. We describe the distinct subcellular localization of AIF1L to actin stress fibers, focal adhesion complexes and the nuclear compartment of podocytes <i>in vitro</i>. Genetic deletion of <i>AIF1L</i> in immortalized human podocytes resulted in an increased formation of filopodial extensions and decreased actomyosin contractility. By the use of SILAC based quantitative proteomics analysis we describe the podocyte specific AIF1L interactome and identify several components of the actomyosin machinery such as MYL9 and UNC45A as potential AIF1L interaction partners. Together, these findings indicate an involvement of AIF1L in the stabilization of podocyte morphology by titrating actomyosin contractility and membrane dynamics.</p></div

AIF1L prevents formation of filopodial extensions in podocytes.

<p>(a-c) Immunofluorescence studies revealed the presence of numerous thin filopodial extensions evolving prom pre-existing lamellipodial structures in both AIF1L knockouts (dashed boxes indicate areas of higher magnification; white arrows indicate filopodial extensions). (d-e) Quantification of filopodia showed higher numbers per cell in conditions of AIF1L loss; also, measurements of filopodia demonstrated overall increased length in respective AIF1L knockout clones (n = 53 WT, 53 KO-A and 42 KO-B podocytes out of 3 independent experiments were analyzed for filopodia number; filopodia from those cells were measured for length, n = 277 WT, 1175 KO-A and 778 KO-B filopodia; **** p<0.0001). (f-h) Seeding of podocytes on thin fibrillar collagen gels for 3 hours resulted in an overall multi-polar morphology with several membrane protrusions. Numerous filopodia extended from those areas of membrane protrusion in AIF1L knockout cells. (black arrows indicate filopodial extensions; dashed boxes indicate areas of magnification).</p

AIF1L localizes to filamentous actin, adhesion sites and cellular protrusions.

<p>(a) Expression of GFP-AIF1L in wild type podocytes localized to focal adhesion sites, as revealed by co-staining with the FA marker PAXILLIN (PXN—dashed boxes indicate sites of magnification, white arrows indicate FA tips or nascent FAs with less intense co-labeling of AIF1L and PAXILLIN). (b) In co-stainings with the filamentous actin marker phalloidin and the nuclear dye DAPI AIF1L localized to stress fibers and to the nuclear compartment (dashed boxes indicate sites of magnification; white asterisk indicates localization of AIF1L to the nucleus). (c) Filopodial extensions and the leading edge zone showed also an accumulation of AIF1L (white arrows indicate filopodial extensions; white asterisks indicate the leading edge zone; pictures in c were gamma and intensity adjusted to increase filopodia visualization).</p

AIF1L knockout clones exhibit an impaired actomyosin contractility reserve.

<p>(a-c) Western blot experiments and quantification by densitometry demonstrated decreased levels of MYL9 as well as MYH9 in respective AIF1L knockout clones, whereas UNC45A or MYPT were not affected (n = 6 WT and KO WB intensities out of 3 independent experiments; *** p<0.001; **** p<0.0001). (d-g) Treatment of wild type cells and AIF1L knockout clones with the myosin-II inhibitor blebbistatin resulted in a more rapid dissolution of FA complexes in conditions of AIF1L loss, indicating a decreased actomyosin contractility reserve (n = 29 WT, 27 KO-A and 29 KO-B podocytes out of 3 independent experiments were analyzed; * p<0.05; **** p<0.0001). (h-j) Washout experiments for blebbistatin showed that in conditions of reconstituting actomyosin contractility podocytes show a high generation rate of filopodial protrusions (note that these structures are associated with FA sites at the filopodial basis indicated by white arrows; white dashed boxes indicate areas of magnification;(n = 20 podocytes per condition were analyzed; n.s.–non significant, **** p<0.0001)).</p

AIF1L interacts with components of the actomyosin machinery and nuclear proteins.

<p>(a) Schematic depicting the labeling strategy of wild type and AIF1L knockout clones employing the SILAC technology, and the consecutive immunoprecipitation as well as sample preparation for analysis via mass spectrometry. (b) Color-coded tabular presentation of proteins according to their enrichment score (normalized to the AIF1L knockout control) in two replicates. Red colored protein names belong to the non-muscle myosin-II actomyosin machinery. (c) GO-Term mapping of interaction partners from the knockout controlled immunoprecipitation experiments. Aside from proteins belonging to the actomyosin machinery, also ERM proteins and chromatin regulating proteins were mainly detected. (d) Validation experiments employing western blot confirm the association of MYL9, MYH9 and the chaperone UNC45A with AIF1L.</p

Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development, Function or Stress Response

<div><p>The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive <i>Cxadr</i> knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, <i>Cxadr</i> is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of <i>Cxadr</i> for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific <i>Cxadr</i> knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of <i>Cxadr</i> on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of <i>Cxadr</i>.</p></div

Nephrotoxic serum (NTS) enhances podocyte specific CXADR expression, but lack of CXADR does not influence the course of NTS induced disease.

<p><b>(A-C)</b> On both d4 and d5 after NTS injection CXADR was increased in glomerular podocytes as shown in co-labeling experiments with NEPHRIN (control: white arrows—parietal epithelial cells, NTS: arrow heads—podocytes). As can be easily depicted from the NEPHRIN specific panel <b>(A”, B”, C”)</b> NEPHRIN abundance is greatly reduced during the course of the disease, underlining the severity of the chosen stress model. <b>(D)</b> Schematic of the injection scheme and follow-up using urine collections. <b>(E&F)</b> Western blot using decapsulated glomerular lysates was used to quantify CXADR expression which was increased two fold 5 days after NTS injection. <b>(G&H)</b> As shown with immunofluorescence stainings CXADR induction was absent in podocyte specific <i>Cxadr</i> knock-out animals. <b>(I)</b> Proteinuria developed similarly in wild-type and knock-out animals showing no functional differences.</p

Glomerular function, histology and ultrastructure are maintained in podocyte specific <i>Cxadr</i> knock-out animals.

<p><b>(A)</b> Functional assessment only showed a slightly elevated albuminuria in <i>Cxadr</i> deficient animals at P2 which was lost during further maturation of the kidney. <b>(B&C)</b> Histology of glomeruli was normal in podocyte deficient <i>Cxadr</i> animals as was <b>(D&E)</b> ultrastructural assessment by SEM <b>(D–D”</b>, <b>E–E”)</b> and TEM <b>(D”‘</b>, <b>E”‘)</b>.</p

<i>Cxadr</i> is highly expressed during kidney development.

<p><b>(A-D)</b> In-Situ-Hybridization showed intense expression of <i>Cxadr</i> in E15.5 embryos, especially in the nervous system, gut, lung and kidney. <b>(E&F)</b> After birth at P1 and P14 <i>Cxadr</i> could be detected in glomerular cells as well as in the nephron (<i>thick ascending limb of Henle</i>). <b>(G)</b> Western blot confirmed CXADR expression in heart, kidney, lung, brain and more specifically in isolated murine podocytes. <b>(H-K)</b> Developmental assessment of <i>Cxadr</i> expression during kidney development started in the S-shaped body phase where podocytes and parietal epithelial cells formed a continuous stretch of cells. <b>(L&M)</b> In both, podocytes (arrows) and parietal epithelial cells (asterisk) CXADR could be robustly detected in the early capillary loop stage, while mature glomeruli only possessed reduced amounts of CXADR in podocytes.</p

Adriamycin enhances podocyte specific CXADR expression, but lack of CXADR does not influence the course of Adriamycin induced FSGS.

<p><b>(A)</b> In adult wild-type animals CXADR was restricted to parietal epithelial cells (white arrows). <b>(B)</b> Administration of Adriamycin led to an increased glomerular CXADR expression which could be localized to glomerular podocytes using co-labeling experiments with NEPHRIN (white arrow heads). <b>(C)</b> Adriamycin injection scheme and subsequent urine measurements. <b>(D&E)</b> Western blots of decapsulated glomerular lysates were used to quantify CXADR expression in podocytes which indeed was upregulated two-fold. We next assessed abundance and distribution of the slit diaphragm molecules PODOCIN and NEPHRIN after adriamycin injection, and could not discern any differences between the two genotypes <b>(F-F”, G-G”</b> dashed lines-podocyte compartment<b>)</b>. In contrast to NEPHRIN the pattern of ZO-1 expression was largely maintained in both genotypes. <b>(H-H”, I-I”</b> dashed lines—podocyte compartment<b>).</b> We next assessed functional consequences on two different mouse genetic background strains. <b>(J)</b> Wild-type and knock-out animals on a C57Bl6/NCrl were analysed to check for increased susceptibility of knock-out animals while <b>(K)</b> wild-type and knock-out animals on an ICR background were used to test for a protective effect of podocyte specific CXADR deficiency. On both mouse genetic backgrounds we could not discern a difference between wild-type and knock-out mice over a follow-up period of 5 weeks.</p