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 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 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 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