Fig 1: Loss of ARP3 Results in Altered Focal Adhesion Morphology and Increased Actomyosin Activity(A and B) Staining for the focal adhesion (FA) component PAXILLIN (red) revealed confluent FA clusters in primary Arp3 KO podocytes (B-1), which were connected via filamentous actin webs (wild type (A) and KO (B)). F-actin (blue) was stained by Phalloidin.(C and D) Quantification of average focal adhesion area (C) and major axis (D) showed increased values for both parameters in respective KO cells (25 wild-type and 28 Arp3 KO cells were analyzed; ***p < 0.001, ****p < 0.0001).(E and F) Average focal adhesion area (E) and lamellipodium formation (F) was not obviously impaired in primary N-WASP KO podocytes in 2D culture conditions (n = 36 cells per genotype were analyzed, n.s., non significant; FA were stained by PAXILLIN and F-actin by phalloidin; white arrows indicate lamellipodia).(G) Analysis of focal adhesion clusters due to their individual size indicated a shift toward enlarged adhesion clusters in Arp3 KO podocytes (25 WT and 26 Arp3 KO cells were analyzed; **p < 0.01, ****p < 0.0001).(H) This shift in FA size was paralleled by a change in the distribution of individual FA classes: loss of Arp3 resulted in the accumulation of FA plaques (n = 18 cells per genotype were analyzed; n.s., non significant, **p < 0.01).(I and J) Analysis of PAXILLIN fluorescence intensities (FI) in wild type podocytes (I-1) and in respective Arp3 KO cells (I-2) revealed altered intensity patterns and increased mean PAXILLIN intensity (J) per focal adhesion (n = 100 mature FA per genotype were analyzed; ****p < 0.0001).(K and L) Immunofluorescence for NMHC-IIA (K) demonstrated an increased sarcomere-like pattern of actin fibers (L) in respective Arp3 KO podocytes (yellow lines indicate positions for line scan measurements, and black arrows indicate myosin-2 peaks).(M) Immunofluorescence staining for pp-MLC and the focal adhesion marker PAXILLIN illustrated prominent, fiber-like accumulations of pp-MLC linked to FAs in Arp3 KO podocytes.(N) Staining for pp-MLC revealed increased MLC activation levels in Arp3 KO podocytes, condensed in arc regions between pseudopodial protrusions. Also a ring-like distribution was observed in the majority of Arp3 KO podocytes. (Yellow lines indicate representative positions for line scan measurements).(O) Line scan profiles for pp-MLC showed highest activation levels close to the cell edge in Arp3 KO cells (values represent mean intensities of 6 cells per genotype; gray error bars represent SEM).(P) Quantification of fluorescence intensity for pp-MLC on either glass or soft gels revealed higher MLC activation levels for Arp3 KO podocytes (37 wild-type and 41 Arp3 KO cells on glass; 31 wild-type and 32 Arp3 KO cells on gels were analyzed; **p < 0.01, ****p < 0.0001).(Q and R) Staining for pp-MLC in glomeruli from either wild-type (Q-1) or Arp3 KO (Q-2) animals showed high levels of phosphorylated MLC in the podocyte compartment, as visualized with the podocyte-specific marker NEPHRIN (mean podocyte pp-MLC intensity of n = 4 WT and 4 KO animals were statistically analyzed; *p < 0.05; the mean pp-MLC intensity per animal was calculated from mean pp-MLC intensity of the segmented podocyte compartment of at least 20 glomeruli per animal).(S and T) Immunofluorescence staining for the mechanical tension marker YAP in glomeruli from either wild-type or Arp3 KO (S) animals showed increased percentages of YAP-positive podocyte nuclei in KO animals (T). Podocyte nuclei were visualized by the podocyte-specific marker WT1 (the mean percentage of YAP-positive podocyte nuclei per glomerulus of n = 5 WT and 5 KO animals were analyzed; white arrow bars indicate podocyte nuclei with co-occurring positivity for YAP, **p < 0.01; the percentage of YAP positive podocyte nuclei per glomerulus per animal was calculated from at least 20 glomeruli per animal). All data are represented as mean ± SEM.
Fig 2: ARP3-Mediated Actin Polymerization Is Required for Efficient Protrusion Formation(A and B) Primary podocytes in 3D culture conditions form protrusions with increasing levels of branching; immunofluorescence staining revealed that terminally branched protrusions consisted mainly of filamentous actin, whereas major protrusions also showed positivity for tubulin (B). (C) Acute treatment with the actin polymerization inhibitor Latrunculin A markedly suppressed protrusion formation, as shown by a quantifcation for main protrusions (D) (n = 38–41 analyzed cells; ****p < 0.0001).(E) Schematic depicting the generation of a fluorescence-based reporter line: after podocyte-specific Cre-recombination mGFP is selectively expressed in only the glomerular epithelial cell compartment. Single-cell isolation and further FACS-based purification results in a primary culture system with proven genetic origin.(F and G) 3D reconstruction of primary podocytes in 3D culture systems (wild typ podocytes [F]) unmasked the deficiency of Arp3 knockout podocytes (G) to form terminally branched protrusions.(H) Color code describing segmentation of individual protrusion classes into main, primary, and secondary order protrusions.(I) Loss of ARP3 and N-WASP resulted in significantly less cells with branching protrusion phenotypes (at least 150 cells per condition were analyzed, and data were accumulated over at least 3 experiments; n.s., non significant, ***p < 0.001, ****p < 0.0001).(J and K) Reconstructed images of individual z stacks were used as a basis for the segmentation analysis (J) and length measurements of protrusions (K): due to ARP3 and N-WASP deficiency, higher order branched protrusions were diminished and apparent protrusions were significantly shorter compared to control conditions (17 wild-type, 32 Arp3 KO, and 20 N-WASP KO primary podocytes were reconstructed and analyzed; n.s., non significant, *p < 0.05, **p < 0.01, ****p < 0.0001).(L–O) Analysis of the cortical actin cytoskeleton of wild-type (L) and respective KO (M) cells under 3D culture conditions. Representative line scans (N) of filamentous actin intensities in single planes demonstrated altered levels of cortical actin due to loss of ARP3 (19 wild-type and 16 Arp3 KO cells were reconstructed, and ratios between cortical and cytoplasmic actin intensities were measured at multiple localizations (O); orange dashed lines indicate areas of line scan analysis, black arrows mark individual representative cell borders; ****p < 0.0001; MFI, mean fluorescence intensity).(P) Scanning electron microscopy (SEM) detected pronounced simplification, reduced branching and misconfiguration of podocyte processes in Arp3 KO ((P-1) shows a representative image of wild type controls, whereas (P-2) and (P-3) demonstrate representative images from Arp3 KO animals; colorized SEM image (P-3) depicting segmentation of podocyte processes, each colorized dashed line indicates one individual major process; white arrows and red asterisks mark secondary processes). (Q) Podocyte process branching was quantified from semi-planar SEM areas (n = 5 wild-type and 4 Arp3 KO mice were analyzed at p8; ****p < 0.0001). All data are represented as mean ± SEM.
Fig 3: Competition between Different F-Actin Networks Modulates Protrusion Phenotypes and FA Morphology of Podocytes(A and B) Decrease of intracellular tension in Arp3 KO primary podocytes with the myosin-2 inhibitor blebbistatin (Bleb) resulted in the formation of simplified cellular protrusions compared to blebbistatin-treated WT cells (A). Cell morphology and thereby protrusion generation was quantified by circularity scores (B). Quantification showed that co-treatment with blebbistatin results in a decrease of the circularity score in Arp3 as well as N-WASP KO podocytes, reflecting a more protrusive phenotype. Suppression of protrusion formation by SMIFH2-mediated formin inhibition is resistant to co-treatment with blebbistatin. (44 DMSO and 43 blebbistatin treated wild-type cells; 73 DMSO and 70 blebbistatin treated Arp3 KO cells; 50 DMSO and 53 blebbistatin treated N-WASP KO cells; 70 SMIFH2+DMSO and 100 SMIFH2+blebbistatin treated wild-type cells were analyzed; n.s., non significant, ****p < 0.0001).(C) Definition of a branching index (major protrusion per end tips (minor protrusions) allowed a more detailed analysis of protrusion complexity and quality. Quantification for this branching index revealed simplified cellular protrusions with reduced branching levels for blebbistatin treated Arp3 KO cell podocytes compared to blebbistatin treated WT cells. (35 WT and 36 Arp3 KO cells were analyzed; ****p < 0.0001).(D–J) Inhibition of myosin-2 via blebbistatin resulted in a dose-dependent decrease in focal adhesion (FA) size in Arp3 knockout podocytes (WT - (D-F) and Arp3 KO - (G-I)), indicating that FA plaques represent myosin-2-dependent structures in these cells. Quantification of FA area was performed ([J]; white arrows indicating FA sites; 20 cells per condition were analyzed; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).(K) Correlation of average focal adhesion (FA) size to the level of lamellipodium formation shows an inverse correlation between these cellular processes, indicating also an inverse correlation between Arp2/3 and (acto-) myosin-2 activity as an underlying mechanism. Calyculin-A and blebbistatin were used, respectively, to either activate or inhibit myosin-2 activity. Rac1 inhibition by NSC-23766 or CK-869 was used to inhibit Arp2/3-mediated actin nucleation; Nocodazol was used to stabilize focal adhesions (FA) in a myosin-independent fashion. (24 to 44 immortalized human podocytes were analyzed per condition; *p < 0.05, **p < 0.01; ****p > 0.0001).(L–P) Treatment with the formin inhibitor SMIFH2 resulted in a dissolution of mature FAs, which was accompanied by an increase in the number of nascent adhesions in wild-type cells (M) and a loss of focal adhesion formation in Arp3 KO podocytes (N) (compared to untreated Arp3 KO cells - (L); n.s., non significant; **p < 0.01, ***p < 0.001, ****p < 0.0001). (O) Focal adhesion area and (P) FA number were quantified.(Q and R) Washout experiments employing cytochalasin-D and co-treatments with inhibitors for myosin-2 activity (blebbistatin), formin nucleation (SMIFH2), or Arp2/3 nucleation (CK-869) revealed specific actin network activities as a prerequisite for the inverse correlation between dynamic focal adhesion maturation (Q) and cell size spreading (R) of podocytes (31–104 cells per condition were analyzed for FA maturation and 40–215 cells were analyzed for cell size; *p < 0.05; ***p < 0.001; ****p > 0.0001). All data are represented as mean ± SEM.
Fig 4: Loss of ARP3 Leads to Decreased Adhesive Properties of Podocytes under Mechanical Stress Conditions(A) Schematic illustrating the application of cyclic stretch on primary podocytes by culture on a flexible membrane.(B–E) Immunofluorescence staining of the actin cytoskeleton in wild-type (B and C) and knockout podocytes (D and E) analyzed under non-stressed and stressed conditions, respectively.(F) Quantification of stress fibers under stressed conditions revealed an adaptive deficit of actin filaments in Arp3 knockout podocytes when compared to wild-type controls (at least 50 cells per condition were analyzed; n.s., non significant, **p < 0.01;****p < 0.0001).(G–L) Analysis of focal adhesion morphology ((G and H) depict wild type podocyte FAs; (I and J) depict Arp3 KO podocytes) under stressed conditions indicated an increase in focal adhesion size in wild-type cells ((K) - FA area quantification and (I) - FA major axis quantification for each condition), whereas no changes were observed in respective knockout podocytes (at least 11 cells per condition and genotype, n.s., non significant, *p < 0.05; **p < 0.01).(M and N) Quantification of traction force microscopy (M) on collagen-coated soft gel matrices showed increased traction forces in Arp3 KO podocyte and increased strain energy (N) (22 WT and 32 KO cells, ***p < 0.001, ****p < 0.0001).(O) Pseudo-colored force maps of wild-type and Arp3 KO podocytes on soft matrices (note difference in individual traction scale between wild-type and KO cells).(P) Immunofluorescence staining of Arp2/3 inhibited podocytes under stressed conditions. (Q) Quantification of adherent cells under stressed conditions (n = 3 individual experiments; ***p < 0.001). All data are represented as mean ± SEM.
Fig 5: ARP3 Is Required for Efficient Podocyte Adhesion and Maintenance of Foot Process Morphology(A and B) Immunfluorescence for WT1-positive podocytes in wild type and Arp3 knockout animals.(C) Quantification of podocyte number per glomerulus a pronounced decrease in conditional Arp3 knockout animals, indicating significant detachment of podocytes (at least 106 glomeruli of 3 mice per genotype and age; n.s., non significant, ****p < 0.0001).(D) Western blot for WT-1 positive cells in urine detected only detached podocytes in respective knockout animals.(E) Schematic illustrating the crossing strategy for the generation of tetracycline inducible Arp3fl/fl*hNphs2*rtTA*TetOCre knockout animals.(F) Immunofluorescence for WT1-positive podocytes in Arp3 inducible knockout animals.(G) Quantification of podocyte numbers in inducible Arp3 knockout animals.(H) Albumin to creatinine ratio (mg/mg) measurements detected increased levels of proteinuria already in the second week of doxycycline induction: levels increased furthermore over a period of 4 weeks (3–11 animals per time point and genotype were analyzed; *p < 0.05, **p < 0.01, ****p < 0.0001).(I) PAS staining demonstrated focal segmental glomerulosclerosis in inducible Arp3 KO animals.(J) Scanning electron microscopy demonstrates simplified FP morphology in Arp3 inducible knockout animals.(K and L) SIM revealed aberrant kidney filtration morphology correlating to simplified FPs in Arp3 inducible knockout animals (measurements were accumulated over 2 individual animals per genotype; ***p < 0.001).(M–O) Graphical summary of proposed Arp2/3-mediated functions in podocytes (M): nucleation of dendritic actin networks by the Arp2/3 complex promotes FA assembly at cell protrusion sites and controls FA maturation by limiting non-muscle myosin II (NM-II) complex activity (N). (O) Arp2/3 allows the development of podocyte protrusions by modulating NM-II activity and directly promotes complex arborization of podocyte protrusions in vitro and in vivo. All data are represented as mean ± SEM.
Supplier Page from Abcam for Anti-Arp3 antibody [EPR10428(B)]