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Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation

† † † Changkyu Gu,* Ha Won Lee, Garrett Garborcauskas,* Jochen Reiser, Vineet Gupta, and Sanja Sever*

*Department of Medicine, Harvard Medical School, Division of Nephrology, Massachusetts General Hospital, Charlestown, Massachusetts; and †Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois

ABSTRACT Rho family GTPases, the prototypical members of which are Cdc42, Rac1, and RhoA, in podocytes via a parallel signaling path- are molecular switches best known for regulating the actin cytoskeleton. In addition way to RhoA. to the canonical small GTPases, the large GTPase dynamin has been implicated in To induce actin polymerization, dy- regulating the actin cytoskeleton via direct dynamin-actin interactions. The physio- naminmustformDynOLIGO.12 The logic role of dynamin in regulating the actin cytoskeleton has been linked to the availability of Bis-T-23 (Aberjona Labo- maintenance of the kidney filtration barrier. Additionally, the small molecule Bis-T- ratories, Inc., Woburn, MA) allowed us 23, which promotes actin–dependent dynamin oligomerization and thus, increases to examine whether DynOLIGO–induced actin polymerization, improved renal health in diverse models of CKD, implicating actin polymerization affects the forma- dynamin as a potential therapeutic target for the treatment of CKD. Here, we show tion of FAs and stress fibers in podocytes. that treating cultured mouse podocytes with Bis-T-23 promoted stress fiber forma- The effect of Bis-T-23 on the actin cyto- tion and focal adhesion maturation in a dynamin-dependent manner. Furthermore, skeleton in mouse podocytes (Figure 1A) Bis-T-23 induced the formation of focal adhesions and stress fibers in cells in which was examined using a fully automated the RhoA signaling pathway was downregulated by multiple experimental ap- high–throughput assay that measures proaches. Our study suggests that dynamin regulates focal adhesion maturation distinct parameters in thousands of cells by a mechanism parallel to and synergistic with the RhoA signaling pathway. Iden- (each diamond in Figure 1, B–D repre- tification of dynamin as one of the essential and autonomous regulators of focal sents data averaged from one well with adhesion maturation suggests a molecular mechanism that underlies the beneficial 200–300 cells; six wells were used per effect of Bis-T-23 on podocyte physiology. condition). Bis-T-23 resulted in the dou- bling of the amount of stress fibers per J Am Soc Nephrol 28: 446–451, 2017. doi: 10.1681/ASN.2016010008 cell (Figure 1B), the doubling of their thickness (Figure 1C), and an increase in the intensity staining for paxillin at Cells exhibit the extraordinary ability to activation cycles of the Rho family GTPa- FAs (Figure 1D). The EC for all three adjust their cytoskeletal organization ses Cdc42, Rac1, and RhoA play an essential 50 parameters was approximately 13.5 mM, with regard to changes in their immedi- role during FA formation and maturation.6 approximately equivalent to the EC50 for ate surroundings.1,2 Because podocytes Interestingly, whereas mice lacking Cdc42 OLIGO Dyn as determined in vitro (EC is are exposed to filtration forces, their in podocytes developed proteinuria and 50 approximately 12.5 mM).14 Maturation of ability to efficiently adhere to the base- foot process effacement and ultimately focal complexes into stress fiber–bound ment membrane is critical for their role died as a result of renal failure,7,8 mice in maintenance of the kidney filtration lacking Rac1 or RhoA were overtly normal 7,8 barrier. As such, podocyte detachment and lived to adulthood. Additionally, a Received January 4, 2016. Accepted June 1, 2016. is a key factor for CKD progression.3–5 largeGTPasedynaminwasalsofound – 9,10 Published online ahead of print. Publication date Focal adhesions (FAs), integrin based to be essential for kidney function. Al- available at www.jasn.org. multiprotein complexes tightly associ- though dynamin is best known for its ated with the actin cytoskeleton, com- role in endocytosis,11 dynamin also pro- Correspondence: Dr. Changkyu Gu or Dr. Sanja via Sever, 149 13th Street, Charlestown, MA 02129. prise the cellular machinery essential motes actin polymerization direct Email: [email protected] or ssever@ for the interactions of the cells with dynamin-actin interactions.12,13 In this mgh.harvard.edu their environment. Synergistic integrin- study, we examined the possibility that Copyright © 2017 by the American Society of syndecan signaling and alternating dynamin might regulate FA maturation Nephrology

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Figure 1. DynOLIGO induces formation of FAs and stress fibers. (A) Wild–type or Dyn2KD mouse podocytes were treated with 30 mMBis-T- 23 or 0.1% DMSO (vehicle control) for 30 minutes. FAs and actin filaments were visualized with anti-paxillin antibody and rhodamine- phalloidin, respectively. Scale bar, 20 mm. (B–D) Dose-response curves showing the effects of increasing concentrations of Bis-T-23 on (B) the number of actin fibers, (C) the thickness of actin fibers, and (D) paxillin intensity. Each data point represents the value calculated from a single well (approximately 1500 cells per well). (E) Western blot analyses of dynamin and FA proteins in wild–type or Dyn2KD mouse podocytes after indicated treatments and (F) bar graphs depicting the ratio of phosphorylated proteins to total proteins normalized to the control cells. The data are plotted as means6SD (n=3). **P,0.01. (G) Bar graphs depicting distribution of FAs on the basis of their size. Data represent measurements of .50 cells and are plotted as means6SD (n=3). ***P,0.001. (H and I) Bar graphs depicting the numbers of (H) FAs and (I) actin fibers under the conditions indicated. Data represent measurements of .50 cells and are plotted as means6SD (n=3). ***P,0.001.

FAs is associated with changes in protein (Figure 1, E and F), whereas the addition of dynamin-rich centers (Supplemental composition and tyrosine phosphoryla- of the dynamin inhibitor Dynole (Abcam, Figure 1, E, red circles and F).17 Previously, tion and a corresponding increase in Inc., Cambridge, MA)16 exhibits the op- we have shown that dynamin clustered size.15 In accordance with immunofluo- posite effect (Figure 1, E and F). Bis-T-23 with paxillin on actin filaments, marking rescence data (Figure 1D), Bis-T-23 leads also markedly augments the intensity of those clusters as FAs.12 Finally, analysis of to an approximately 1.5-fold increase in p-paxillin, zyxin, and dynamin at FAs thesizeofFAsusingconfocalmicroscopy the levels of phosphorylated paxillin (Supplemental Figure 1, A–C). Electron reveals a decrease in the number of imma- (p-paxillin) and phosphorylated FAK microscopy of podocytes treated with ture focal complexes and a subsequent in- without affecting their overall levels Bis-T-23 shows an increase in the number crease in extralarge FAs (Figure 1G) that

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Figure 2. DynOLIGO regulates actin cytoskeleton independently from RhoA signaling. (A) Schematic diagram of parallel signaling pathways mediated by two GTPases, RhoA and dynamin, for the formation of FAs. (B and C) Western blot analyses of (B) total RhoA and (C) Rac1 and Cdc42 levels in podocytes. (D) Bar graphs depicting the amount of GTP-bound RhoA, Rac1, and Cdc42 in podocytes. The data are plotted as means6SD (n=3). **P,0.01; ***P,0.001. (E) Organization of the actin cytoskeleton and FAs in podocytes in which Rho- dependent signaling has been downregulated by diverse strategies. RhoAKD,mDia1KD, or GelsolinKD cells were infected with

448 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 446–451, 2017 www.jasn.org BRIEF COMMUNICATION containanincreaseinp-paxillin(Supple- oligomerization in podocytes is compara- forms of RhoA, Rac1, and Cdc42 (Figure mental Figure 1, B and D). ble with that observed for Dyn1 in the 2, B–D). In these ELISA-based assays, se- To test whether the effect of Bis-T-23 presence of Bis-T-23. Thus, the observed rumandEGFwereusedtoactivateRhoA on the FAs was dynamin dependent, Bis- effects on the stress fibers and FAs shared and Rac1/Cdc42, respectively (Figure 2D). T-23 was added to cells in which the by Dyn1E/K and Bis-T-23 and the inability Together, these data suggest that the effect ubiquitously expressed dynamin 2 of Bis-T-23 to promote oligomerization of of Bis-T-23 in cells is not associated with (Dyn2) isoform had been downregulated Dyn1K/E and Dyn1I690K in cells together other GTPases. using a short hairpin RNA.12 Downregu- support the hypothesis that actin- We next examined whether Bis-T-23 lation of endogenous Dyn2 (Dyn2KD) dependent DynOLIGO plays a role in the could induce the formation of FAs and results in a striking loss of both FAs formation of FAs and stress fibers. Of stress fibers, even after RhoA signaling and stress fibers, with concomitant dra- note, Dyn1E/K and Dyn1K/E exhibited no has been impaired. Downregulation of matically altered cell shape (Figure 1, A, effect on endocytosis,12 which is consistent RhoA (RhoAKD) (Supplemental Figure H, and I).12 Bis-T-23 fails to restore both with the wild–type biochemical character- 3, A and B) or one of its downstream FAs and stress fibers in Dyn2KD cells istics of the mutants.12,14 In addition, Bis- effectors mDia1 (mDia1KD) (Supple- (Figure1,A,H,andI).Furthermore, T-23 has no effect on lipid–dependent mental Figure 3, C and D) using short in Dyn2KD cells, Bis-T-23 is unable to dynamin oligomerization.14 Furthermore, hairpin RNA results in a loss of FAs and stimulate the phosphorylation of FA the dynamin mutant impaired in interac- stress fibers (Figure 2, E–G). Bis-T-23 proteins (Figure 1, E and F). Of note, tions with known actin binding proteins, partially rescues, whereas dynamin in- although the neuronal isoform, Dyn1,11 such as cortactin, was originally shown to hibitor Dynole further decreases both is expressed in podocytes at low level, be able to rescue formation of FAs and of the parameters (Figure 2, F and G). it seemed to be insufficient to support stress fibers in Dyn2KD cells.12 Finally, the Furthermore, the inhibition of ROCK by the Bis-T-23–induced FA formation, inability of Bis-T-23 to rescue domi- Y-27632 or myosin II activity by blebbis- suggesting that Bis-T-23 required the nant negative phenotypes of Dyn1K/E and tatin significantly lowers the number of presence of Dyn2 to exert its effect on Dyn1I690K in cell culture is consistent with FAs and stress fibers within the cell body the FAs. the failure of Bis-T-23 to ameliorate pro- but increases the number of both param- We next examined the effects of Bis- teinuria in zebrafish expressing Dyn1K/E or eters in the vicinity of the membrane (in T-23 on stress fibers and FAs in cells Dyn1I690K.13 Together, these data suggest Figure 2E, white arrowheads mark FAs overexpressing similar levels of various that DynOLIGO regulates the formation of located in the membrane vicinity, and dynamin mutants (Supplemental Figure stress fibers and FAs independent from its the black arrowhead marks actin arcs). 2, A and B). Dyn1K/E, a mutant impaired role in endocytosis. Once again, the addition of Bis-T-23 in actin binding, exhibited a dramatic We next asked whether the observed partially restores both parameters within loss of FAs and stress fibers.12 The addi- effects of Bis-T-23 in the cell were dyna- the cell body (Figure 2, E–G). In cells treat- tion of Bis-T-23 failed to rescue these min autonomous or whether dynamin ed with Y-27632, Bis-T-23 increases the phenotypes (Supplemental Figure 2, acted, in part, by altering RhoA signaling. formation of free barbed ends (Supple- C–E), despite the fact that Bis-T-23 in- As depicted in Figure 2A, RhoA plays mental Figure 3, E–G), concomitant with duced its oligomerization in vitro.14 a role in the assembly of stress fibers increased level of p-paxillin without affect- Similar phenotypes were observed in and FAs by stimulating its downstream ing its total level (Supplemental Figure podocytes expressing Dyn1I690K (Supple- effectors: Rho–associated protein ki- 3H). Together, these data provide addi- mental Figure 2, C–E), an oligomerization- nase (ROCK) and mammalian Dia 1 tional evidence that DynOLIGO plays a ma- incompetent mutant.18 Conversely, (mDia1).6 Dynamin’sabilitytoinduce jor role in FA maturation, most likely by Dyn1E/K, a mutant with an increased affin- actin polymerization by displacing gel- stimulating actin polymerization. ity for actin, increases the amount of stress solin and its ability to crosslink actin fil- Experiments using pure proteins sug- fibers and FAs (Supplemental Figure 2, C–E). aments12 suggest a parallel pathway for gested that DynOLIGO induces actin po- We have previously shown using fluores- FA maturation (Figure 2A). Although lymerization by displacing gelsolin from cence lifetime imaging microscopy that the Bis-T-23–induced phenotype was the barbed ends.12 Downregulation of Dyn1E/K exhibited a heightened propen- similar to that of the activated form of gelsolin (GsnKD) (Supplemental Figure sity for actin-dependent oligomerization RhoA,19 Bis-T-23 treatment did not alter 4, A and B) increases the number of in podocytes.14 The extent of Dyn1E/K either overall levels or those of GTP-bound FAs and results in thicker stress fibers adenoviruses expressing short hairpin RNA (shRNA) against respective proteins. Where indicated, cells were treated with blebbistatin (20 mM) or Y-27632 (10 mM) for 30 minutes. Bis-T-23 (30 mM) was subsequently added for 30 minutes before fixation. FAs and stress fibers were visualized with anti-paxillin antibody and rhodamine-phalloidin, respectively. White arrowheads in panels 4 and 5 indicate FAs formed at the cell membrane, and the black arrowhead in panel 4 indicates actin arcs. Scale bars, 20 mm. (F and G) Bar graphs depicting the numbers of (F) FAs and (G) stress fibers after the RhoA signaling has been impaired. Of note, FAs formed at the plasma membrane were not counted. Data represent measurements of .50 cells as shown in E and are plotted as means6SD. *P,0.05; **P,0.01; ***P,0.001.

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(Figure 2, E–G), suggesting that gelsolin type specificity for the same major sig- REFERENCES regulates actin turnover in podocytes. Of naling pathways. The ability of dynamin note, gelsolin has been implicated in ac- to bypass multiple actin binding and 1. Geiger B, Spatz JP, Bershadsky AD: Envi- tin turnover in platelets.20 Bis-T-23 does regulatory proteins involved in the ca- ronmental sensing through focal adhesions. – not exhibit any additional effects on FAs nonical pathways, such as RhoA (this Nat Rev Mol Cell Biol 10: 21 33, 2009 KD – 2. Sachs N, Sonnenberg A: Cell-matrix adhe- in Gsn cells (Figure 2, E G), suggest- study) and maybe even Cdc42 and sion of podocytes in physiology and disease. OLIGO ing that Dyn and gelsolin act on the Rac1, makes dynamin a unique pharma- Nat Rev Nephrol 9: 200–210, 2013 same pathway. Finally, Bis-T-23 induces cologic target for altering actin cytoskel- 3. 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Interestingly, our study shows The authors thank Tristan Hays for his technical Moeckel G, Cremona O, Holzman LB, De that, despite the lack of phenotype in an- help and discussion. Camilli P, Ishibe S: Role of dynamin, synaptojanin, imals lacking RhoA, impairment in the This work was supported by National and endophilin in podocyte foot processes. J Clin Invest 122: 4401–4411, 2012 RhoA downstream effectors, such as Institutes of Health grant R01DK093773 11. Ferguson SM, De Camilli P: Dynamin, a ROCK or mDia1, resulted in pronounced (to S.S.). fi membrane-remodelling GTPase. Nat Rev loss of FAs and stress bers. Rho/mDia Mol Cell Biol 13: 75–88, 2012 signaling has been associated with for- 12. Gu C, Yaddanapudi S, Weins A, Osborn T, mation of lamellipoda, which in podo- DISCLOSURES Reiser J, Pollak M, Hartwig J, Sever S: Direct cytes, required interactions between dynamin-actin interactions regulate the actin J.R., V.G., and S.S. have pending or issued patents cytoskeleton. EMBO J 29: 3593– 3606, 2010 mDia and inverted formin 2.28 Because – on novel kidney protective therapies that have been 13. Schiffer M, Teng B, Gu C, Shchedrina VA, mutations in inverted formin 2 are a outlicensed to Trisaq Inc. (Miami, FL), in which they Kasaikina M, Pham VA, Hanke N, Rong S, common cause of familial FSGS, to- have financial interest. In addition, they stand to gain Gueler F, Schroder P, Tossidou I, Park JK, gether, these insights emphasize the cell royalties from their commercialization. Staggs L, Haller H, Erschow S, Hilfiker-Kleiner

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