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Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation

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Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation BRIEF COMMUNICATION www.jasn.org 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 446 ISSN : 1046-6673/2802-446 J Am Soc Nephrol 28: 446–451, 2017 www.jasn.org BRIEF COMMUNICATION 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 J Am Soc Nephrol 28: 446–451, 2017 Dynamin and Focal Adhesions 447 BRIEF COMMUNICATION www.jasn.org 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).
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