Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness

Nigro, Elisa Agnese; Distefano, Gianfranco; Chiaravalli, Marco; Matafora, Vittoria; Castelli, Maddalena; Pesenti Gritti, Angela; Bachi, Angela; Boletta, Alessandra

doi:10.1038/s41598-019-53061-0

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Published: 12 November 2019

Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness

Elisa Agnese Nigro¹,
Gianfranco Distefano¹,
Marco Chiaravalli¹,
Vittoria Matafora²,
Maddalena Castelli¹,
Angela Pesenti Gritti¹,
Angela Bachi² &
…
Alessandra Boletta¹

Scientific Reports volume 9, Article number: 16640 (2019) Cite this article

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Abstract

Polycystin-1 (PC-1) and 2 (PC-2) are the products of the PKD1 and PKD2 genes, which are mutated in Autosomal Dominant Polycystic Kidney Disease (ADPKD). They form a receptor/channel complex that has been suggested to function as a mechanosensor, possibly activated by ciliary bending in the renal tubule, and resulting in calcium influx. This model has recently been challenged, leaving the question as to which mechanical stimuli activate the polycystins still open. Here, we used a SILAC/Mass-Spec approach to identify intracellular binding partners of tagged-endogenous PC-1 whereby we detected a class of interactors mediating regulation of cellular actomyosin contraction. Accordingly, using gain and loss-of-function cellular systems we found that PC-1 negatively regulates cellular contraction and YAP activation in response to extracellular stiffness. Thus, PC-1 enables cells to sense the rigidity of the extracellular milieu and to respond appropriately. Of note, in an orthologous murine model of PKD we found evidence of increased actomyosin contraction, leading to enhanced YAP nuclear translocation and transcriptional activity. Finally, we show that inhibition of ROCK-dependent actomyosin contraction by Fasudil reversed YAP activation and significantly improved disease progression, in line with recent studies. Our data suggest a possible direct role of PC-1 as a mechanosensor of extracellular stiffness.

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Introduction

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a relatively common monogenic disorder, affecting approximately 1:2000 individuals^1,2. The disease is due to mutation in either the PKD1 or the PKD2 genes, encoding for two large membrane proteins, Polycystin-1 (PC-1) and Polycystin-2 (PC-2), respectively. PC-1 is a transmembrane protein with a long extracellular domain, 11 transmembrane domains and a short intracellular C-tail^2,3,4. PC-1 C-terminal interacts through a coiled-coil domain with the C-terminal of PC-2, a cation channel of the TRPP family, to form the PC-1/PC-2 complex, likely with an intrinsic channel activity^5,6. The PC-1/PC-2 complex localizes to cilia, as well as to sites of cell-cell/matrix interaction^2,3,7,8,9. Although the PC-1/PC-2 complex has been described to play a role in several molecular pathways, its function remains to be understood². One key activity of the polycystins receptor/channel complex has been ascribed to the capability of PC-1 to regulate key morphogenetic programs such as renal tubular morphogenesis both in vitro^10,11 and in vivo^12,13 as well as lymphatics development¹⁴. Importantly, overexpression of PC-1 was reported to drive a program of cell migration^15,16 and front-rear polarity via its role in regulating focal adhesions¹⁷ and adherent junctions¹⁵ turnover as well as by regulating the actin cytoskeleton and the function of a Par3/aPKC complex^12,13,16. The molecular mechanisms involved in this specific function of PC-1, however, remain largely elusive. Likewise, the mechanism of activation of this receptor/channel complex remains unclear. Chemical stimuli such as Extracellular Matrix (ECM) components and Wnt proteins have been shown to interact with PC-1 and to mediate its activation¹⁸, highlighting the possibility of this receptor being regulated by specific chemical ligands. In addition to this property, a possible role as a mechanosensor for PC-1 has been postulated on the basis of the fact that the predicted structure of PC-1 is very similar to proteins with structural and mechanical roles^{19,20,21,22,23}. Specifically, the PC-1 extracellular region, rich in extensible Ig-like domains, shows elastic properties suggesting its role as a potential mechanosensor²². Furthermore, PC-1 has been shown to sense and transduce mechanical forces into biochemical signals^{24,25,26,27,28,29,30}. In particular, based on its localization to cilia, PC-1 has been long believed to be the mechanosensor enabling renal epithelial cells to respond to the bending of cilia caused by urine flow resulting in calcium influx²⁴. However, recent studies have questioned this PC-1 and PC-2 function³¹. Finally, evidence of PC-1 classification as a mechanosensor are based on its interaction with both components of the ECM and the cytoskeleton via intermediate filaments^8,32,33. Of note, it has been shown that inactivation of integrin β1 in collecting ducts, a known positive mechanosensor of extracellular stiffness, led to inhibition of Pkd1-dependent cystogenesis in vivo³⁴.

However, while the role of PC-1 extracellular domain has long been postulated to be that of a mechanical sensor, the precise signal that activates the mechanical properties of the receptor remains unknown. Polycystins are expressed in tissues other than kidney⁹, such as bone where matrix stiffness has an important impact³⁵. In line with this, a recent study suggests that PC-1 has a role in regulating osteoblastogenesis and adipogenesis through interaction with the transcriptional coactivator with PDZ-binding motif (TAZ)^29,36. Mechanical forces, such as ECM stiffness activate the PC-1/PC-2 complex leading to induction of PC-1- C-Terminal Tail (PC-1-CTT) cleavage and TAZ nuclear translocation to enhance osteoblast gene transcription and to inhibit PPARγ and adipogenesis. Supporting the role of PC-1 and TAZ in regulating bone development, Zebrafish injected with morpholinos against Pkd1 show bone calcification defects that can be rescued by the injection of either PC-1-CTT or dominant-active TAZ mRNA³⁶. Both TAZ and Yes-Associated Protein (YAP) are known as the primary downstream effectors of the Hippo pathway, which has been previously found de-regulated in ADPKD³⁷, and other unrelated cystic kidney disorders³⁸. More recent work has firmly established that YAP can also be activated in response to mechanical stimuli, and more precisely by extracellular stiffness generated either by the matrix³⁹, or by cell density in epithelia⁴⁰. Notably, an elegant recent study provides strong evidence that a RhoA–YAP–c-Myc axis is a direct downstream target of PKD1 mutations⁴¹. Thus, PKD1 deficiency causes constitutive activation of YAP/TAZ during kidney cystogenesis^37,41. Here, we show that in response to the stiffness of the extracellular environment, PC-1 activates an inhibitory signal which decreases the contraction of actomyosin fibers, ultimately leading to regulation of YAP shuttling and transcriptional activity, previously shown to play an important role in renal cystogenesis³⁷. Importantly, we show that PC-1 interacts in a complex of proteins regulating actomyosin contraction. Thus, we provide a direct physical and functional link between PC-1 and the inhibition of a pro-cystogenic pathway. Importantly, our data seem to suggest that the physical properties of the extracellular milieau, rather than its chemical composition, might play a key role in regulating the PC-1 receptor function, opening a new perspective on the mechanosensory activity of this receptor and the potential mechanism of pathogenesis.

Results

Generation of the first PC-1 interactome reveals its central role in regulation of the actomyosin machinery

PC-1 has been proposed to play a role in multiple signaling pathways but its main physiological function is far from being understood. To gather an overview of a possible central function of PC-1, we set out to generate the interactome of the endogenous protein under stringent conditions. We isolated mouse embryonic fibroblasts (MEFs) from a Pkd1^HA/HA mouse model, in which an HA tag has been inserted in-frame in the last exon of the Pkd1 gene resulting in a fully functional endogenous PC-1 protein⁴². The presence of the HA tag at the C-terminus of PC-1 allowed the immunoprecipitation (IP) of the endogenous protein using commercial beads covalently linked to an anti-HA antibody⁴². As a control, we used MEFs derived from Pkd1^WT/WT littermate embryos expressing equal levels of untagged PC-1. Two IP coupled to Mass Spectrometry approaches have been used, both based on SILAC (Stable Isotopes Labeling by Aminoacids in Cell culture). Briefly, Pkd1^HA/HA and Pkd1^WT/WT MEFs were grown in the presence of amino acids labeled with different isotopes (heavy-¹³C and light-¹²C, respectively) and, upon HA IP, samples were mixed and analyzed together (Fig. 1a,b). In the first approach, named SILAC1, we run the immunoprecipitated proteins on the gel and we performed in-gel proteins digestion followed by liquid chromatography mass spectrometry; in the second approach, named SILAC2, the digestion of the proteins was performed directly on the beads. While soluble proteins are better digested in solution, membrane proteins, such as PC-1, are better digested if completely denatured in the gel; therefore, a combination of the above strategies is the best choice to increase interactome coverage as much as possible. For each method two technical replicates were performed. Globally, we identified 316 proteins, 30 of them significantly enriched in the IP versus the control (Fig. 1c,d and Supplementary Table 1), including the bait itself PC-1. A scatter plot of the interactors with their heavy/light ratio is reported (Fig. 1c) together with the functional protein association networks, based on STRING database (Fig. 1e). When inspecting the list of interactors, we were pleased to see that a few known interactors were represented in the list, including the Gαi subunit of heterotrimeric G proteins, previously demonstrated to interact with PC-1^43,44. Much to our surprise the list of interactors and the STRING analysis pointed to a single network of functional regulators of vascular smooth muscle contraction, regulation of actin cytoskeleton machinery and focal adhesions (Fig. 1e).

PC-1 regulates actomyosin contraction

Our Mass Spectrometry data indicate that several interactors of PC-1 are proteins involved in the regulation of the actomyosin machinery. Therefore, we investigated the phosphorylation state of myosin light chain (pMLC), the main regulator of actomyosin contraction, in Pkd1 gain and loss of function cellular models. We found that the levels of pMLC are decreased in Madine Darby Canine Kidney overexpressing PC-1 (MDCK^PKD1Zeo)¹⁰ cells compared to controls (Fig. 2a and Supplementary Fig. 1). Conversely, MEFs isolated from Pkd1^−/− mice showed an increased phosphorylation state of pMLC (Fig. 2b and Supplementary Fig. 1)⁴⁵. These results suggest that PC-1 is a negative regulator of the phosphorylation state of MLC. We therefore tested whether PC-1 can effectively regulate the contractility of cells performing a collagen contraction assay⁴⁶. In this assay, cells are plated on top of collagen discs that are allowed to polymerize in multiwells. Collagen discs are then mechanically detached from the border of the wells, and followed over time. The contractility of cells causes a progressive reduction of the area covered by the discs, thus disc-area is a direct measure of contractility and it is inversely correlated with the capability of cells to contract. Collagen discs in the absence of cells were used as controls to define a complete absence of contraction. Consistent with their pMLC levels, MDCK^PKD1Zeo cells retracted collagen discs to a lesser extent, compared to control MDCK^Zeo cells (Fig. 2c,d). On the contrary we observed a fast and remarkable contraction capability of Pkd1^−/− MEFs (Fig. 2c,d) and PC-1 silenced Inner Medullary Collecting Duct (IMCD^shPkd1) cells, compared to their controls (Fig. 2e). Thus, the expression levels of PC-1 inversely correlate with the pMLC levels and the capability of cells to contract. Next, we wondered which molecules upstream of the pMLC could play a role in the Pkd1^−/− cells. The phosphorylation levels of MLC are regulated at multiple levels, and in particular Rho kinase (ROCK) and Myosin Light Chain Kinase (MLCK)⁴⁷ can both enhance the levels of pMLC. In addition, ROCK inhibits the phosphatase responsible for dephosphorylation of MLC (MYPT1) ultimately enhancing the contraction capability of myosin (Fig. 3a). Notably, MYPT1 appears among the direct interactors of PC-1 (PPP1r12a gene in Fig. 1d). Interestingly, both inhibitors of ROCK (Y27632) and MLCK (ML-7) were able to decrease the phosphorylation of MLC to control levels in Pkd1^−/− cells (Fig. 3b and Supplementary Fig. 1). In line with this, both inhibitors prevented the enhanced contraction observed in the mutant cells (Fig. 3c).

PC-1 regulates the cellular response to extracellular stiffness

The actomyosin pathway plays a central role in the response of cells to mechanical stimuli such as ECM elasticity or cell density, and it has been shown to regulate the activity of the transcription factor YAP in response to mechanical stress^39,48. In particular, cells plated on stiff substrates or in sparse condition, show active nuclear localization of YAP that promotes cells proliferation and differentiation^39,49. On the contrary in cells cultured on soft substrates or in confluent condition, YAP is retained in the cytoplasm becoming functionally inactive (Fig. 4a)³⁹.

In the previous section we showed that PC-1 regulates myosin phosphorylation (Fig. 2a,b) and cell contractility (Fig. 2c,d,e). Furthermore, PC-1 structure suggests a possible role as a mechanosensor. We therefore wondered whether this receptor could be involved in sensing the rigidity of the extracellular environment. Given the central role of YAP localization in this type of response³⁹, we used YAP localization as a proximal marker to measure the cellular response to extracellular stiffness in the presence or absence of PC-1.

To test this hypothesis, we plated cells on hydrogels mimicking different stiffness of the matrix with an elasticity ranging from 0.5 KPa to 40 KPa as recently reported^29,39. Consistent with their low level of myosin phosphorylation, MDCK^PKD1Zeo cells show an attenuated response to the increased stiffness displaying higher retention of YAP in the cytoplasm, compared to control MDCK^Zeo cells (Fig. 4b). We observed a comparable response when plating MDCK^PKD1Zeo cells at low density since in epithelial cells the modulation of cell density recapitulates the response of the cell to the extracellular stiffness (Fig. 4c)⁴⁰. As a consequence, expression of the YAP target genes Ankrd1 and Cyr61 is down-regulated (Fig. 4d). This is in line with PC-1 localization at the site of cell-cell and cell-matrix junction other than at the primary cilium^3,4,9,50. According to a possible role of PC-1 in attenuating the response to extracellular stiffness, IMCD^shPkd1 and Pkd1^−/− MEFs fail to decrease YAP activation in soft conditions compared to controls (Fig. 4e,f), in line with a recent work^29,41. Furthermore, we tested whether in epithelial cells silencing of Pkd1 conferred a lack of response to soft conditions generated by increased cell density⁴⁰. Indeed, we found that IMCD^shPkd1 show enhanced YAP nuclear translocation even when plated in confluent and hyper-confluent conditions (Fig. 4e).

The dysfunctional sensitivity of extracellular stiffness results in deregulated actomyosin in vivo

To address whether the role of PC-1 as a regulator of myosin activity is conserved in vivo, we examined pMLC levels in Pkd1 kidney-specific conditional mutants (Pkd1^flox/−:KspCre), displaying a cystic phenotype. Consistent with data in cells, we found increased phosphorylation of MLC in cystic kidneys at both P5 and P12, compared to controls (Fig. 5a and Supplementary Fig. 1). Furthermore, immunohistochemistry (IHC) analysis of kidney tissues showed the presence of nuclear YAP in the majority of epithelial cells forming cysts, whereas YAP localization was mainly cytoplasmic in non-cystic renal cells (Fig. 5b). Quantification using a semi-automated system confirmed the significant increase in cells with YAP nuclear translocation in mutant kidneys (Fig. 5c) in line with previous studies³⁷. In line with this observation, we also found an enhanced expression of YAP target genes in cystic kidneys at different ages, both in a neonatal PKD model and in the adult compared to controls (Fig. 5d). Importantly, treatment of mice with the ROCK inhibitor Fasudil led to a decrease in MLC phosphorylation (Fig. 6a and Supplementary Fig. 1), YAP nuclear translocation (Fig. 6b,c) and expression of YAP target genes Ctgf, Ankrd1, Cyr61 (Fig. 6d). These results demonstrate that in the cystic epithelia the increased YAP nuclear translocation and activity is mainly dependent on the actomyosin contraction machinery, supporting the hypothesis that cystic epithelia fail to sense and activate a proper cellular response to extracellular rigidity. Of note, treatment of mice with the inhibitor Fasudil by intra-peritoneal injection (i.p.) for three consequent days was also sufficient to ameliorate the cystic phenotype. Mice showed a reduced kidney/body weight (Fig. 6e) with a decrease of the cystic index due to the increment of normal tubules (Fig. 6f). Furthermore, analysis of proliferation by Ki67 staining showed a significant decrease in proliferation (Fig. 6g), whereas analysis of apoptosis by TUNEL did not show any difference between untreated and treated cystic kidneys (Fig. 6h). Thus, we conclude that Fasudil achieves improvement of the phenotype by reducing the proliferation rates of the cystic epithelia.

Discussion

In this study, we performed a controlled screening for intracellular interactors of endogenous PC-1 and identified a new role for this receptor as a mechanosensor of the cellular response to extracellular stiffness. Of interest, PC-1 appears to act as a negative, rather than a positive, regulator of the cellular response to rigidity (Fig. 7).

The mass-spectrometry analysis of mouse embryonic fibroblasts carrying tagged endogenous PC-1 revealed that PC-1 interacts with proteins that regulate the actomyosin contraction machinery. Accordingly, we found that overexpression of PC-1 decreases pMLC levels and cellular contraction, while cells knocked-out or silenced for Pkd1 have both effects upregulated. We further found increased pMLC in polycystic kidneys generated by kidney-specific inactivation of Pkd1. The actomyosin pathway plays a central role in the response of cells to extracellular rigidity. To study whether PC-1 could have a role in regulating this process we have used YAP translocation into the nucleus as a read-out of this response. Plating cells on hydrogels that mimic the different stiffness of the ECM, we have observed a reduced YAP nuclear translocation in cells overexpressing PC-1 in stiff conditions while Pkd1 mutant cells partially failed to respond to soft condition showing an enhanced nuclear localization of YAP. Furthermore, Pkd1 mutant kidneys display enhanced actomyosin-dependent YAP nuclear translocation and transcriptional activity. It is interesting to note here that to date only positive regulators of the extracellular response to stiffness have been identified³⁹, whereas our data seem to indicate that PC-1 might be acting as a receptor for extracellular stiffness able to inhibit the cellular response via inhibition of phosphorylation of myosin. Indeed, our studies are supported by evidence that inactivation of a positive regulator of the pathway, integrin β1, results in a drastic improvement of the cystic phenotype in Pkd1 mutant mice³⁴. Our data are also in agreement with the previously reported role of PC-1 in the regulation of cell adhesion and in particular as a positive regulator of focal adhesions turnover^17,51. It remains to be established whether PC-1 exerts this novel function via its localization at focal adhesions and cell-cell contacts or, on the contrary, whether this new function of PC-1 is also modulated by the pool of proteins present in primary cilia and future work should focus on understanding this aspect. Irrespective of the subcellular compartment in which PC-1 is active to inhibit this pathway, it is important to note here that our data might potentially explain a very interesting recent finding observed in human-derived iPS cells. Indeed, it was shown that patient derived cells carrying a PKD1 mutation can originate a very aggressive form of cystogenesis when cultured in vitro, but only when the mechanical constraints are lacking, i.e. when cells are grown in suspension⁵². Based on our findings we speculate that this might be due to an inability of the cells to respond to the extracellular mechanical force given the absence of a key mechanoreceptor such as PC-1. Our data are also potentially in agreement with studies carried out in Zebrafish⁵³. Indeed, silencing of Pkd1 by morpholinos in the fish resulted in increased collagen matrix deposition and tail curvature⁵³. We speculate that in this context as well, absence of PC-1 might result in a failure to sense the correct amount of collagen deposition and matrix rigidity, ultimately leading to a constitutive and excessive deposition of matrix⁵³, a phenotype also observed in patient specimens^54,55,56. Given the fact that deletion of integrin β1 in PKD murine models results in phenotype improvement, it would be interesting to test whether morpholinos directed against the same integrin subunit in the fish improves also this specific phenotype. Importantly, the fact that an increased matrix deposition was also observed in patient-derived tissues, points to the potential central importance of the process for the pathophysiology of ADPKD⁵⁴.

Finally, it should be considered that, irrespectively of whether or not deregulation of actomyosin contraction is a primary event resulting in cystogenesis or, viceversa, it is deregulated once cysts have formed, it is important to highlight the potential of targeting this pathway as a new therapeutic approach. Indeed, we have found that the in vivo inhibition of the ROCK pathway using the compound Fasudil reduced YAP nuclear translocation and ameliorates PKD progression. These data are in agreement with recent work showing that the inhibitor Y27632 was able to retard disease progression in animal models⁴¹. While it is difficult to think that Y27632 could be used in the clinic, this provides a very important proof of principle. Our data confirm these findings and in addition show that a compound available on the market for targeting a different medical condition could offer a good opportunity for drug repurposing.

Finally, while our data do not clarify the precise mechanism by which PC-1 regulates the actomyosin contraction, they indicate that both ROCK and MLC kinase inhibitors are able to revert the pMLC levels in mutant cells. This is relevant because MLC is a Ca++ dependent kinase⁴⁷ that might be altered primarily or secondarily in ADPKD tissues due to PC-2 dysfunction and this might concur in regulation of pMLC levels and cellular proliferation in the cystic epithelia.

In conclusion, our results indicate that applying a very stringent method for the identification of PC-1 interactors allowed the identification of few molecules pointing to a central role for PC-1 in regulation of the cellular response to the physical nature of the extracellular environment, such as the rigidity of the milieu. Even if this property is a novel central function of PC-1 potentially involved in inducing cystogenesis, it might be difficult to be corrected. However, inhibition of a downstream pathway involved in the cellular response might contribute to retard disease progression and might be explored as a potential new therapy.

Materials and Methods

Cell lines

MDCK cell lines stably transfected either with a full-length human PKD1 cDNA construct (MDCK^PKD1Zeo clones C8/68, G3/11) or with an empty vector (MDCK^Zeo, clones F2, F6) are described in Boletta et al.¹⁰. Pkd1^+/+ (clone 11) and Pkd1^−/− (clone 14) MEFs are described in Distefano et al.⁴⁵. Murine IMCD cells transduced with viral vectors expressing shRNA encoding the Pkd1-targeting shRNA sequence (shPkd1) (clones C12 and C16) or the scrambled sequences (shScr) (control clones M3 and M4) are already described¹⁷. All the cell lines were grown in 37 °C, 5% CO2 incubators, in high glucose DMEM medium (GIBCO, #41965-062), 10% FBS, 1% Penicillin-Streptomycin (GIBCO, #15070-063); for MDCK, medium was supplied with 0.5 μg/ml Geneticin (GIBCO, #11811-064) and 0.05 μg/ml Zeocin (Invitrogen, #R25001). For IMCD clones, medium was supplemented with 1 μg/ml Puromycin (Invitrogen, #A11138-03).

Mice

The generation of both Pkd1^flox/−:KspCre and Pkd1^flox/−:TmCre mice in a pure C57BL/6N genetic background has been previously described^42,57. Intra-litter Pkd1^flox/+:KspCre and Pkd1^flox/+:TmCre mice respectively were used as controls. For the Pkd1^flox/−:TmCre model the Cre recombinase activity was induced by a single i.p. injection at P12 of Tamoxifen (Sigma-Aldrich, #T5648; 10 mg/40 g) dissolved in corn oil (Sigma-Aldrich, #C8267) by continuous shaking at 37 °C for 8 h. For all animal work, the female to male ratio was 1:1, and mice were randomized for each experiment. All animal care and all protocols used were carried out according to the Institutional regulations and specifically approved by the institutional care and use ethical committee at the San Raffaele Scientific Institute, further approved by the Italian Ministry of Health (IACUC #736).

In vivo treatments

We intra-peritoneally (i.p.) injected the Rho kinase inhibitor Fasudil (LC Laboratories, #F-4660) or vehicle (NaCl) daily from P6 until P8 at 25 mg/kg of body weight. For boost treatment we i.p. injected Fasudil or vehicle (NaCl) at P8 five times every hour at 10 mg/kg of body weight.

PC-1 immunprecipitation

Pkd1^HA/HA and Pkd1^WT/WT MEFs were grown in presence of aminoacids labeled with different isotopes (heavy-¹³C and light-¹²C, respectively). PC-1 target proteins were immunoprecipitated using anti-HA antibody. Briefly, Pkd1^HA/HA and Pkd1^WT/WT MEFs were lysate by using lysis Buffer 1% triton X-100 (Sigma-Aldrich, #1002324354). 10 mg of total extract (1 ml final volume) was incubated for 2 h with 50 μl of agarose beads at 4 °C to pre-clear the samples. The clarified supernatants were incubated overnight with anti-HA beads for the IP. Then, samples were washed 5 times with Buffer 1% triton.

Depending on the type of SILAC experiment, the heavy and light proteins immunoprecipitated were mixed 1:1 after the IP, then, for SILAC1, PC-1 IP eluates were separated by 4–12% SDS-PAGE (Life Technologies, # NP0335BOX), stained with Coomassie Brilliant Blue (Sigma-Aldrich, #B-0149) and excised in eight slices for LC-MS/MS analysis. For SILAC2, beads were resuspended in Urea 6M, Tris HCl 10 mM, proteins were digested with Lys C overnight, Urea was then diluted to 2M and trypsin digestion was performed for 3 h.

Mass spectrometry and data analysis

Mass spectrometry analysis was performed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer (ThermoScientific, Bremen, Germany). Tryptic digests were cleaned using Stage Tips as described previously⁵⁸ and then injected in a capillary chromatographic system (EasyLC, Proxeon Biosystems, Odense, Denmark). Peptides separation occurred on a homemade column obtained with a 10-cm fused silica capillary (75 m inner diameter and 360 m outer diameter; Proxeon Biosystems) filled with Reprosil-Pur C18 3-m resin (Dr Maisch GmbH, Ammerbuch-Entringen, Germany) using a pressurized ‘packing bomb’. A gradient of eluents A [distilled water with 2% (v/v) acetonitrile, 0.1% (v/v) formic acid] and B [acetonitrile, 2% (v/v) distilled water with 0.1% (v/v) formic acid] was used to achieve separation from 8% B (at 0 min, 0.2 ml/min flow rate) to 50% B (at 80 min for SILAC1 and 180 min for SILAC2, 0.2 ml/min flow rate). The LC system was connected to the orbitrap equipped with a nanoelectrospray ion source (Proxeon Biosystems). Full-scan mass spectra were acquired in the LTQ-Orbitrap mass spectrometer in the mass range m/z 350–1500 Da and with the resolution set to 60000. The ‘lock-mass’ option was used for accurate mass measurements. The 10 most intense doubly and triply charged ions were automatically selected and fragmented in the ion trap. Target ions already selected for the MS/MS were dynamically excluded for 60 s. Protein identification and quantification were achieved using the MaxQuant software version 1.1.1.36⁵⁹. Mass spectra were analyzed against a concatenated forward and reversed database version of uniprot_cp_mus_2011_03. The initial mass tolerance in MS mode was set to 7 ppm and MS/MS mass tolerance was 0.5 Da. Cysteine carbamidomethylation was searched as a fixed modification, whereas N-acetyl protein and oxidized methionine were searched as variable modifications. Labeled arginine and lysine were also specified as variable modifications. SILAC peptides and proteins quantification were performed automatically with MaxQuant using default settings as parameters. Peptides and proteins were accepted with a false-discovery rate of 0.01, minimum one unique peptide identified per protein. To identify PC-1 interactors, we analyzed the Gaussian distribution and we used the standard deviation of the sample as a cut-off to identify outliers. Outliers were selected based on p < 0.05 as the most stringent criteria and a cutoff ratio H/L > mean + 2 SD considering the Gaussian distribution.

Antibodies and inhibitors

For Western blot analysis we used the following antibodies: PC-1 LRR (Santa Cruz Biotechnology, #sc-130554; 1:1000), HA High Affinity (Roche, #11867423001; 1:1000) p-MLC^Thr18/Ser19 (Cell Signaling, #3674S; 1:1000), MLC (Cell Signaling, #3672; 1:1000). HRP-conjugated secondary antibodies were from GE Healthcare, anti-rabbit IgG HRP linked (#934V) anti-mouse IgG HRP linked (#NA9310V) and anti-rat IgG HRP linked (#NA935V). For IF and IHC staining we used YAP (Santa Cruz Biotechnology, #sc-101199; 1:200 or Cell Signaling, #4912; 1:50 respectively), Ki67 (Thermo Fisher Scientific, #RM-9106; 1:100) antibodies and DAPI (Santa Cruz Biotechnology, #sc-3598; 1:5000). Alexa-488 secondary anti mouse antibody were from Invitrogen (#A11001). For IP we used anti-HA affinity matrix (Roche, #11815016001). Rho kinase inhibitor Y27632 and MLCK inhibitor ML-7 were from Tocris (#1254 and #4310, respectively).

Immunohistochemistry

For histological analysis mice were sacrificed at the indicated time, kidneys were collected, weighted and fixed in formalin 10% (BioOptica, #05-01005Q), included in paraffin and cut 5 μm/slides. Kidney sections were air-dried and rehydrated in PBS (Sigma-Aldrich, #P4417). For YAP staining kidney sections were incubated with YAP antibody and 5 min in Eosin G (BioOptica, #05-10002/L); For Ki67 staining kidney sections were incubated with Ki67 antibody and 2 min in Hematoxylin (BioOptica, #05-06015/L). Sections were then washed, processed through a dehydration alcohol scale and mounted in DPX (Sigma-Aldrich, #06522). For the semi-automated quantification, slides were acquired with Aperio AT2 digital scanner at magnification of 40 × (Leica Biosystems) and analyzed with Imagescope (Leica Biosystem). YAP and Ki67 nuclear staining per cyst was performed by Aperio Image analysis software (Leica Biosystems) and counted manually.

Cystic index

To quantify the Cystic Index we manually counted at least 1000 tubular structures in cortical sections of kidneys stained with Hematoxylin-Eosin. To quantify the rate of cystic kidneys we applied a grid of squares 13.625 μm large to sections of kidneys stained with Hematoxylin-Eosin. We marked each cross with a dot and we manually counted the number of dots inside the lumen on three litters. We determined the degree of dilatation according to 1 dot: normal tubules; >2 dots: dilated tubules and cysts as in⁶⁰.

Immunofluorescence

For immunofluorescence staining, cells seeded on a glass or on a hydrogels-covered glass were washed in PBS, fixed 10 min in 4% Paraformaldehyde and permeabilized in 0.2% Triton X-100 in PBS. After 1 h blocking in 3% BSA (Sigma-Aldrich, #A7906) in PBS at RT, cells were incubated over night at 4 °C with primary antibody diluted in 3% BSA in PBS. Cells were then incubated with secondary antibody diluted in 3% BSA in PBS for 1 h at RT and nuclei were stained with DAPI. Glasses were then mounted with Fluorescence Mounting medium (Dako, #S3023). For the apoptotic assay, kidney sections were analyzed by the DeadEnd Flurometric transferase-mediated dUTP nick-end labeling (TUNEL) system kit (Promega, #G3250). Images were obtained using Zeiss Axio Observer.Z1 microscope. Quantification of both YAP and TUNEL nuclear staining was performed manually.

Real-time pcr analysis

Total RNA was isolated from cells or kidneys using the RNAspin Mini kit (GE Healthcare, #25-0500-72). cDNA was obtained by reverse transcription of extracted RNA using Oligo(dt)₁₅ primers (Promega, #C1101) and ImProm-II Reverse Transcriptase (Promega, #A3802). Quantitative Real Time PCR analysis was performed on technical duplicates using SYBR Green I master mix (Roche, #04887352001) on LightCycler 480 Instrument (Roche). Primer sequences for qRT-PCR:

mHprt1 fw 5′-TTATGTCCCCCGTTGACTGA-3′

mHprt1 rev 3′-ACATTGTGGCCCTCTGTGTG-5′

mCtgf fw 5′-GCTTGGCGATTTTAGGTGTC-3′

mCtgf rev 3′-CAGACTGGAGAAGCAGAGCC-5′

mAnkrd1 fw 5′-CAGTGCAACACCAGATCCAT-3′

mAnkrd1 rev 3′-ATGCCAAGGACAGAGAAGGA-5′

mCyr61 fw 5′-TTTACAGTTGGGCTGGAAGC-3′

mCyr61rev 3′-AAGGGGTTGGAATGCAATTT-5′

Canine Hprt1 fw 5′-ACACTGGGAAAACAATGCAGAC-3′

Canine Hprt1 rev 3′-TCAGGTTTATAGCCAACACTTCG-5′

Canine Ankrd1 fw 5′-TGGAGCCCAGATCGAATTCC-3′

Canine Ankrd1rev 3′-CACTGAGTCATGCAGAGGGG-5′

Canine Cyr61 fw 5′-TCTGTGACGACGATGATGCC-3′

Canine Cyr61 rev 3′-GTACAGGATTCGAGGCTCCG-5′

Western blot analysis

For Western blot analysis cells or kidneys were lysed in lysis buffer solution of 150 mM NaCl (Sigma-Aldrich, #s9625), 20 mM Na₂HPO₄ (BDH, #10494L)/NaH₂PO₄ (BDH, #102455S), 10% Glycerol (Sigma-Aldrich, #G7757), 1% Triton X-100 (pH 7.2), complete protease inhibitor cocktail (Roche, #11836145001) and phosphatase inhibitors [1 mM final concentration of glycerophosphate (Sigma-Aldrich, #G9891), sodium orthovanadate (Sigma-Aldrich, #S6508) and sodium fluoride (Sigma-Aldrich, #S6521)]. Total lysates were then quantified with Bio-Rad Protein Assay Dye reagent (Bio-Rad, #500–0006) and Laemmli buffer at a final concentration 2x was added to the samples. Proteins were next resolved in 4–12% Tris-Glycine gradient gels (Life Technologies, # NP0335BOX) and then transferred onto Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore, #IPVH00010). We then blocked membranes with 5% milk in Tris-buffered saline, Tween 20 (Sigma-Aldrich, #P1379) (TBS-T). All the primary antibodies for Western blot analysis were diluted in 3% BSA in TBS-T. HRP-conjugated secondary antibodies were diluted 1:10000 in 5% milk, TBS-T and detection was made with ECL (GE Healthcare, #RPN2106) alone or supplied with 10% SuperSignal West Femto (Thermo Fisher Scientific, #34095) when necessary.

Hydrogels of polyacrylamide

In order to test the cellular response to the mechanical properties of the extra-cellular matrix we have prepared hydrogels of polyacrylamide with modulable rigidity. The protocol described by Tse et al.⁶¹ has been adapted as follows: 15 mm of diameter round cover glasses were incubated with 250 μl 0.1M NaOH (Riedel de Haen, #30620) and placed at 80 °C until the liquid has evaporated. Glasses were then incubated with 125 μl of 3-aminopropyltriethoxysilane (APES) (Thermoscience, #80370) for 5 min and then placed into a multi-well plate and washed with ddH2O three times for 5 min. Glasses were then incubated with 250 μl of 0.5% glutaraldehyde (Sigma-Aldrich, #G6257) in PBS for 30 min. After removing the glutaraldehyde and allow glasses to dry, we treated the glass surface of slide glasses with 100 μl of Diclorodimethylsilane (DCDMS) (Merck, #8034520250) for 5 min and then washed with ddH2O. We prepared acrylamide/bis-acrylamide (Sigma-Aldrich, #A2792) solutions at different proportions according to the desired rigidity and added 10% APS (1:100) and TEMED (1:1000) (BDH, #4430836) quickly before pipetting 10 μl of solution onto the slide glass. We put the cover glasses with the side treated towards the drop and let the gel polymerized for 10 min. We then washed the obtained hydrogels with ddH2O and store them at 4 °C in PBS. The day before plating cells, we treated the hydrogels with 500 μl of sulfo-SANPAH (0.2 mg/ml sulfosuccinimidyl-6-(4’-azido-2’-nitrophenylamino-hexanoate) (Thermo Fisher Scientific, #22589) under UV (365 nm) for 10 min. We washed three times with HEPES (Sigma-Aldrich #H3375) 50 mM pH 8.5 and coated the hydrogels with 50 μl of Collagen type 1 Rat tail solution (Corning, #354236) in HEPES 0.10 mg/ml at 37 °C O/N. The following day, we transfered the hydrogels into a 12-well plates and washed them three times with PBS. After treating the hydrogels for 30 min with UV, we seeded 35000 cells/hydrogels.

Contraction assay

To monitor cellular contractility in vitro, cells were plated on thin collagen gels. Cellular contraction caused a decrease in the collagen area that can be quantified to estimate cells contractility during time⁴⁶. The day before plating the cells we coated a 12-well plate with 500 μl/well of a Collagen solution in PBS at 37 °C O/N: the solution consist in 7 Volumes of Collagen Type 1 (1 mg/ml), 2 Volumes of DMEM (GIBCO, #52100-039) and 1 Volume of Neutralizing Buffer (2.2 gr NaHCO3 (Carlo Erba Reagents, #478537), 4.77 gr HEPES, NaOH 0.05M in a final volume of 100 ml), FBS 5%. The day after, we seeded 150000 cells/well and waited the cells to adhere for about 4 h. We then detached the edges of the gel form the well and monitor the changes in the gel area taking pictures over time. The analysis of the images of the gel area/well area were performed with the ImageJ software.

Statistical analysis

Differences between averages were established with Student’s t-test or one way ANOVA analysis of variance as indicated in the figure legends; Bonferroni’s post-test was carried out for multiple comparisons.

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Acknowledgements

The authors are grateful to other members of the Boletta lab for helpful discussions. The work was funded by Telethon Italy (GGP12183), by Fondazione Cariplo (2010-0769) and by the PKD Foundation to A. Boletta.

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Authors and Affiliations

IRCCS San Raffaele Scientific Institute, Molecular Basis of Cystic Kidney Disorders, Division of Genetics and Cell Biology, Milan, Italy
Elisa Agnese Nigro, Gianfranco Distefano, Marco Chiaravalli, Maddalena Castelli, Angela Pesenti Gritti & Alessandra Boletta
IFOM-FIRC Institute of Molecular Oncology, Milan, Italy
Vittoria Matafora & Angela Bachi

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Elisa Agnese Nigro
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Gianfranco Distefano
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Marco Chiaravalli
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Vittoria Matafora
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Maddalena Castelli
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Angela Pesenti Gritti
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Angela Bachi
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Alessandra Boletta
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Contributions

E.A.N., designed and performed most studies, analyzed data, prepared the figures including drawings and wrote the manuscript; G.D., performed the labeling and IP from cells, conceived the initial idea of the study and performed Western blot experiments; M.Chiaravalli, performed animal crossings and in vivo experiments and analyzed histological data; V.M., designed, performed and interpreted the Mass Spectrometry experiments; M.Castelli, set up the collagen contraction assay experiments; A.P.G, set up the hydrogels experiments; A.Bachi, supervised the Mass Spectrometry analysis and wrote the manuscript; A.Boletta, supervised the work and wrote the manuscript.

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Correspondence to Alessandra Boletta.

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Nigro, E.A., Distefano, G., Chiaravalli, M. et al. Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness. Sci Rep 9, 16640 (2019). https://doi.org/10.1038/s41598-019-53061-0

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Received: 01 May 2019
Accepted: 24 October 2019
Published: 12 November 2019
DOI: https://doi.org/10.1038/s41598-019-53061-0

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