A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation

  1. Charles E MacKay
  2. Miranda Floen
  3. M Dennis Leo
  4. Raquibul Hasan
  5. Tessa AC Garrud
  6. Carlos Fernández-Peña
  7. Purnima Singh
  8. Kafait U Malik
  9. Jonathan H Jaggar  Is a corresponding author
  1. Department of Physiology, University of Tennessee Health Science Center, United States
6 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Flow stimulates PC-1-mediated, Ca2+-dependent currents in mesenteric artery endothelial cells (ECs) that elicit arterial hyperpolarization.

(A) Representative Western blots illustrating PC-1, PC-2, SK3, IK, TRPV4, Piezo1, eNOS, GPR68, and actin proteins in mesenteric arteries of Pkd1fl/fl and Pkd1 ecKO mice. (B) Mean data for PC-1, …

Figure 1—figure supplement 1
Genomic PCR indicating that tamoxifen stimulated Cre-recombination in mesenteric arteries of Pkd1fl/fl: Cdh5(PAC)-CreERT2 mice.

Representative of four separate experiments.

Figure 2 with 1 supplement
Endothelial cell PC-1 stimulates vasodilation via eNOS, IK channel, and SK channel activation.

(A) Representative traces illustrating reversible flow-mediated dilation in pressurized (80 mmHg) mesenteric arteries of Pkd1fl/fl and Pkd1 ecKO mice. (B) Mean dilation to flow (15 dyn/cm2) or ACh …

Figure 2—figure supplement 1
Depolarization-induced vasoconstriction and passive diameter are similar in Pkd1fl/fl and Pkd1 ecKO arteries.

(A) Representative traces illustrating dilations to ACh (10 µM) and Ca2+-free bath solution in pressurized (80 mmHg) Pkd1fl/fl and Pkd1 ecKO arteries. (B) PC-1 knockout attenuates flow-mediated …

Figure 3 with 1 supplement
Pkd1 ecKO mice are hypertensive with normal kidney anatomy.

(A) Blood pressure recordings obtained over 24 hr in a Pkd1fl/fl and Pkd1 ecKO mouse. (B) Mean arterial pressures (MAPs) in Pkd1fl/fl (n=5) and Pkd1 ecKO (n=7) mice. *p<0.05 versus Pkd1fl/fl. (C) …

Figure 3—figure supplement 1
Diastolic and systolic blood pressures are higher in Pkd1 ecKO mice.

(A) Mean diastolic and systolic blood pressures for Pkd1fl/fl (n=5) and Pkd1 ecKO (n=7) mice. *=p<0.05 versus Pkd1fl/fl. (B) Mean data for Pkd1fl/fl (n=5) and Pkd1 ecKO (n=7) mouse activity.

Figure 4 with 1 supplement
PC-1 and PC-2 coassemble and colocalize in endothelial cells (ECs).

(A) Representative Western blots illustrating the immunoblot (IB) detection of both PC-1 and PC-2 in PC-2 immunoprecipitate (IP) (n=5). (B) PC-1 (Alexa546) and PC-2 (Alexa488) antibody labeling …

Figure 4—figure supplement 1
PC-1 or PC-2 knockout abolishes immunoFRET in endothelial cells (ECs).

PC-1 (Alexa546) and PC-2 (Alexa488) antibody labeling and FRET in mesenteric artery ECs of Pkd1 ecKO (A) and Pkd2 ecKO (B) mice. Scale bars=10 µm. immunoFRET, immunofluorescence energy transfer.

Figure 5 with 4 supplements
Plasma membrane PC-1 and PC-2 clusters colocalize in endothelial cells (ECs).

(A) TIRF-SMLM images of PC-1 and PC-2 surface clusters in a Pkd1fl/fl and Pkd1 ecKO EC. Scale bars=5 µm. (B) Mean data for PC-1 and PC-2 cluster density measured in Pkd1fl/fl and Pkd1 ecKO ECs under …

Figure 5—figure supplement 1
Localization precision of fluorophores used in SMLM experiments.

Mean data illustrating the localization precision (FWHM) of Alexa Fluor 488 (A)- and Alexa Fluor 647 (B)-tagged secondary antibodies in ECs under all conditions when imaged using SMLM. (A) …

Figure 5—figure supplement 2
Properties of PC-1 and PC-2 clusters and their spatial proximity and overlap in Pkd1fl/fl and Pkd1 ecKO endothelial cells (ECs).

(A) Histogram illustrating PC-1 cluster size distribution in a Pkd1fl/fl (static) and Pkd1 ecKO (static) EC. (B) Histogram illustrating PC-2 cluster sizes in a Pkd1fl/fl (static) and Pkd1 ecKO …

Figure 5—figure supplement 3
Properties of PC-1 and PC-2 surface clusters in endothelial cells (ECs) of Pkd2fl/fl and Pkd2 ecKO mice.

(A) TIRF-SMLM images of PC-1 and PC-2 surface clusters in a Pkd2fl/fl and Pkd2 ecKO EC. Scale bars = 5 µm. (B) Mean data for PC-1 and PC-2 cluster density measured in Pkd2fl/fl and Pkd2 ecKO ECs …

Figure 5—figure supplement 4
Analysis of PC-1 and PC-2 cluster overlap in endothelial cells of Pkd2fl/fl and Pkd2 ecKO mice.

(A) TIRF-SMLM images of PC-1 and PC-2 clusters, overlap of PC-1 and PC-2 data and overlap of PC-1 and PC-2 data following Costes’ randomization simulation. Scale bars = 5 µm. (B) Mean experimental …

Figure 6 with 2 supplements
PC-1 and PC-2 are interdependent for flow-mediated vasodilation and ICat activation in mesenteric artery endothelial cells (ECs).

(A) Representative Western blots of PC-1, PC-2, and eNOS in mesenteric arteries of Pkd1fl/fl/Pkd2fl/fl and Pkd1/Pkd2 ecKO mice. (B) Mean data. *p<0.05 versus Pkd1fl/fl/Pkd2fl/fl. n=5 for each data …

Figure 6—figure supplement 1
Smooth muscle-specific vasoconstriction and vasodilation and passive diameter are unaltered in Pkd1/Pkd2 ecKO mouse arteries.

(A) Representative traces illustrating 60 mM-induced K+ constriction in a pressurized (10 mmHg) Pkd1fl/fl/Pkd2fl/fl and Pkd1/Pkd2 ecKO artery. (B) Mean data for 60 mM K+-induced constriction. n=6 …

Figure 6—figure supplement 2
Scrambled PC-1 and PC-2 peptides do not alter flow-activated ICat in endothelial cells (ECs).

Original recordings illustrating that intracellular introduction via the patch pipette of either a PC-1 scrambled (scram) peptide or a PC-2 scrambled peptide does not alter flow (10 …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)Pkd1fl/flBaltimore PKD Core CenterPMID:15579506MGI: 3617325Mice with Pkd1 gene flanked by loxP regions.
Strain, strain background (M. musculus)Pkd2fl/flBaltimore PKD Core CenterPMID:20862291MGI: 4843127Mice with Pkd2 gene flanked by loxP regions.
Strain, strain background (M. musculus)Pkd1fl/fl /Pkd2fl/flThis paperMouse line created in-house by mating Pkd1fl/fl with Pkd2fl/fl. Mice with Pkd1 and Pkd2 genes flanked by loxP regions.
Strain, strain background (M. musculus)Cdh5(PAC)-CreERT2Cancer Research UKRRID: MGI: 3848982Mice with tamoxifen-inducible Cre recombinase that is expressed specifically in endothelial cells.
Strain, strain background (M. musculus)Pkd1fl/fl: Cdh5(PAC)-CreERT2This paperMouse line created in-house by mating Pkd1fl/fl with Cdh5(PAC)-CreERT2. Mice with inducible endothelial cell-specific deletion of PC-1.
Strain, strain background (M. musculus)Pkd2fl/fl: Cdh5(PAC)-CreERT2PMID:32364494Mouse line created in-house by mating Pkd2fl/fl with Cdh5(PAC)-CreERT2. Mice with inducible endothelial cell-specific deletion of PC-2.
Strain, strain background (M. musculus)Pkd1fl/fl/Pkd2fl/fl: Cdh5(PAC)-CreERT2This paperMouse line created in-house by mating Pkd1fl/fl Cdh5(PAC)-CreERT2 with Pkd2fl/fl Cdh5(PAC)-CreERT2. Mice with inducible endothelial cell-specific deletion both PC-1 and PC-2.
AntibodyAnti-PC-1 (mouse monoclonal)Baltimore PKD CoreMouse mAB SF4AZ E3IF (1:100)Lattice SIM (1:100)SMLM (1:100)
AntibodyAnti-PC-2 (rabbit monoclonal)Baltimore PKD CoreRabbit mAB 3374 CT-14/4IF (1:100)Lattice SIM (1:100)SMLM (1:100)
AntibodyAnti-CD31 (rat monoclonal)AbcamCat. #: Ab7388RRID: AB_305905IF (1:100)Lattice SIM (1:100)SMLM (1:100)
AntibodyAnti-PC-1 (H-260)(rabbit polyclonal)Santa Cruz BiotechnologyCat. #: sc-25570,
RRID: AB_2163357
N-FRET (1:100)
AntibodyAnti-PC-2 (mouse monoclonal)Santa Cruz BiotechnologyCat. #: sc-28331,
RRID: AB_672377
N-FRET (1:100)
AntibodyAlexa 488-conjugated anti-rat IgG (donkey polyclonal)Thermo Fisher ScientificCat. #: A-21208,RRID: AB_2535794IF (1:500)
AntibodyAlexa 546-conjugated anti-mouse IgG (donkey polyclonal)Thermo Fisher ScientificCat. #: A-10036,RRID: AB_2534012IF (1:500)
AntibodyAlexa 488-conjugated anti-mouse IgG (donkey polyclonal)Thermo Fisher ScientificCat. #: A-21202,RRID: AB_141607Lattice SIM (1:500) SMLM (1:500)
AntibodyAlexa 546-conjugated anti-rabbit IgG (donkey polyclonal)Thermo Fisher ScientificCat. #: A-10040,RRID: AB_2534016Lattice SIM(1:500)
AntibodyAlexa 647-conjugated anti-rat IgG (goat polyclonal)Thermo Fisher ScientificCat. #: A-21247,RRID: AB_141778Lattice SIM(1:500)
AntibodyAlexa 555-conjugated anti-rat IgG (goat polyclonal)Thermo Fisher ScientificCat. #: A-21434,RRID: AB_2535855SMLM (1:500)
AntibodyAlexa 647-conjugated anti-rabbit IgG (donkey polyclonal)Thermo Fisher ScientificCat. #: A-31573,RRID: AB_2536183SMLM (1:500)
AntibodyAlexa Fluor 488-conjugated anti-mouse (goat polyclonal)Thermo Fisher ScientificCat. #: A-11001,
RRID: AB_2534069
N-FRET (1:100)
AntibodyAlexa Fluor 555-conjugated anti-rabbit (donkey polyclonal)Thermo Fisher ScientificCat. #: A-31572,
RRID: AB_162543
N-FRET (1:100)
AntibodyAnti-PC-1 (mouse monoclonal)Santa Cruz BiotechnologyCat. #: sc-130554,RRID: AB_2163355WB (1:100)
AntibodyAnti-PC-2 (mouse monoclonal)Santa Cruz BiotechnologyCat. #: sc-47734,RRID: AB_672380WB (1:100)IP (1:20)
AntibodyAnti-IK1 (D-5) (mouse monoclonal)Santa Cruz BiotechnologyCat. #: sc-365265,RRID: AB_10841432WB (1:100)
AntibodyAnti-SK3(rabbit polyclonal)AbcamCat. #: ab28631,RRID: AB_775888WB (1:100)
AntibodyAnti-eNOS [M221](mouse monoclonal)AbcamCat. #: ab76198,RRID: AB_1310183WB (1:100)
AntibodyAnti-TRPV4(clone 1B2.6) (mouse monoclonal)Millipore SigmaCat. #: MABS466WB (1:100)
AntibodyAnti-Actin (clone C4)(mouse monoclonal)MilliporeSigmaCat. #: MAB1501,RRID: AB_2223041WB (1:1000)
AntibodyAnti-p-eNOS (rabbit polyclonal)Cell Signaling TechnologyCat. #: 9571,RRID: AB_329837WB (1:100)
AntibodyAnti-GPR68(rabbit polyclonal)NOVUS BiologicalsCat. #: NBP2-32747WB (1:100)
AntibodyAnti-Piezo1 (rabbit polyclonal)ProteinTechCat. #: 15939-1-AP,RRID: AB_2231460WB (1:100)
AntibodyMouse IgG control (mouse polyclonal)MilliporeSigmaCat. #: 12-371,RRID: AB_145840IP (1:20)
Peptide, recombinant proteinPC-1 coiled-coil domain peptideGenscriptFDRLNQATE
DVYQLEQQL
1 µM
Peptide, recombinant proteinPC-1 scrambled peptideGenscriptQLNDLFQTE
VAEDLQRYQ
1 µM
Peptide, recombinant proteinPC-2 coiled-coil domain peptideGenscriptKRREVLGRLL1 µM
Peptide, recombinant proteinPC-2 scrambled peptideGenscriptVLKLLRRRGE1 µM
Commercial assay or kitPierce crosslink magnetic IP/co-IP KitThermo Fisher ScientificCat. #: 88805
OtherDAPI stainThermo FisherScientificCat. #: 3571RRID: AB_23074451:1000

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