Phase transitions of multivalent proteins can promote clustering of membrane receptors
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
Reconstitution of p-Nephrin clusters on supported lipid bilayers.
(A) Cartoon illustrating the interaction of triply-phosphorylated His8-tagged Nephrin (p-Nephrin) with its partners Nck and N-WASP. Top panel illustrates p-Nephrin attached to bilayers. Bottom panel …
Figure 1—figure supplement 1
p-Nephrin, Nck and N-WASP colocalize to clusters formed on fluid supported lipid bilayers.
(A) Fluorescence recovery after photobleaching (FRAP) on a supported bilayer with p-Nephrin shows full recovery with exponential recovery time constant τ = 1.3 s. Line shows fit to a …
Figure 2 with 2 supplements
Nephrin clusters are created via a two-dimensional phase-transition.
(A) Fractional intensity in clusters (blue symbols, left ordinate) and signal variance (red symbols, right ordinate) of p-Nephrin fluorescence on a DOPC bilayer as a function of Nck concentration …
Figure 2—figure supplement 1
Quantitative analysis of the measurement and control of His8-p-Nephrin density on supported lipid bilayers.
(A) Fluorescence intensity as a function of fluorescent lipid (OG-DHPE) concentration for a solution of small unilamellar vesicles. Fluorescence of liposomes (in solution) containing OG-DHPE were …
Figure 2—figure supplement 2
Quantification of average p-Nephrin density on the bilayer for every titration point shown in Figure 2A.
Y-axis represents p-Nephrin density and x-axis represents the different Nck concentrations of the titration as in Figure 2A. Densities are averages of five different areas of each bilayer. Error …
Figure 3 with 3 supplements
Clusters are dynamic.
(A) Time-lapse TIRF imaging of bilayers containing ∼3100 molecules/µm2 Alexa488-labeled p-Nephrin after addition of 1 µM Nck and 1 µM N-WASP. Images represent time intervals of 2 min and show …
Figure 3—figure supplement 1
Cluster size-distribution analyses at different times suggest exponential behavior at lower densities.
(A) Log-linear plot of cluster number vs size at p-Nephrin density of ∼2500 μm2, 1 μM Nck, and 1 μM N-WASP. The distributions are plotted for times between 2 and 20 min. (B) Log–log plot of the same …
Figure 3—figure supplement 2
Cluster size-distribution analyses suggest power law behavior at higher densities.
(A) Log-linear plot of the size distribution of clusters at a Nephrin density of ∼4000 μm2, 1 μM Nck, and 1 μM N-WASP, recorded 60 min after clustering was initiated. (B) Log–log plot of the size …
Figure 3—figure supplement 3
Average cluster size is dependent on molecular density.
Samples contained either low (∼2000 molecules/µm2, red dots) or high (∼3500 molecules/µm2, blue dots) density of p-Nephrin. Clustering was initiated by addition of 1 μM Nck and 1 μM N-WASP. Images …
Figure 4 with 1 supplement
Clustering is dependent upon the valency of the interacting motifs.
Plots show fractional intensity of fluorescent Nephrin proteins in clusters as a function of Nck protein concentrations for 500 nM N-WASP. (A) Top, middle, and bottom panels show data for p-Nephrin …
Figure 4—figure supplement 1
Di-valent molecules are stronger clustering agents than mono-valent molecules.
(A) In the presence of 5 µM Nck and 2 μM N-WASP, p-Nephrin 2pY forms clusters (right panel). Even in the presence of 10 µM Nck and 5 μM N-WASP, p-Nephrin 1pY does not form clusters (left panel). …
Figure 5 with 1 supplement
Molecular affinities affect macroscopic clustering.
(A) Fractional intensity of fluorescent pTyr proteins in clusters as a function of SH3 (module) concentrations for 500 nM N-WASP. Left and right panels show data for a p-TIR and p-Nephrin, whose …
Figure 5—figure supplement 1
Measurement of the affinity of Nck for p-TIR and p-Nephrin.
Isothermal titration calorimetry analysis of Nck binding to p-Nephrin and p-TIR. Nck (150 µM) in the syringe was titrated into 5 μM of either (A) p-Nephrin (3pY) or (B) p-TIR (3pY). Both datasets …
Figure 6
Mono-valent pTyr peptide can eliminate clusters.
(A) Time course following addition of 10 µM of a monovalent pTyr peptide derived from TIR (with KD of 40 nM for the Nck SH2 domain) to clusters formed from p-Nephrin /(SH3)3/N-WASP. (B) Time course …
Figure 7 with 2 supplements
Actin assembles specifically on p-Nephrin/Nck/N-WASP clusters.
(A) Alexa488-labeled p-Nephrin (2200 molecules/µm2) was clustered by addition of 2 μM N-WASP and 1 μM Nck. Images show time course of p-Nephrin (top row), actin (middle row) and merge (bottom row) …
Figure 7—figure supplement 1
Actin localizes to and assembles on the clusters in an Arp2/3 dependent manner.
(A) Images of clusters formed by p-Nephrin (2173 molecules/μm2 on the supported lipid bilayer) plus soluble 1 μM Nck, 2 μM N-WASP, 10 nM Arp2/3 complex, and 1 μM actin (10% rhodamine labeled) at 0 …
Figure 7—figure supplement 2
Actin assembly reorganizes p-Nephrin clusters.
Enlarged actin assembly images from Figure 7, including more time points between 36 and 45 min. Top, middle, and bottom rows show p-Nephrin Alexa488, rhodamine-actin and merge, respectively.
Videos
Video 1
Addition of Nck and N-WASP to p-Nephrin produces macroscopic clusters on supported bilayers.
Time-lapse images taken immediately after adding 1 μM Nck and 1 μM N-WASP to p-Nephrin Alexa488. Images were captured every minute.
Video 2
Clusters are dynamic.
Time-lapse of clusters made from 1 μM Nck and 1 μM N-WASP with p-Nephrin Alexa488 on the membrane. Images were captured every 30 s. In addition to fusion events, the clusters also occasionally …
Video 3
Mono-valent peptide dissolves clusters.
Addition of 10 μM 1pY—TIR causes the clusters of 1 μM (SH3)3 and 500 nM N-WASP to dissipate. Images were captured every 30 s.
Tables
Table 1
Information on the protein constructs used in this study
| Proteins | Sequence information | Notes |
|---|---|---|
| Nck | GHMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKCSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYCIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS | Human, WT, residues 1–377 |
| N-WASP BPVCA | GSEFKEKKKGKAKKKRAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPHPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVPASSGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVSDGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDDDDEEDFEDDDEWED | Rat, residues 183–193 fused to 273–501 |
| Nck (cysteine-modified) | GHMCMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS | Human, residues 1–377, with mutations: C139S, C232A, C266S, C340S |
| Nephrin3Y | GGSLEHHHHHHHHGGSCGGSGGSGGSGGSHLYDEVERTFPPSGAWGPLYDEVQMGPWDLHWPEDTFQDPRGIYDQVAGD | Human, residues 1174–1223, with mutations: Y1183F, Y1210F |
| Nephrin2Y | GGSLEHHHHHHHHGGSCGGSGGSGGSGGSHLFDEVERTFPPSGAWGPLYDEVQMGPWDLHWPEDTFQDPRGIYDQVAGD | Human, residues 1174–1223, with mutations: Y1176F, Y1183F, Y1210F |
| Nephrin1Y | GGSLEHHHHHHHHGGSCGGSGGSGGSGGSHLFDEVERTFPPSGAWGPLYDEVQMGPWDLHWPEDTFQDPRGIFDQVAGD | Human, residues 1174–1223, with mutations: Y1176F, Y1183F, Y1210F, Y1217F |
| TIR3Y | GGSLEHHHHHHHHGGSCGGSGGSGGSGGSHMHIYDEVAADPPPSGAWGHIYDEVAADPWDLHWPEDTFQDPRHIYDEVAADP | Human Nephrin, with pTyr sites replaced by those in EPEC Tir protein (underlined) |
| (SH3)3 | GHMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYCIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS | Human, three repeats of the second Nck SH3 domain, plus the Nck SH2 domain |
| (SH3)2 | GHMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYCIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS | Human, two repeats of the second Nck SH3 domain, plus the Nck SH2 domain |
| (SH3)1 | GHMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYCIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS | Human, one repeat of the second Nck SH3 domain, plus the Nck SH2 domain |
| TIR-1pY | EEHIpYDEVAADPGGSWGGSC | N-terminal rhodamine labeled single pTyr motif from EPEC Tir protein |
| Lck | ANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHDLVRHYTNASDGLCTKLSRPCQTQKPQKPWWEDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHPRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLNVNKLLDMAAQIAEGMAFIEEQNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYHLMMLCWKERPEDRPTFDYLRSVLDDFFTATEGQFQPQP | Human, 119–509, Y505F |
Table 2
Statistics of fitting for FRAP data
| p-Nephrin | Nck (with p-Nephrin) | Nck (with p-TIR) | N-WASP | |
|---|---|---|---|---|
| Null hypothesis | Single Exp. | Single Exp. | Single Exp. | Single Exp. |
| Alternate hypothesis | Double Exp. | Double Exp. | Double Exp. | Double Exp. |
| p value | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
| Conclusion (alpha = 0.05) | Reject null hypo. | Reject null hypo. | Reject null hypo. | Reject null hypo. |
| Preferred model | Double Exp. | Double Exp. | Double Exp. | Double Exp. |
| F (DFn, DFd) | 64.16 (2282) | 47.33 (2635) | 46.72 (2379) | 48.64 (2379) |
