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Received 3 Jun 2013 | Accepted 28 Aug 2013 | Published 27 Sep 2013

reveals that this region harbours a non-canonical pleckstrin homology (PH) domain connected to a 16-leucine-rich repeat domain. Lipid binding is mediated by the PH domain, but is further enhanced by a central helical domain. Small-angle X-ray scattering reveals that the helical domain mediates antiparallel dimerization, properly positioning the PH domains for simultaneous membrane interaction. In cells, deletion of the PH domain impairs leading edge localization. The results support a direct membrane-binding mechanism for CARMIL localization at the leading edge, where it regulates cytoskeletal effectors and motility.

DOI: 10.1038/ncomms3523

CARMIL leading edge localization dependson a non-canonical PH domain and dimerization

Adam Zwolak1, Changsong Yang2, Elizabeth A. Feeser1, E. Michael Ostap1, Tatyana Svitkina2

& Roberto Dominguez1

1 Department of Physiology, Perelman School of Medicine, University of Pennsylvania, 728 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. 2 221 Leidy Laboratory, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. Correspondence and requests for materials should be addressed to R.D. (email: mailto:[email protected]

Web End [email protected] ).

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Actin-based cell motility underlies many fundamental biological processes, such as dendritic spine formation, immune response and tumour metastasis13. During these

processes, actin laments form highly dynamic networks at the leading edge of motile cells, with their fast-growing barbed (or )

ends directed towards the plasma membrane (PM), providing the forces for cell motility4. As a consequence, actin barbed-end polymerization is tightly regulated in cells via membrane-associated proteins. An important and ubiquitous regulator of barbed-end dynamics is capping protein (CP)5. CP binds tightly to the barbed end, which prevents actin monomer addition or dissociation6. In this way, CP promotes the formation of actin networks consisting of shorter and more densely branched laments, thought to be necessary for efcient lamellipodial protrusion4,7. CP is one of a small subset of cytoskeletal proteins required for reconstitution of actin-based motility in vitro8, and is a crucial component of the dendritic actin polymerization model4. Despite its critical role in the regulation of actin dynamics, CP lacks a mechanism for direct membrane association and is itself regulated by multiple proteins that could provide a link to the membrane5,7. The proteins that regulate CP, including CARMIL (CP Arp2/3 complex myosin-I linker), CD2AP, CKIP-1, CapZIP and FAM21, are generally unrelated, but most use a similar allosteric mechanism; they contain a conserved B30-aa CP interaction (CPI) motif that binds to CP on the opposite side from the actin lament, lowering its afnity for the barbed end9,10. CARMIL contains, in addition to the CPI motif, a B14-aa so-called CARMIL-specic interaction motif that also participates in interaction with CP10.

CARMIL is a large (B1,370-aa) multi-domain protein originally identied as a binding partner of the SH3 domain of myosin-I (refs 1114). The interaction with myosin-I has been mapped to the carboxy-terminal proline-rich domain (PRD) of CARMIL12, which features six canonical SH3-binding PxxP sites (Fig. 1a). Vertebrates express three CARMIL isoforms (CARMIL13) that have non-overlapping roles in cell motility15. CARMIL3 is an oncofetal protein, whose overexpression promotes cell proliferation and tumour growth in adult mice16. CARMIL2, which is downregulated in patients affected by psoriasis17, colocalizes with the vimentin lament network, and loss of its function impairs cell polarity and motility15. CARMIL1 (referred to here simply as CARMIL) has been more extensively studied; it colocalizes with CP at the cell leading edge and its knockdown results in loss of lamellipodial actin and strong inhibition of cell motility and macropinocytosis15,18. In neuronal cells, CARMIL negatively regulates Trio (UNC-73), a Rho-family GTPase guanine nucleotide exchange factor19, resulting in inhibition of axon growth cone migration15,20. Because of its role in the regulation of actin dynamics, CARMIL is considered a key factor in normal cell motility and tissue development, as well as in aberrant migration of metastatic cancer cells7. Yet, with the exception of its interaction with CP10,2125, the structural and biochemical properties of this large cytoskeletal effector and its mechanism of leading edge localization are poorly understood.

Here we advance our understanding of CARMIL structure function by identifying two previously unknown domains: a pleckstrin homology (PH) domain implicated in direct interaction with the PM, and a central helical domain (HD) that mediates antiparallel dimerization and enhances membrane binding by positioning the PH domains for optimal interaction with the membrane. Cellular studies conrm the role of the PH domain in leading edge localization. When combined, the results lead to a new model of CARMIL localization and function at the leading edge.

ResultsCARMIL binds lipid membranes through its amino-terminal region. We sought to determine the mechanism by which

CARMIL localizes to the PM in a CP-independent manner15,18. Prior studies had identied three domains in CARMIL: the leucine-rich repeat (LRR) domain, whose exact boundaries remained unclear, the CPICARMIL-specic interaction motif and the carboxy-terminal PRD. Through sequence analysis, we identied what appeared to be two additional domains, a B150-aa

N-terminal domain clearly distinguished from the LRR domain by its predicted high b-strand content, and a predicted HD (residues 689878 of mouse CARMIL1, the species studied here) positioned between the LRR and CPI domains (Fig. 1a). We expressed fragments corresponding to each of these individual domains as well as combinations of domains (Fig. 1a). CARMIL1154 had

limited solubility on its own, and was expressed as a maltose-binding protein (MBP) fusion, which increased its solubility. To test whether CARMIL could directly bind lipid membranes, we assessed the ability of each construct to cosediment with liposomes derived from bovine brain lipid extracts (Folch fraction I). In the absence of lipids, all the constructs were soluble and did not sediment (Fig. 1a and Supplementary Fig. S1). In the presence of lipids, constructs containing the N-terminal domain cosedimented with lipids (Fig. 1a). Interestingly, CARMIL1878, including the

HD, cosedimented more abundantly than MBP-CARMIL1154 and

CARMIL1668 (80% versus 55%). This was a surprising result, as

the isolated HD, CARMIL689878, did not cosediment with lipids.

When combined, these ndings suggested that the N-terminal region of CARMIL directly binds lipid membranes, whereas the HD contributes indirectly to this activity (addressed below).

Crystal structure of CARMIL1668. The N-terminal domain of

CARMIL implicated above in lipid binding had no detectable sequence similarity with any known structure. Therefore, to understand the molecular basis for lipid binding, we sought to determine its structure. Multiple N-terminal fragments were expressed, but they all had poor solubility. The isolated LRR domain also had low solubility. In contrast, CARMIL1668, com

prising the N-terminal and LRR domains, was highly soluble, suggesting that these two domains were structurally interconnected. CARMIL1668 was crystallized and its structure was determined using the single-wavelength anomalous dispersion method (Fig. 1b, Table 1, Supplementary Fig. S2 and Supplementary Movie 1). The structure was rened to 2.9 resolution using four-fold noncrystallographic symmetry constraints, and revealed two major domains: a PH domain (residues 25118) and a 16-LRR domain (residues 191638).

The PH domain is a recognized lipid-binding fold26, which is consistent with the ability of the N-terminal region of CARMIL to cosediment with lipid extracts. It is not surprising, however, that the PH domain of CARMIL had remained undetected from sequence, as it displays multiple non-canonical features. The most closely related canonical PH domains identied with the programme Dali27 share r12% sequence identity with that of

CARMIL (Supplementary Fig. S3). The PH domain fold consists of a seven-strand b-barrel, capped at one end by a C-terminal a-helix, whereas the other end of the b-barrel hosts the lipid-binding pocket28. Although these features are conserved in the PH domain of CARMIL, key residues in the lipid-binding pocket are not conserved (Supplementary Fig. S3). Moreover, the PH domain of CARMIL is tightly integrated with N- and C-terminal structural elements that do not form part of the canonical PH fold. These include an a-helix at the N terminus (residues 1020), referred to here as the N-helix, and a b-strand followed by an a-helix at the C terminus (residues 129147), referred to here as the Linker region (Fig. 1b and Supplementary Fig. S2b). The b-strand in the Linker region is incorporated as an additional strand into the b-barrel of the PH domain. The Linker region is

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Linker

CPI-CSI PRD

1,039 1,085 1,374

1878

N-helix N-cap C-cap

PH LRR

25 119 155 191 639 668 689 878 975

100

58 80

S P

1668

MBP-1154

MBP

158878

689878

S S

P P

0.2 0.4 0.6 0.8 1.0 Lipid + Lipid Fraction bound

Linker

N N-helix

LRR

PH N-cap

C-cap

1 2 3 4 5

6 7 2

119

237

355

473

591 668

3 4

14 15

1 2 1

7 7

8 8

10 10

13 13 14

15 16

1 2

Figure 1 | Identication of a lipid-binding domain and structure of CARMIL1668. (a) Domain organization of CARMIL, design of protein constructs and sedimentation with brain lipid extracts. CARMIL was known to contain an LRR domain, although its size and boundaries were unknown, a CPICARMIL-specic interaction (CSI) motif and a PRD. Here, through sequence and structural analyses, we have dened the exact boundaries of the LRR domain and identied previously unknown N- and C-terminal caps of the LRR, and two additional domains, a PH domain and a HD. The PH domain is interconnected with an N-terminal helix (N-helix) and a C-terminal linker (Linker). CARMIL constructs (5 mM) were tested for their ability to cosediment with brain lipid extracts (1 mg ml 1). MBP was used as a control. Proteins were centrifuged in the absence or presence of lipids. Positions of molecular weight markers are indicated. Coomassie-stained gel bands were quantied. Error bars represent s.d. from three independent experiments.

(b) Two perpendicular representations of the crystal structure of CARMIL1668, with domains coloured as in a. A representation of the secondary structure and domain assignments resulting from the structure is shown above the sequence of CARMIL1668, coloured according to a.

conserved within each CARMIL isoform, but not across isoforms (Supplementary Fig. S4), suggesting that it might perform isoform-specic functions in addition to linking the PH and LRR domains.

The LRR domain has an overall planar horseshoe shape with an inner radius (r) of B19 and spans an arc angle (f) of B232 (Supplementary Fig. S5a)29. The LRR domain of CARMIL is most closely related to that of ribonuclease inhibitor30, but has a somewhat more elliptical shape. Each LRR motif consists

of a b-strand, occupying the inner side of the LRR domain, and an a-helix on the outer side of the domain. The loop connecting these two secondary structure elements is called the ascending loop, whereas that connecting one repeat to the next is called the descending loop. In CARMIL, only repeat 6 displays a fully canonical LRR sequence (LxxLxLxxN/CxL) (Supplementary Fig. S5b) and was thus used as a reference in comparisons with the other repeats. Repeats 315 overlay well with repeat 6, with an average root-mean-square displacement (RMSD) for equivalent

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Table 1 | Data collection and renement.

Data collection (Se-Met derivative)Space group P1*Cell dimensionsa, b, c () 57.17, 67.99, 212.15 a, b, g () 92.73, 96.95, 110.15

Wavelength () 0.9784 Resolution () 2.950.0 (2.93.0)w Rmerge 10.2 (48.9)

I/s 7.5 (1.9) Completeness (%) 98.6 (83.5)

Redundancy 13.8 (10.0)

RenementResolution () 2.950.0 (2.93.0) No. reections 53,184 Rwork/Rfree 21.5/25.9

No. atoms

Protein 20,364 Ligand 17 B-factors (2)

Protein 56.5 Ligand 54.2 Root-mean-square displacement

Bond lengths () 0.02 Bond angles () 2.1

*Data from a single crystal.

wValues in parentheses are for highest resolution shell.

Ca-atoms of 0.65 , whereas repeats 1, 2 and the incomplete repeat 16 diverge more signicantly (Supplementary Fig. S5c).

The LRR of CARMIL is capped at the N- and C-terminal ends by helix-loop-helix motifs. These caps are highly conserved among CARMIL isoforms, particularly the N-terminal cap (Supplementary Fig. S4). Both caps contain a helix that runs diagonally to the rst (or the last) LRR motif, such that it shields the hydrophobic core of the LRR domain from solvent exposure (Supplementary Fig. S5d,e). Although such caps are common among LRR-containing proteins and are thought to stabilize their structures29, those of CARMIL are distinct from other known LRR capping motifs. Only one other protein, tropomodulin31, contains a helix within its C-terminal cap that superimposes well with that of CARMIL (Supplementary Fig. S5f).

The PH domain and lipid binding. PH domains are often implicated in phospholipid binding26. Therefore, we asked whether the PH domain of CARMIL bound specic phospholipids, accounting for the cosedimentation observed with brain lipid membranes (Fig. 1a). Using a lipid strip assay32, we found that CARMIL1878, the fragment that cosedimented

most abundantly with lipid membranes, showed specicity for phosphatidylinositol, phosphatidylserine (PS) and monophosphorylated phosphatidylinositides (PtdIns(3)P, PtdIns(4)P and PtdIns(5)P) (Fig. 2a). However, contrary to most canonical PH domains, CARMIL1878 did not appear to bind polypho

sphorylated phosphatidylinositides. PS accounts for B12% of the lipid content of the inner leaet of the PM33, suggesting that CARMIL could bind directly to the PM through its PH domain. Accordingly, CARMIL158878, in which the PH domain was

deleted, showed no lipid-binding activity (Fig. 2a), which is consistent with the inability of this fragment to cosediment with lipid membranes (Fig. 1a).

Most PH domains that bind phospholipids with high afnity tend to have conserved residues that form a lipid-binding pocket

on one side of the b-barrel and display an overall basic charge around the lipid-binding pocket26,28. Such is the case of DAPP1, a canonical PH domain identied by Dali27 as one of the closest structural relatives of CARMILs PH domain (Supplementary Fig. S3), which was crystallized with bound PtdIns(1,3,4,5)P4 (ref. 28 and Fig. 2b). In contrast, the PH domain of CARMIL lacks most of these residues (Fig. 2c). However, on the opposite side of the PH domain, at the interface between the N-helix and the PH domain, CARMIL has a prominent basic pocket formed by residues R10, K17, R22, K23, K25, K29, K30, K31 and K33 (Fig. 2c). As some PH domains bind lipids through sites other than the classical pocket34, we suspected that this pocket in CARMIL could harbour a lipid-binding site. However, a CARMIL1878 mutant in which ve of these residues (K17, K23,

K29, K31 and K33) were all substituted by glutamic acid bound lipids similarly to the wild-type protein in the lipid strip assay (Fig. 2a). As this basic pocket had no apparent role in lipid binding, we tested whether the canonical lipid-binding pocket was functional. Three basic residues surround this pocket in CARMIL (K37, R40 and K44). A CARMIL1878 mutant in which

these three residues were replaced by glutamic acid, and E42 in the middle of the pocket was substituted by serine, did not bind lipids in the strip assay. We thus conclude that despite its unusual properties the lipid-binding pocket in the PH domain of CARMIL is still functional, but contrary to most PH domains it appears to have specicity for PS and monophosphorylated lipids. To quantitatively test this possibility, we measured the afnity of CARMIL1878 for different types of lipids using a cosedimentation assay (Fig. 2d). This assay quanties the amount of CARMIL1878

cosedimenting with large unilamellar vesicles containing lipids identied in the strip assay35. Consistent with the strip assays, CARMIL1878 did not bind phosphatidylcholine, cosedimented very weakly with PtdIns(4,5)P2, but bound DOPS (1,2-dioleoylsn-glycero-3-phospho-L-serine) and PtdIns(5)P with B10 mM afnity. These results suggest that membrane binding by the PH domain of CARMIL might be dominated by binding to PS, which is highly abundant in the PM.

A central HD mediates antiparallel dimerization. CARMIL1878

cosedimented B25% more abundantly with brain lipids than CARMIL1668. These two fragments differ only in that CARMIL1878

includes the predicted HD, which had no lipid-binding activity on its own (Fig. 1a). Consistent with the secondary structure prediction (Supplementary Fig. S4), the circular dichroism spectrum of CARMIL689878 displayed minima at 208 and 222 nm, characteristic of an all-helical structure (Fig. 3a). Analysis of CARMIL constructs by size exclusion chromatography (SEC) showed that fragments containing the HD eluted with molecular weight characteristics of dimers, whereas fragments lacking the HD were monomeric (Fig. 3b and Table 2). Note that each mass measurement was carried out with at least two different methods, to account for uncertainties in the mass estimates of elongated proteins derived from SEC (Table 2). At higher concentrations the isolated HD showed limited solubility, but a fusion protein with MBP (MBP-CARMIL689878)

was highly soluble. MBP-CARMIL689878 formed a dimer in

solution, as determined by analytical SEC and multi-angle light scattering (SECMALS), whereas MBP alone was monomeric (Fig. 3b,c and Table 2). These results conrmed that the HD was sufcient for dimerization.

To investigate the mechanism of dimerization, we used small-angle X-ray scattering (SAXS). Data collected at several concentrations of MBP-CARMIL689878 showed a linear depen

dence of the scattering intensity with protein concentration (Fig. 3d), conrming that this construct does not aggregate even at high concentrations (up to 40 mg ml 1). The molecular mass

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CARMIL1878

CARMIL1878 LPA S1P

PI3,4P2 PI3,5P2

PI4,5P2 PI3,4,5P3

PA PS Blank

K17E, K23E,

K29E, K31E,

K33E

CARMIL158878

K37E, R40S,

E42S, K44E

LPC

PI3P PI4P

PI5P

PE PC

100% DOPC

1.0

0.5

1.0

0.5

0.2

0.4 0.6 0.8

Keff = 9 M

1.0

DAPP1

80% DOPC, 20% DOPS

R235

T180

R206

Y195

K197

R184 K182

Fraction bound

0.2

0.4 0.6 0.8 1.0

CARMIL

95% DOPC, 5% Ptdlns(5)P

95% DOPC, 5% Ptdlns(4,5)P2

N92

K37

R40

1.0

0.5

1.0

0.5

L66

L34

E65

S57 K44 E42

R59 N

Keff = 12 M

Keff > 1,000 M

0.2 0.4 0.6 0.8

1.0

180

K25

K23

0.2 0.4 0.6 0.8 1.0 [Lipid]outer leaflet (mM)

K30 K29 K17

R10

R22 N

K33 K31

Figure 2 | Specicity and afnity of lipid binding. (a) Lipid-binding specicity of CARMIL constructs and PH domain mutants (60 nM) using a lipid overlay assay (Echelon Biosciences). Each experiment was repeated three times. (b) Surface charge distribution and cartoon representation of the canonical PH domain of DAPP1 with bound PtdIns(1,3,4,5)P4 (PDB ID: 1FAO), used as reference for comparison with the PH domain of CARMIL. Residues important for ligand binding are shown. (c) Two views of the PH domain of CARMIL, including the N-helix (magenta) and Linker (orange), whichform a structural unit with the PH domain. The top view is the same as for DAPP1 and highlights differences in the canonical lipid-binding pocket. The bottom view shows a highly basic pocket formed at the interface between the PH domain and the N-helix. Residues of both pockets mutated in a are circled. (d) Binding of CARMIL1878 to large unilamellar vesicle (LUVs) with the indicated lipid composition. The fraction of CARMIL1878 (5 mM)

cosedimenting with LUVs is plotted as a function of outer leaet lipid concentration. Error bars represent s.d. from three independent experiments. Solid lines represent the best t of the data. Effective lipid-binding afnities are indicated.

estimated from the scattering intensity at zero angle, I(0), and using glucose isomerase as a standard, was 129 kDa (theoretical mass, 61 kDa), corresponding to a dimer in solution (Table 2). The distance distribution plot generated from the scattering data showed a bimodal distribution, indicative of a dumbbell-shaped molecule (Fig. 3e). A molecular envelope was obtained by averaging 20 ab initio structures calculated with the programme DAMMIF36. As we had established that MBP-CARMIL689878

was dimeric, two-fold symmetry constraints were imposed during the calculations, which substantially improves the quality of the envelope37. Consistent with the distance distribution plot, the envelope displayed two distal lobes (Fig. 3f). These lobes t well

two globular MBP molecules (molecular mass, 40 kDa). The remaining density corresponded to the HD dimer (molecular mass, 42 kDa), which seemed to have a narrow and elongated shape. The MBP molecules were distally disposed in the envelope, suggesting that the HD associates in an antiparallel manner.

To determine the mechanism by which the HD enhances lipid binding by the PH domain, we analysed the structure of CARMIL1878 by SAXS. At high concentrations, CARMIL1878

was prone to aggregation. Therefore, the scattering data was collected at a single concentration, from protein separated by SEC immediately before data collection. The molecular mass of CARMIL1878 estimated from the scattering intensity was 226 kDa,

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[] (deg cm2dmol1)

20 689878

Standard 1878

198 kDa 115 kDa

158 kDa

81 kDa

1.0

0.8

MBP-689878 1668

280(a.u.)

0.6

670 kDa

200 220

[afii9838] (nm)

240

44 kDa

A 0.4

121 kDa

MBP

1.0 0.2

100

MBP-689878

120

Molecular mass (kDa)

0.8

A 280(a.u.)

0.6

0.4

100 80 60 40 20

Volume (ml)

200

43 kDa

0.2

Volume (ml)

12.5

I(0)

200

100

Conc. (mg ml1)

1.0

I(q)

100

1.0

0.8

0.1

P(r)

0.6

0.4

0.2

0.1 0.2 q (1)

50 100 r ()

90 90

Figure 3 | CARMIL dimerization. (a) The circular dichroism spectrum of CARMIL689878 displays minima at 208 and 222 nm, characteristic of all-helical structures. (b) Molecular mass estimates from gel ltration. (c) Measurement of the masses of MBP and MBP-CARMIL689878 by SECMALS. (d) SAXS

intensity plotted versus momentum transfer for different MBP-CARMIL689878 concentrations (1, 2.5, 5, 7.5, 10, 20, 30 and 40 mg ml 1). The I(0) versus concentration plot shown in the inset is linear, demonstrating lack of protein aggregation. (e) Normalized distance distribution function of

MBP-CARMIL689878. (f) Three perpendicular views of the average SAXS envelope of MBP-CARMIL689878 t with two molecules of MBP (PDB ID: 3Q25). The dimeric HD, whose structure is unknown, is schematically represented by two crossing cylinders (purple). (g) Three perpendicular views of the average SAXS envelope of CARMIL1878 t with two copies of the structure of CARMIL1668 and a schematic representation of the HD.

consistent with a dimer, whereas that of CARMIL1668 was

80 kDa, consistent with a monomer (Table 2). The distance distribution plots for these two fragments had the characteristic shape of elongated globular proteins37 (Supplementary Fig. S6a,b). An ab initio SAXS envelope of CARMIL1878 was generated by

imposing two-fold symmetry constraints. The crystal structure of

CARMIL1668 (Fig. 1b) was manually t as a dimer into the envelope, which could only be accomplished in a single orientation (Fig. 3g and Supplementary Movie 1). The remaining unlled density was assigned to the antiparallel HD and had approximately the same shape and size as determined above from the envelope of MBP-CARMIL689878 (Supplementary Fig. S6c).

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In the resulting model of the dimer, the PH domains project out in the same orientation, such that their lipid-binding pockets can simultaneously interact with the PM.

The PH domain mediates leading edge localization in cells. The ensemble of our in vitro results implicated the PH domain of CARMIL in membrane binding. To test whether this was also its role in cells, we expressed CARMILFL-GFP or CARMILDPH-GFP (lacking residues 1157) in B16F1 cells and compared the ability of these two constructs to localize to the leading edge (Fig. 4). The localization of the CARMIL constructs was quantitatively compared with each other and with that of green uorescent protein (GFP), after normalization against red uorescence protein (RFP) uorescence. Quantication of PM localization was carried out using the PM index38, which characterizes the localization along the entire leading edge and avoids errors due to volume effects. Both GFP and RFP displayed diffuse localization (PM index 0).

The PM indexes of CARMILFL-GFP and CARMILDPH-GFP were1.41 and 0.61, respectively (Fig. 4), corresponding to more than a twofold reduction in PM localization due to deletion of the PH domain. This result indicates that in cells the PH domain functions to recruit CARMIL to the leading edge through direct interaction with the membrane.

DiscussionCARMIL is an important factor in normal cell motility and tissue development, as well as aberrant migration of metastatic cancer cells7. However, although signicant effort has been devoted to understanding the interaction of CARMIL with CP and its effect on barbed-end dynamics10,2125, the structural and functional properties of the other domains of this large cytoskeletal effector remained poorly understood. This study advances our knowledge of CARMIL function, including the identication and characterization of two previously unknown domains N- and C-terminal to the LRR domain: a PH domain involved in direct binding to the PM and a HD responsible for antiparallel dimerization and enhancement of CARMILs membrane-binding activity.

The PH domain of CARMIL had remained undetected because of its low sequence identity with canonical PH domains (Supplementary Fig. S3). Structural features also set it apart, including its tight integration with N- and C-terminal structural elements (N-helix and Linker), which are not part of the canonical PH fold, and a lipid-binding pocket with substitutions of most of the conserved residues in canonical PH domains26. At the biochemical level, these differences translate into the rather unusual lipid specicity of this PH domain, adapted for interaction with monophosphorylated lipids, particularly PS (Fig. 2). Given the relative abundance of PS at the PM33, it appears that CARMILs PH domain mediates non-specic binding to the membrane, in contrast to other PH domains that bind polyphosphorylated phosphatidylinositides, which are thought to function as signalling lipids35. Signalling lipids exist transiently in the membrane, are spatially segregated and are present in low amounts39. In contrast, PS is uniformly distributed, such that the observed enrichment of CARMIL at the leading edge (Fig. 4) must be due in part to interactions with other factors. One such factor could be myosin-I, whose SH3 domain binds to the C-terminal PRD of CARMIL1114, and which also contains a PH domain that allows it to bind independently to the PM40. Another binding partner of CARMIL, Trio15,20, also contains two PH

Table 2 | Molecular masses (kDa) estimated by different methods.

Construct Theoretical SEC MALS SAXS Oligomer 1668 74 81 80 Monomer 1878 97 198 226 Dimer MBP-689878 61 115 121 129 Dimer MBP 42 43 Monomer

Abbreviations: MALS, multi-angle light scattering; MBP, maltose-binding protein; SEC, size

exclusion chromatography; SAXS, small-angle X-ray scattering.

RFP

GFP Merge Quantification

Background

Membrane

PH FL GFP

GFP GFP-CARMIL

GFP-CARMIL PH

Cytosol

N=10

PM index

N=14

N=10

Figure 4 | Role of the PH domain in leading edge localization of CARMIL. Wide-eld uorescence microscopy of B16F1 cells co-expressing RFP and either GFP, GFP-CARMILFL or GFP-CARMILDPH (residues 1581,374). Scale bars, 10 mm. Regions highlighted by dashed boxes are enlarged in insets. Solid boxes indicate areas used to calculate the background uorescence. The inset shown on the top right corner illustrates the method used to denethe background, leading edge and cytosol regions for averaging of uorescence intensities. Quantications of the PM index of each construct are given as box-and-whisker plots: box length represents values from low to upper quartile, whiskers encompass 595th percentile, boxes are divided by the median. The number of cells analysed for each construct was: GFP, 10; GFP-CARMILFL, 10; and GFP-CARMILDPH, 14. The statistical signicance of the difference between GFP-CARMILFL and GFP-CARMILDPH, based on the unpaired Students t-test, is indicated by asterisks (**Po0.01).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3523

domains and interacts with GTPases at the membrane through its two guanine-nucleotide exchange factor (GEF) domains19. Because of its large modular structure, other binding partners of CARMIL are likely to emerge, offering alternative mechanisms for leading edge localization and regulation of its activity. The PH domain, in particular, is more than a lipid-binding fold, as it frequently becomes involved in proteinprotein interactions with membrane-bound partners41, enhancing membrane localization by a mechanism known as coincidence detection42.

Using sedimentation equilibrium, it was previously reported that Acanthamoeba castellanii CARMIL existed in monomer dimer equilibrium with an association constant of 1.0 106 M 1,

and it was further suggested that binding to CP promotes CARMIL dimerization to form a 1:2 CP:CARMIL complex43. The mammalian isoform studied here appears to function as a constitutive dimer, independent of interaction with CP, as we did not observe any evidence of dissociation. As the binding site for CP is fully contained within a CARMIL monomer10, our results also predict that the stoichiometry of the CP:CARMIL complex in cell is 2:2 (one CARMIL dimer to a two CP heterodimers). We further determined the molecular mechanism of dimerization, which is mediated by a B200-aa central domain that is mostly helical and associates in an antiparallel manner (Fig. 3). These characteristics and the overall dimensions of the HD, which is relatively narrow in two directions and elongated in the third direction, are shared with two other folds that are abundant among cytoskeletal proteins and consist of antiparallel helical bundles, the spectrin repeat and the BAR domain (Supplementary Fig. S6c). Through dimerization, all the interactions of CARMIL, including with CP, myosin-I and membranes, are duplicated. In the case of membrane binding, dimerization not only works by duplication of the membrane-binding afnity of the PH domain, as frequently observed with membrane-binding modules42, but also by optimally orienting the PH domains such that they can simultaneously bind the membrane (Fig. 5), probably explaining why CARMIL1878 cosedimented B25% more abundantly with membranes than CARMIL1668.

Although the HD is sufcient for dimerization, other parts of the CARMIL molecule may participate in dimerization

interactions. Indeed, CARMIL1668 and CARMIL1878 were

monomeric and dimeric, respectively, whereas the isolated HD (MBP-CARMIL689878) was dimeric. Yet, the SAXS envelope of

CARMIL1878 suggests that the LRR domain may also contribute to the dimerization interface (Fig. 3g). Coincidentally, the descending surface of the LRR displays two distinct areas of high sequence conservation at the ends, coinciding with the LRRLRR contact interface in the dimer (Supplementary Fig. S2a). On the other hand, the ascending surface and N-cap regions of the LRR are more uniformly conserved and fully exposed in the dimer, suggesting that these surfaces may be involved in proteinprotein interactions, which is the general function of LRR proteins29. Large LRR domains typically bind more than one target through different surfaces; CARMILs LRR with 16 repeats is also likely to provide a platform for multiple proteinprotein interactions.

The involvement of CARMILs PH domain in membrane localization was demonstrated here in cells, where deletion of the PH domain results in more than a 50% reduction in leading edge localization (Fig. 4). This value probably underestimates the role of the PH domain in membrane localization, as CARMILDPH could have cross-dimerized with endogenous CARMIL in cells, articially increasing the localization of CARMILDPH at the leading edge. Our results contrast with a previous qualitative analysis, suggesting that a 594-aa N-terminal fragment of CARMIL did not localize to the leading edge18. Possible causes for this discrepancy are the inability of this construct to dimerize, a factor shown here to have a key role in leading edge localization, and potential misfolding due to an incomplete LRR domain lacking the C-terminal cap.

Whether directly through its PH domain or indirectly through partners, the cellular functions of CARMIL, specically CARMIL1, are localized at the leading edge of the cell where it controls cell motility15,18. Interestingly, other CPI-containing proteins are also localized at the membrane, including CD2AP44, Fam21 (ref.45) and CKIP-1 (ref. 46). In particular, CKIP-1 contains a PH domain, which, similar to that of CARMIL, binds preferentially monophosphorylated lipids and PS46. Furthermore, CD2AP, whose leading edge localization depends on interaction with cortactin, was recently shown to recruit CP to the leading edge44. It is therefore emerging that proteins that regulate the activity of CP may also regulate its localization at the interface between lament barbed ends and membranes. The ensemble of our results support a model in which the HD and PH domains work synergistically to enhance CARMILs localization at the leading edge (Fig. 5), where it interacts with partners such as Trio and myosin-I, and exerts its regulatory function on CP and cell motility.

Methods

Proteins. Mouse CARMIL1 (UniProt ID: Q6EDY6) fragments (see Fig. 1a for details) were cloned into plasmids pMAL-c2E (New England Biolabs) or pRSFDuet-1 (EMD Millipore). The specic primers used for each construct are listed in Supplementary Table S1. Proteins were expressed in BL21 (DE3)-RIPL cells (Agilent Technologies). Cells were lysed using a Microuidizer (Micro-uidics). Proteins were puried by afnity chromatography (amylose or Ni-NTA resin), followed by additional purication steps through mono-Q and gel ltration columns. Unless otherwise specied, the afnity purication tags (MBP or hexahistidine) were cleaved using TEV protease.

Lipid-binding assays. Cosedimentation assays with lipids were performed as described47, by mixing proteins at 5 mM with 0.6 mg ml 1 brain lipids (Folch fraction I, Sigma), followed by centrifugation at 150,000 g. The supernatant was removed and the pellet was washed with buffer and resuspended. Supernatant and pellet fractions were analysed by SDSPAGE. The intensities of the bands in the gel were quantied using ImageJ software48.

Lipid overlay assays were performed using PIP strips (Echelon Biosciences). Membranes were incubated with 60 nM proteins. Proteins were detected with 1,000 diluted rabbit anti-CARMIL antibodies (Santa Cruz Biotechnology).

Membrane-bound

partners (for example, myosin-I)

Barbed end

Arp2/3 complex

F-actin

Linker N-cap C-cap CPI-CSI PRD

N-helix

PH 118

LRR

878 1,039 1,374

668

Figure 5 | CARMIL functional model. CP caps the barbed ends of actin laments in dendritic networks nucleated by the Arp2/3 complex, resulting in short, densely branched laments required for cell motility. CARMIL allosterically inhibits the interaction of CP with barbed ends and may also mediate CPs localization to the leading edge. CARMILs own localization to the leading edge likely involves a combination of direct membrane binding by the PH domain, dimerization through the HD and proteinprotein interactions with partners such as myosin-I mediated by its other domains.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3523 ARTICLE

Membranes were then incubated with anti-rabbit antibody-conjugated horseradish peroxidase (GE Healthcare Life Sciences). Bound CARMIL was then detected using an enhanced chemiluminescence kit (GE Healthcare Life Sciences).

Vesicle-binding assays were carried out using large unilamellar vesicles prepared as described49. Chloroform-solvated lipids consisting of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and either 20% DOPS, 5% PtdIns(5)P or 5% brain PtdIns(4,5)P2 (mole fraction) were dried under a nitrogen stream. Note that DOPC and DOPS are commonly used substitutes for naturally occurring PC and PS, which can have varying fatty acid compositions. Lipids were resuspended in 20 mM HEPES pH 7.5 and 176 mM sucrose to a nal concentration of 5 mM. Lipid solutions were then subjected to ten cycles of freezing and thawing, bath sonication for 1 min and extrusion through a 100-nm lter using a Mini-Extruder (Avanti Polar Lipids). Lipids were then dialysed in 20 mM HEPES pH 7.5, 200 mM NaCl and 1 mM dithiothreitol (DTT). Vesicle-binding assays were performed as described previously49. CARMIL1878 at 5 mM was mixed with lipids at various concentrations ranging from 0 to 2 mM and was centrifuged at 150,000 g for 30 min at 20 C. Supernatant and pellet fractions were analysed by SDSPAGE and gel band intensities were quantied using ImageJ. Data were t to a single-site binding equation using Grace software. The apparent afnity constants (Keff) were

expressed in terms of the total outer membrane lipid concentration.

Circular dichroism. The far ultraviolet spectrum of CARMIL689878 was obtained at a protein concentration of 10 mM in 20 mM phosphate buffer pH 7.5, 200 mM NaCl and 1 mM DTT, using an Aviv Model 410 spectrometer. Measurements were taken at 25 C. The nal normalized circular dichroism spectrum was obtained by subtracting the average of ten buffer-only scans from the average of ten protein scans.

SEC and light scattering. Samples (10 mg ml 1) were fractionated by SEC using a HiLoad 26/60 Superdex 200 column and molecular weights were estimated by comparison with a molecular weight standard (Bio-Rad).

For SECMALS, samples (5 mg ml 1) were separated using a TSK-gel Super SW2000 column (Tosoh Bioscience). Light-scattering measurements were performed using a DAWN HELEOS MALS detector and an Optilab Rex refractive index detector, and molecular masses were estimated using Astra software (Wyatt Technology).

Small-angle X-ray scattering. Scattering data collection was carried out at the Cornell High Energy Synchrotron Source (CHESS) beamline F2 at 20 C. CARMIL samples were analysed in 20 mM Tris HCl pH 8.0, 100 mM NaCl, 2 mM DTT. To limit radiation damage, the samples were continuously oscillated inside the cuvette during the 30-s exposures. Six exposures were typically collected from each sample before radiation damage became apparent, as estimated from comparisons of each measurement to the rst. Independent measurements were collected from each sample at different concentrations, checking the linear dependence of the scattering intensity at zero angle as a function of concentration to ensure lack of aggregation. One of the samples, CARMIL1878, was prone to aggregation. This sample was pre-concentrated to 12 mg ml 1 and run through a SD200 gel ltration column, and data were collected immediately after elution of the dimeric peak. Data normalization, solvent subtraction and Guinier analysis were done using the BioXTAS RAW software. Data analysis was carried out using the ATSAS software suite, including the programmes GNOM50, used to calculate the distance distribution function P(r), and DAMMIF36, used in automated bead modelling for shape determination. To generate the models of the dimeric samples MBPCARMIL689878 and CARMIL1878, two-fold symmetry constraints were imposed,

which produces more reliable envelopes by reducing noise37. For each sample, 20 independent shape models calculated with DAMMIF were averaged using DAMAVER51 to produce the nal ab initio envelopes.

Crystallography. Crystals of CARMIL1668 (10 mg ml 1) in 20 mM Tris HCl pH8.0, 100 mM NaCl and 2 mM DTT were obtained at 20 C in hanging drops containing a 1:0.5:0.5 (v/v) mixture of protein solution, well solution (130 mM Li2SO4 and 16% PEG 3350) and Silver Bullets 71 (Hampton Research). Crystals belonging to space group P1 (Table 1) grew to their nal size after 48 h. Crystals of a Se-Met derivative were grown under similar conditions by mixing the protein solution at 30:1 (v/v) ratio with a seeding solution of crushed native crystals in crystallization buffer. For data collection, crystals were ash frozen in liquid nitrogen from a cryosolution consisting of crystallization buffer supplemented with 11% PEG 1,000 and 11% PEG 400.

X-ray data collection was carried out at National Synchrotron Light Source beamline X6A. Data were indexed and scaled using HKL2000 (ref. 52). Experimental phases were determined using the single-wavelength anomalous dispersion method. SnB53 was the only programme capable of nding 56 correct solutions out of the 76 selenium atoms expected for the four molecules in the P1 unit cell. The positions of the selenium atoms were rened with the programme Phenix54. Phenix also detected the four-fold non-crystallographic symmetry operators, found additional sites, removed six wrong sites and automatically built parts of the structure into the fourfold averaged electron density map. Additional model building was carried out manually, using the programme Coot55. The statistics of the nal Phenix-rened model are given in Table 1.

Expression and visualization of CARMIL constructs in cells. B16F1 mouse melanoma cells were cultured as described56. Cells were cotransfected with RFP and either GFP, GFP-CARMILFL or GFP-CARMILDPH using Lipofectamine LTX and Plus reagents (Invitrogen). Light microscopy was performed using a Nikon

Eclipse TE2000U Inverted Microscope equipped with a Planapo 100 1.3

numerical aperture objective and a Cascade 512B CCD camera (Photometrics) driven by the MetaMorph imaging software (Molecular Devices). The average uorescence intensity of a 0.46-mm wide line drawn along the entire leading edge was compared with the intensity in the cytosol, dened as a 3-mm band separated by 3 mm from the leading edge (as depicted in Fig. 4a). Membrane rufes were excluded from this analysis. Intensities were measured using ImageJ48. The background uorescence intensity, measured from a rectangle outside the cell, was subtracted from the average intensities of the leading edge and the cytosol. The expression of RFP was used to control for volume uctuation in the cell. The PM index, a measure of the membrane-bound fraction of each construct, was calculated as described38, using the equation: PM index ((GFPm/GFPc)/(RFPm/RFPc)) 1,

where GFPm, GFPc, RFPm and RFPc are the average uorescence intensities of GFP and RFP at the leading edge or in the cytosol after subtraction of the background intensity. The statistical signicance of the measurements was determined using the Students t-test, with a two-tailed non-paired comparison.

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Acknowledgements

This work was supported by the National Institute of Health (NIH) grants R01 MH087950 and R01 GM073791 to R.D. T.S. and E.M.O. were supported by NIH grants GM070898 and GM057247, respectively. E.A.F. was supported by NIH/NRSA fellowship GM090551. X-ray data collection at beamline X6A of the National Synchrotron Light Source was supported by NIH grant GM-0080 and DOE contract DE-AC02 98CH10886. SAXS data collection at CHESS beamline F2 was supported by NSF grant DMR-0936384 and NIH grant GM103485. We thank Vivian Stojanoff, Jean Jakoncic and Edwin Lazo for help with synchrotron data collection. We thank Richard Gillilanat MacCHESS for help with SAXS data collection. We thank Thomas Terwilliger(Los Alamos National Laboratory) for helpful discussions during determination ofthe structure.

Author contributions

A.Z. expressed proteins, carried out lipid-binding experiments, crystallization, structure determination, SAXS, biochemical experiments and participated in cellular studies. C.Y. and T.S. performed cellular studies. E.A.F. and E.M.O. participated in lipid-binding experiments. R.D. solved the crystal structure, supervised the research and wrote the manuscript together with A.Z.

Additional information

Accession codes: The atomic coordinates and structure factors for the structure of CARMIL1668 have been deposited in the Protein Data Bank with accession code 4K17.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: Zwolak, A. et al. CARMIL leading edge localization depends on a non-canonical PH domain and dimerization. Nat. Commun. 4:2523doi: 10.1038/ncomms3523 (2013).

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Abstract

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CARMIL is an approximately 1,370-amino-acid cytoskeletal scaffold that has crucial roles in cell motility and tissue development through interactions with cytoskeletal effectors and regulation of capping protein at the leading edge. However, the mechanism of CARMIL leading edge localization is unknown. Here we show that CARMIL interacts directly with the plasma membrane through its amino-terminal region. The crystal structure of CARMIL_1-668 reveals that this region harbours a non-canonical pleckstrin homology (PH) domain connected to a 16-leucine-rich repeat domain. Lipid binding is mediated by the PH domain, but is further enhanced by a central helical domain. Small-angle X-ray scattering reveals that the helical domain mediates antiparallel dimerization, properly positioning the PH domains for simultaneous membrane interaction. In cells, deletion of the PH domain impairs leading edge localization. The results support a direct membrane-binding mechanism for CARMIL localization at the leading edge, where it regulates cytoskeletal effectors and motility.

Details

Title

CARMIL leading edge localization depends on a non-canonical PH domain and dimerization

Author

Zwolak, Adam; Yang, Changsong; Feeser, Elizabeth A; Michael Ostap, E; Svitkina, Tatyana; Dominguez, Roberto

Pages

2523

Publication year

2013

Publication date

Sep 2013

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms3523

ProQuest document ID

1437156474

CARMIL leading edge localization depends on a non-canonical PH domain and dimerization

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