Molecular Phylogenetics and Evolution 80 (2014) 193–204

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Tracing the evolution of FERM domain of Kindlins Raja Hashim Ali a,b,1, Ammad Aslam Khan c,d,⇑,1 a

KTH Royal Institute of Technology, School of Computer Science and Communication, Department of Computational Biology, 100 44 Stockholm, Sweden Science for Life Laboratories, Karolinska Institute Science Park, 171 65 Solna, Sweden c Quaid-i-Azam University, Department of Animal Sciences, Islamabad, Pakistan d Karolinska Institutet, Center for Biosciences, Department of Biosciences and Nutrition, Novum, 141 83 Huddinge, Sweden b

a r t i c l e

i n f o

Article history: Received 31 May 2014 Revised 31 July 2014 Accepted 8 August 2014 Available online 20 August 2014 Keywords: FERM domain Kindlins Protein domain evolution Evolutionary trace analysis Focal adhesions PH domain

a b s t r a c t Kindlin proteins represent a novel family of evolutionarily conserved FERM domain containing proteins (FDCPs) and are members of B4.1 superfamily. Kindlins consist of three conserved protein homologs in vertebrates: Kindlin-1, Kindlin-2 and Kindlin-3. All three homologs are associated with focal adhesions and are involved in Integrin activation. FERM domain of each Kindlin is bipartite and plays a key role in Integrin activation. A single ancestral Kindlin protein can be traced back to earliest metazoans, e.g., to Parazoa. This protein underwent multiple rounds of duplication in vertebrates, leading to the present Kindlin family. In this study, we trace phylogenetic and evolutionary history of Kindlin FERM domain with respect to FERM domain of other FDCPs. We show that FERM domain in Kindlin homologs is conserved among Kindlins but amount of conservation is less in comparison with FERM domain of other members in B4.1 superfamily. Furthermore, insertion of Pleckstrin Homology like domain in Kindlin FERM domain has important evolutionary and functional consequences. Important residues in Kindlins are traced and ranked according to their evolutionary significance. The structural and functional significance of high ranked residues is highlighted and validated by their known involvement in Kindlin associated diseases. In light of these findings, we hypothesize that FERM domain originated from a proto-Talin protein in unicellular or proto-multicellular organism and advent of multi-cellularity was accompanied by burst of FDCPs, which supported multi-cellularity functions required for complex organisms. This study helps in developing a better understanding of evolutionary history of FERM domain of FDCPs and the role of FERM domain in metazoan evolution. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Kindlins are functionally important members of FERM domain containing protein (FDCP) superfamily. Three Kindlin paralogs exist in vertebrates namely Kindlin-1, Kindlin-2 and Kindlin-3 (Siegel et al., 2003). All three Kindlin paralogs exhibit differential gene expression in Homo sapiens despite sharing a strong structural and sequence similarity, i.e., Kindlin-2 is expressed in multiple tissues, Kindlin-1 is expressed mainly in epithelial tissues and Kindlin-3 exhibits exclusive hematopoietic system specific expression (Ussar et al., 2006). Kindlins are found in all metazoan species indicating their importance in metazoans (Khan et al., 2011). The ability to activate Integrin is one of the most important and well-studied feature of Kindlins, which plays a significant role in Integrins inside out signaling. Integrin activation is known to be ⇑ Corresponding author at: Quaid-i-Azam University, Department of Animal Sciences, Islamabad, Pakistan. E-mail address: [email protected] (A.A. Khan). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.ympev.2014.08.008 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

central in many important functions, e.g., cell-to-cell contact, cell spreading, cell migration and embryonic development (Rogalski et al., 2000; Shi et al., 2007; Ye and Petrich, 2011). Kindler syndrome is the first disease associated with Kindlins, which is a skin disease caused by mutation in Kindlin-1 coding region (Kindler, 1954; Has et al., 2011). Kindlin-3 is involved in leukocyte adhesion deficiency III (LAD-III), a rare disease of hematopoietic system, which features combined dysfunction of b1, b2 and b3 Integrin families on platelets and leukocytes, and is characterized by excessive bleeding and abnormal immune response (Svensson et al., 2009; Wang et al., 2010). Kindlin-2 has not been associated with any specific pathological phenotype but the indispensability of this gene for development of organism is indicated by embryonic knockout of Kindlin-2, which proves lethal for embryo (Montanez et al., 2008). Different types of cancers abnormally express all three Kindlins although no rigorous mechanistic studies have been done to see the role of Kindlins in cancer progression (Lai-Cheong et al., 2010; Zhan et al., 2012; Gao et al., 2013). Presence of FERM domain is the structural and functional hallmark of Kindlins. The name FERM implies presence of this domain

194

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

in Band4.1 protein and its three homologs Erzin, Radixin and Merlin (ERM) (Chishti et al., 1998). FERM domain is composed of three subdomains namely F1, F2 and F3. These subdomains are arranged in a cloverleaf shape (Han et al., 2000). FERM domain of Kindlins is significantly different in structure from FERM domain of other FDCPs because F2 (also known as FERM-M domain) in Kindlins is divided in two portions due to insertion of Pleckstrin Homology (PH) domain (Liu et al., 2012) unlike the single F2 subdomain observed in other FDCPs. FDCPs are characteristically known as linker molecules between plasma membrane and cytoskeletal structures. In addition to Kindlin and ERM proteins, Talin, Myosin, Krev interaction trapped (KRIT), Focal adhesion kinase (FAK), Janus kinase (JAK) and Guanine nucleotide exchange factors (GEFs) are important FDCPs. They have been found in diverse group of organisms, both in vertebrates and invertebrates, belonging to a whole range of metazoans. FDCPs are mainly involved in protein–protein and protein–lipid interactions through their FERM domains. These interactions regulate their subcellular localizations, activity and their recruitment into macromolecular complexes. The important binding molecules of FERM domain are Integrin, Actin, IP3, PIP2 and PIP3 (Hamada et al., 2000; Dunty et al., 2004; Guy Tanentzapf, 2006). In a previous study, we showed that Kindlin-2 is the most conserved protein among Kindlin homologs and all three homologs evolved under purifying selection (Khan et al., 2011). However, evolutionary history of Kindlin FERM domain and its relationship with FERM domains of other FDCPs was not analyzed. Also, effect of insertion of PH domain in Kindlin FERM domain had not been discussed in comparison with FERM domain of other FDCPs from an evolutionary point of view. Furthermore, a detailed phylogenetic analysis had not been performed for FERM domains constituting FERM domains from important FDCPs. In this study, we elucidate evolutionary history of FERM domains of Kindlins with respect to other FDCPs and perform a detailed phylogenetic analysis for this purpose. We show that insertion of PH domain in Kindlins has important evolutionary and functional implications. We also show important class specific and FDCPs specific residues by employing Evolutionary Trace Analysis (ETA). We determine similarity between Kindlin FERM domain and FERM domains of other FDCPs. In our knowledge, this is the first comprehensive attempt on exploring the evolutionary history of FERM domains of Kindlin homologs in particular and FDCPs in general and the first to present a detailed phylogenetic analysis of FERM domain tracing back to earliest metazoans.

2. Material and methods 2.1. FDCP identification and FERM sequence extraction Important FDCP families in Eukaryotes were shortlisted from literature and 14 proteins were identified in Homo sapiens representing each FDCP family. The representative protein for each FDCP family was selected on basis of more conservation among its paralogs, e.g., Kindlin-2 from Kindlin gene family, Myosin-VII from Myosin gene family and Talin-1 from Talin gene family. Important species representing important events and splits in Unikonta species tree were identified, e.g., from Unikonta (represented by Dictyostelium discoideum) to metazoa (represented by Amphimedon Queenslandica), etc. In this way, we identified 15 species important for performing phylogenetic analysis ranging from amoebazoa to primates. Then Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) search was carried out using Homo sapiens representative FDCP as seed sequence to get orthologous sequence of this protein in each species from NCBI (protein

IDs in Supplementary Material). Proteins with partial sequences were rejected. In total, 185 FDCP sequences from 15 different species were used for making a comprehensive phylogeny of FERM domains. FERM domain sequence for each FDCP was extracted using Batch search tool available on NCBI Conserved Domain Database (CDD) (Marchler-Bauer and Bryant, 2004) and Superfamily web tool available on HMM Library and Genome Assignment Server (Gough et al., 2001). Myosin has two FERM domains, one in middle of protein sequence and the other towards the C-terminal. Therefore, each FERM domain in Myosin was named Myosin and ‘‘Myosin2’’ to differentiate between them. As convention, each FERM domain used in this work is represented by protein name (from which it is extracted) and initials of the species (to which the protein belongs) separated by underscore, e.g., Myosin2_D-d represents second FERM domain in Myosin for Dictyostelium discoideum and Kindlin2_H-s represents FERM domain in Kindlin2 for Homo sapiens.

2.2. Phylogenetic analysis Phylogenetic analyses were performed using a pipeline consisting of Mega5 (Tamura et al., 2011), TimeTree (Hedges et al., 2006), DLRS in JPrIMe (Akerborg et al., 2009; Sjostrand et al., 2012) and VMCMC (not published yet). Briefly, FERM domain sequences were aligned by using ClustalW tool employed in MEGA software with affine gap penalties of 5 for gap opening and 1 for gap extension (Chenna et al., 2003). Species tree (Supplementary Material), required for Markov Chain Monte Carlo (MCMC) analysis in JPrIMe, was retrieved from TimeTree using the expert suggestions for probable divergence times between species. 1,000,000 MCMC iterations were run on JPrIME-DLRS with default parameter settings and Maximum A Posteriori (MAP) tree was determined by analyzing output tree distribution using Visual Markov Chain Monte Carlo (VMCMC) tool.

2.3. Evolutionary Trace Analysis (ETA) Evolutionary Trace Analysis (ETA) (http://www.mammoth.bcm.tmc.edu/) was employed to identify evolutionarily conserved residues (called traces) in FDCPs. ETA defines two classes of sequences; superclass consisting of all sequences and subclass consisting of a selected sub-clade of input sequences. Conserved residues in superclass determine globally important residues and ones conserved in subclass but not in superclass define the class specific residues (Lichtarge et al., 1996). In this study, ETA was used to determine class-specific and superfamily-specific residues in Kindlin FERM domains. On the basis of conservation profile of FERM domains, FERM domain superfamily was divided into four groups (Supplementary Data). In this approach, Kindlin was used as subclass while for super-class, the protein closest to Kindlin FERM domain from each of the four groups of FDCPs was selected. In super-class analysis, completely conserved residues (columns) along the tree were given the maximum rank, 1. Same rank was given to residues in subclass, if they were conserved in all members of subclass. Class-specific residues were obtained by subtracting the highest ranked residues in subclass from those in superclass. For structural analysis, pyetv tool was used, which is a modified version of pymol employed in ETA webserver (Lua and Lichtarge, 2010). pyetv clusters superclass or subclass specific residues on experimentally resolved structures and uses a template protein structure. Therefore, our query sequence for ETA was always sequence of the protein whose structure was used as template for pyetv.

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

3. Results 3.1. FERM domain containing proteins originated in Unikonta We traced FERM domain in tree of life through NCBI BLAST and PSI-BLAST. Some interesting results were obtained regarding origins of FERM domain in proteins. FDCPs originated in Unikonta, a subgroup of Eukarya and FERM domain is not found in archea or bacteria (Fig. 2). None of the FDCPs was found in plants, the other major group of Eukarya. In Unikonta, FERM domain is first observed in Dictyostelium discoideum, an amoebazoa and protozoa, in Myosin and Talin proteins. A large group of FDCPs is observed in metazoa in particular parazoa like Amphimedon Queenslandica (sponges) that suggests functional importance of FERM domain in events leading to multicellular organisms from unicellular organisms (protozoa). Kindlin, Radixin, Band4.1, FAK1 and E3Mylip appear at this stage. The remaining FDCPs originate in eumetazoa, which is divided into two groups: proteins originating in cnideria, i.e., FERM–RhoGEF in Hydra magnipapillata and remaining 6 FDCPs in bilateria, e.g., Merlin and KRIT in Drosophila melanogaster.

3.2. Phylogenetic analysis reveals four major groupings in FDCPs An important step in predicting the structure and function of proteins is to explore the evolutionary history of domains contained in the proteins and to determine the underlying clades of

195

homologs through phylogenetic analysis. Therefore, the evolutionary history of FERM domain in FDCPs was elucidated using a pipeline for phylogenetic analysis. Using one gene representative for each FDCP family and for each species, a comprehensive Maximum A Posteriori (MAP) tree for 185 protein sequences representing 200 FERM domains in total was constructed (Fig. 1). The MAP tree obtained here can be divided into four major groups implying presence of four groups in FERM domain of FDCPs based on overall similarity profile of FDCPs. Kindlin, Talin, Myosin, Myosin2 and Krit form one group. FERM–PDZ, JAK and FAK constitute second group. Third group consists of Band4.1, FERM–RhoGEF, FDCP, tpp, Merlin and Radixin while E3-Mylip is the only member in fourth group. It is interesting to note that Kindlin FERM domain is in the same clade with Talin FERM domain and Kinases like FERM–PDZ, JAK and FAK are also observed in same group implying a common origin of their FERM domains. 3.3. Kindlin FERM domain shows closest homology with Talin and farthest with FAK1 Kindlins are the most recently discovered members of FDCPs. Not much is known about evolution of FERM domain and the relationship between FERM domains of different proteins. In our previous work, a brief overview of evolution of Kindlin family of proteins was presented (Khan et al., 2011). In this work, the evolution of FERM domain of Kindlins with respect to other FDCPs is explored. A thorough analysis of FERM domain sequences of FDCPs reflected some interesting insights in the evolution of these

Fig. 1. Maximum A Posteriori (MAP) gene tree of FERM domains. MAP gene tree of FERM domains of 185 FDCPs from 15 species generated by using Markov Chain Monte Carlo (MCMC) method available from JPrIME and extracted by using VMCMC. Color of the branches represents the classification of FDCPs on the basis of their origin, as shown in Fig. 2.

196

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

proteins. First, comparison of Kindlin protein sequences reconfirmed our previous finding that Kindlin-2 is the most conserved protein in the Kindlin family while Kindlin-1 and Kindlin-3 are relatively more diverged (data not shown here). Kindlin FERM domain shows closest homology with the domain of another important Integrin interacting protein, Talin-1 (17%) followed by the second FERM domain of Myosin (14%) (Table 1). Kindlin FERM domain shows least homology with FAK1 (8%) (Fig. 3A). In terms of overall percentage similarity with other members of the FERM domain superfamily, Radixin and Merlin showed the maximum conservation (28%). Kindlin shows considerably less conservation (17%) while both the kinases in the group i.e. JAK2 and FAK1 showed the least overall conservation (16%) (Fig. 3B). Not much variation was seen in the orthologous sequences for each protein, where all the proteins showed more than 85% conservation within their respective orthologous sequences. The exceptions were JAK2 and TPP, which showed 82% and 78% conservation respectively, slightly less than the other FERM domain superfamily members (Fig. 3C).

3.4. Insertion of PH domain changes evolutionary rate of Kindlins FERM-C Unlike its closest homolog Talin and other FDCPs, Kindlins FERM domain have a unique disruption because of insertion of a PH domain. Thus, Kindlins have two PH-like domain modules; one because of insertion and other already present in third subdomain (also known as FERM-C or F3 subdomain) (Fig. 4). This suggests that presence of two PH-like folds relaxes functional constraints on one PH containing regions in Kindlin protein. Calculation of substitution based distances in FERM-M and FERM-C

domains in orthologous Kindlins in Kindlin-1 and Kindlin-2 showed that FERM-C subdomain has higher substitution rate as compared to FERM-M. Same analysis was performed on Talin where FERM-C in Talin is more conserved than FERM-M subdomain. Thus insertion of PH domain in Kindlins leads to an important evolutionary event, which had its structural as well as functional implications (Fig. 5).

3.5. Evolutionary trace analysis of Kindlins with respect to other FDCPs Evolutionary Trace Analysis (ETA) is a phylogenomic method to identify evolutionarily conserved residues in protein sequences. This is a theoretical validation of experimental mutations. The basic crux of ETA is that mutations occurring in distant branches are more important than those occurring at closely related branches. ETA determines globally conserved (superclass-specific) residues of proteins and also pinpoints class-specific residues important in defining unique properties of our protein of interest. In this direction, trace analysis between Kindlins themselves was conducted to know which residues in Kindlins are important for Kindlin class as a whole and which ones define each of the Kindlin paralog individually. At superclass level, 68 highly ranked residues (Table 2) were found. Of these, 46 (68%) residues belonging to FERM-M subdomain (F1 + F2) while remaining 32% belonged to FERM-C subdomain (F3). This showed relative degeneration of FERM-C domain of Kindlins as compared to FERM-C of Talin, as discussed in previous section. Certain residues are conserved at superclass level and are attributed to phenotypes or functions. For instance Gln615, Try612 and Try616 have been attributed to bind Kindlin-2 FERM domain with cytoplasmic tail of Integrin, which

Fig. 2. Origin of FDCPs in metazoa. A schematic dendrogram showing the origin of different FDCPs. The dendrogram above the species images represents the species tree showing major taxonomic groups while the one below the images represents schematic gene tree showing the organisms in which FDCPs have had their origin.

197

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204 Table 1 Percentage similarity of Kindlin FERM domain with respect to other FDCPs.

Kindlin Radixin Merlin Band-4.1 FERM–RhoGEF E3-MYLIP Tpp FDCP Myosin2 Talin Myosin Krit FERM–PDZ FAK JAK

Kindlin

Radixin

Merlin

Band-4.1

FERM–RhoGEF

E3-MYLIP

tpp

FDCP

Myosin2

Talin

Myosin

Krit1

FERM–PDZ

FAK

– 12 11 12 10 13 13 10 14 17 10 10 11 8 9

– – 64 34 30 29 24 23 15 19 17 16 14 12 10

– – – 34 29 27 26 23 18 19 14 17 14 11 12

– – – – 43 26 28 19 16 18 14 13 13 10 10

– – – – – 28 28 18 17 19 13 12 13 10 9

– – – – – – 24 18 16 16 14 14 14 10 10

– – – – – – – 21 16 18 15 14 14 10 9

– – – – – – – – 14 15 13 15 15 7 10

– – – – – – – – – 18 20 15 17 11 11

– – – – – – – – – – 18 16 23 13 12

– – – – – – – – – – – 14 15 11 11

– – – – – – – – – – – – 10 9 11

– – – – – – – – – – – – – 16 11

– – – – – – – – – – – – – – 9

leads to Integrin activation (Ma et al., 2008). It is interesting to note that as these residues are conserved all along superclass, it can be claimed that they also participate in Kindlin-1 and Kindlin-3 based activation of Integrin making the PTB binding domain globally important in Kindlins for Integrin activation. Similarly, mutations in Lys610 and Ile651 in Kindlin-1 reduce recruitment of Kindlin1 in focal adhesions (Harburger et al., 2009). Again, it is predicted that both these residues should also be important for Kindlin-2 and Kindlin-3. An interesting mutation of Glu304 in Kindlin-1 leads to Kindler syndrome (Ashton et al., 2004). ETA analysis showed that Glu304 is Kindlin-1 specific and therefore it is specifically associated with Kindler syndrome. However, Leu302 and Try616, both of which are conserved in Kindlin superclass, are known as a

variant mutation in Kindler syndrome patients. This shows that some highly ranked Kindler syndrome causing variations can be associated with same sort of phenotypes by experimental mutation for other two Kindlin paralogs.

3.6. Structural analysis of FERM domain of Kindlins and other FDCPs ETA was employed to determine evolutionarily important traces and map them on a protein structure to identify important structural properties of these conserved residues. As Kindlin FERM domain structure is not experimentally resolved yet, four proteins with known FERM domain structure were selected for structural

Fig. 3. Sequence analysis of FDCPs. (A) Percentage similarity of different human FDCPs with Kindlins showing Talin FERM domain is the closest and FAK1 FERM domain is the farthest to that of Kindlin’s FERM domain. (B) Histogram showing the ranking of different FDCPs in terms of percentage over-all similarity of their respective FERM domain. The histogram shows that Radixin and Merlin have the FERM domain, which is the common most in FDCPs. (C) Ranking of FDCPS on the basis of percentage conservation with their orthologs showing that FERM domain of Radixin is the most conserved in all FDCPs while that of tpp is the least.

198

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

Fig. 4. Domain architecture of FDCPs. A schematic representation of architecture of FERM domain and the presence of other domain super families in FDCPs.

analysis based upon their similarity with Kindlins (see material method for detail). Talin FERM domain, most similar to Kindlins FERM domain, is the first in our analysis. FERM domain sequence of Gallus gallus Talin was used as query sequence and its crystallographic structure (PDB ID 1MIX) was used for structural mapping. 48 superclassspecific residues (residues completely conserved in FERM domain of both Talin and Kindlin) and 134 Kindlin specific residues (residues conserved only in Kindlins but not in Talin) were found (Supplementary Material). 42% residues were present around F3 subdomain filled with Beta sheets while 58% residues were present around F2 subdomain filled with alpha helices. Surface view for

this imposed structure showed that majority of these highly conserved residues are present in the interior of FERM domain and are not visible on surface, indicating hydrophobic nature of these residues (Fig. 6). A literature review of mutation database on Talin FERM domain gives interesting insights. Garcia-Alvarez et al. mutated four amino acids (K357A, R358A, W359A and I396) to show their importance to facilitate binding between PTB domain of Talin and cytoplasmic domain of Integrin b3 (Garcia-Alvarez et al., 2003). The mutants for these amino acids showed considerable reduction of Integrin–Talin interaction, proving importance of these amino acids. Three out of these four amino acids (K357, W359 and I396) also show maximum rank in our ETA analysis.

Fig. 5. Effect of insertion of a PH domain on FERM domain evolution. (A) Rate of substitution of amino acids in FERM-M and FERM-C domains of Kindlin-1 and Kindlin-2 FERM domain shows that FERMC in Kindlins is undergoing more rapid rate of substitutions. (B) Rate of substitutions in Talin FERM-M and FERM-C subdomain showing much more conserved nature of FERM-C subdomain.

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204 Table 2 Top 20 traces for Kindlin subclass and superclass analysis. The numeric number means position of amino acid in the Kindlin FERM domains. Kindlin-1defining

Kindlin-2defining

Kindlin-3defining

Kindlinsuperclass

N-3 S-5 R-6 S-7 M-9 E-10 E-15 R-22 K-24 Y-25 Y-26 F-29 L-31 N-32 Y-35 A-37 I-40 Q-42 A-47 A-50 L-53

D-3 R-6 S-7 M-9 E-10 D-12 V-13 K-14 E-15 N-16 L-20 R-22 K-24 Y-25 S-27 F-29 L-31 N-32 P-33 Y-35 A-37

S-5 P-33 M-63 I-116 R-122 E-130 I-134 A-135 R-141 G-147 K-151 N-157 M-158 I-167 C-184 Q-46 W-49 L-52 E-54 C-58 T-59

W-1 L-2 S-4 L-8 Q-11 L-19 L-21 F-23 F-28 D-30 K-34 D-36 R-39 L-43 E-45

Among other highly ranked traces, K295 is involved in Talin–Actin binding, K364 in inter-domain interactions (Banno et al., 2012), F370 in Integrin activation (de Pereda et al., 2005), D372 in Talin localization in cells (Anthis et al., 2009), I356 in Integrin activation (Barsukov et al., 2003) and G394 in auto-inhibition of Talin Goksoy et al., 2008 (Fig. 7). Merlin (Neurofibromatosis-2) was second protein considered for ETA with Kindlins and Homo sapiens Merlin structure (PDB ID

199

3U8Z) (Yogesha et al., 2011) was used for structural mapping. ETA analysis for Kindlin–Merlin gave 35 superclass-specific residues and 190 Kindlin2-specific residues which define Kindlin-2 FERM domain relative to FERM domain of B4.1 member proteins (Supplementary Material). Two differences can be observed between superimpositions of Kindlin–Talin and Kindlin–Merlin. First, both F2 and F3 subdomains share almost equal number of highly ranked residues in ETA of Kindlin–Merlin while F3 subdomain showed more conservation than F2 subdomain in ETA of Kindlin–Talin. Second, surface view shows that majority of superclass-specific residues are in protein interior for Kindlin–Talin but are exposed for Kindlin–Merlin to surrounding cytosolic environment due to cavities on surface of FERM domain (Fig. 8). A literature review of mutation in FERM domain of Merlin showed many traces traced by ETA. For instance, Ile301 and Gly302 are involved in inter-domain interaction (Yogesha et al., 2011), K69 in protein trafficking (Bensenor et al., 2010), L275 in subdomain confirmation Shimizu et al. (2002), Phe96 and Glu129 for tumor formation MacCollin et al. (1994), Glu136 and ARG187 in subdomain confirmation Kang et al. (2002), Ile188 in inter-domain interaction and Asn263 in hydrophobic interactions Yogesha et al. (2011) (Fig. 9). Based upon the synergy between published data and ETA analysis, it can be anticipated that aforementioned sites in Kindlins might also be of same importance and can serve as candidate sites for mutational experiments. Similarly, other unpublished traces can act as candidates for these experiments for both Merlin and Kindlin. FAK1 is a focal adhesion molecule like Kindlin and Talin but is farthest in sequence similarity to Kindlins. This dissimilarity is observed at structural level when Kindlin and FAK1 superclass ranks are imposed on Homo sapiens FAK1 FERM domain structure (PDB ID 2AL6). Moreover, this divergence is also observed at functional level. For instance FAK1 is a kinase, which acts exclusively as

Fig. 6. ETA structural analysis of Kindlin/Talin comparison. (A) A cartoon view of the structure of FERM domain of Talin clustered with highly ranked Kindlin/Talin superclass traces. (B) Surface view of the same clearly showing the traces of Kindlins clustering in the interior of FERM domain manifesting the structural nature of these residues. Yellow colored part is the FERM-C domain while greenish spheres represent important traces/residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

200

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

Fig. 7. HMM logos for ETA ranks of Kindlin–Talin. HMM logos for superclass ranks for Kindlin–Talin comparison whose mutations have also been published (A) W359, K364, F370 and D372 (B) K295 and (C) I396. Other highly ranked traces can also been seen along with these published mutations which we predict can play important role and can be a good candidate for future mutation analysis.

a signaling molecule while Kindlin is mainly a structural protein Wehrle-Haller and Imhof (2003) (Supplementary Material and Fig. 10). We conclude that the FERM domain of FAK1 has a higher divergence rate than the FERM domain of other FDCPs.

4. Discussion Kindlin proteins are members of FDCP family, whose major portion of sequence is composed of FERM domain. FERM domain has an important role in cell adhesion and mutations in FERM domain sequence of several proteins are the cause of several diseases in humans. Therefore, it is important and interesting to infer the origin and evolution of FERM domains. For this purpose, important FDCPs were identified from literature and protein sequences for FDCPs in a wide range of species were collected. FDCPs were not found in archea or bacteria. FDCPs are first observed in Dictyostelium discoideum, an amoebazoa and it contains Talin and Myosin proteins. Therefore, the FERM domain of Talin and those of Myosin represent most ancient FERM domains originating in

the last common ancestor (LCA) of Opthisokonta (metazoans) and amoebazoa. Dictyostelium is a transitory group between unicellular and multicellular organisms. FERM domain acts as a linker module between cytoskeleton and plasma membrane providing the mechanical platform for cell to cell contact, cell adhesion and cell migration, which are the characteristic features required for multi-cellularity. Therefore, origin of FERM domains in amoebazoa represents an important event in unicellular to multicellular transition. The simplest organism containing Kindlin is Amphimedon Queenslandica, a sponge, belonging to parazoa. A large number of FDCPs originate in parazoa indicating a vital role of FERM domain in transition from simpler unicellular (non metazoan) to multicellular (metazoan) organization. Interestingly, three proteins related to focal adhesions, i.e., Kindlin, Radixin and FAK1 are first observed in Amphimedon. Focal adhesions are molecular complexes, which play pivotal role in cell-to-cell contact and cell migration WehrleHaller and Imhof (2003), Lock et al. (2008) and Wehrle-Haller (2012). Moreover, Integrin, the major and most well characterized component of focal adhesion complexes Huttenlocher and Horwitz

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

201

Fig. 8. ETA structural analysis of Kindlin/Merlin comparison. (A) A cartoon view of the structure of FERM domain of Merlin (3U8Z) clustered with highly ranked Kindlin/ Merlin superclass traces. (B) Surface view of the same clearly showing cavities in the FERM domain structure of Merlin, making Kindlin traces exposed to external environment.

(2011), also originated at around the same time as Talin (another vital focal adhesion protein). Many other FDCPs that aid and facilitate complexity requirements of higher organisms, are first observed with the transition to metazoans. A second burst of FDCPs is observed with the emergence of bilateral symmetry in metazoans. The bilateral symmetry is accompanied by more complex organism structure due to diverse cellular signaling, higher order nervous system and diverse ways of cellular and organismal locomotion. Thus the emergence of a large number of FDCPs, with more evolved and modified FERM domains, in these organisms indicates importance of FERM domain to fulfill the requirements of complex organization. Phylogeny of FERM domains of all representative members of FDCPs was explored. The resultant Maximum A Posteriori (MAP) tree indicates that the FERM domains of FDCPs can be divided into four major clades. Kindlin and Talin are placed in the same clade validating the homology between Kindlin FERM domain and Talin FERM domain. It also supports the hypothesis that Kindlin evolved from a proto-Talin protein that diverged from it in simplest metazoan organization. Also the presence of FERM domains of Kinases in the same clade indicates a common source of FERM domain for Kinases. Moreover, the clustering together of both Myosin domains indicates a probable duplication of FERM domains in the ancestral Myosin. However, more rigorous phylogenetic analysis, e.g., by reconciling MAP tree with species tree or by tagging all duplication and speciation events in MAP tree, is required to ascertain these deductions. Insertion of a PH domain in the FERM domain of Kindlins is an exclusive feature of Kindlins. A simple sequence based analysis showed that the insertion of a PH domain leads to rapid evolution of Kindlin FERM-C subdomain as FERM-C subdomain has a PH-like fold in it. The presence of two PH-like folds in Kindlins relaxes the functional constraint in one of them. The prominent implication of

this interesting evolutionary event is that Talin and Kindlin differ in terms of their binding affinities for two of the most important membrane phospholipids i.e. Phosphatidylinositol (4,5)-bisphosphate (PIP2) and Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) where Kindlin-2 shows a greater affinity to bind with PIP3 and Talin with PIP2 Liu et al. (2011). This difference can be attributed to the degeneration of PIP2 binding site on corresponding portion of Kindlin FERM domain due to the change in selection pressure imposed by insertion of PH domain. This, in turn, might be the reason of binding of Talin and Kindlin on different locations of Integrin cytoplasmic tail. However, more rigorous analysis and experimental data is needed to prove this hypothesis. Evolutionary trace analysis of Kindlin FERM domains within themselves and with respect to other FDCPs singled out those residues, which were important for individual Kindlins and for superclass. Within Kindlins, Kindlin-2 had most class-specific residues. This is in accordance with our previous finding, which showed that Kindlin-2 is the most conserved protein in Kindlin protein family (Khan et al., 2011). In our previous work, it was speculated that ubiquitous expression of Kindin-2 might be major cause of extra functional constraints on Kindlin-2 but Kindlin-1 and Kindlin-3, being tissue specific in terms of their expression, should evolve faster than Kindlin-2. This becomes prominent in case of Kindlin-3, where only 14 class specific residues are seen, as the expression of Kindlin-3 is exclusively restricted in hematopoietic system. Many class-specific and Kindlin-specific residues are cited in literature as important residues, whose mutations results in either loss of protein function or in disease. This enhances validity of ETA to predict the important components of a protein, which can be used for future mutation analysis. For instance, many highly ranked superclass specific residues were found that have not been mutated yet and results of this study provide them as candidates for future mutation analysis.

202

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

Fig. 9. HMM logos for ETA ranks. HMM logos for superclass ranks for Kindlin–Merlin comparison whose mutations have also been published (A) L75 and F96 (B) E129 and E136 (C) R187 and I88 and finally (D) N263. Other highly ranked traces can also been seen along with these published mutations which we predict can play important role and can be a good candidate for future mutation analysis both in Kindlins as well as in Merlin.

Fig. 10. Heat map for conserved residues. A heat map showing profile of traces obtained from ETA. Numeric on the horizontal axis represents the position of each trace residue.

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

5. Conclusion Kindlin proteins have been recently identified as FERM domain containing proteins (FDCPs) and are member of B4.1 superfamily. The appearance of FDCPs at different important transitional events, i.e., from unicellular to multicellular organization, from parazoa to metazoan and from radial to bilateral symmetry, reflects the importance of FDCPs in evolution of metazoans. The phylogenetic analysis and subsequently constructed MAP tree of all FERM domains suggests four major clades of FERM domains in all FDCPs. Also, Kindlin FERM domain is most similar to Talin1 FERM domain and is farthest from FAK1 FERM domain among FERM domains of member proteins of B4.1 superfamily. The unique disruption of FERM domain of Kindlins by a Pleckstrin Homology domain has affected evolution of these proteins and presence of two PH-like domains has resulted in increased mutation rate in the Kindlin FERM-C domain. Evolutionary trace analysis of Kindlin FERM domain shows the evolutionary history of these proteins and also predicts many residues, which act as putative candidates for Kindlin related phenotypes. In short, the phylogenetic and evolutionary analysis of Kindlin FERM domain suggests the origin of FERM domains and the importance of FERM domain at important transition events in Eukaryotes. Competing interest We have no competing interest for this article. Authors contribution AAK conceived the idea. RHA and AAK extracted data and analyzed results. AAK and RHA wrote the manuscript. Acknowledgments This project was funded by Higher Education Commission of Pakistan and partially funded by Karolinska Institutet and KTH Royal Institute of Technology, Sweden. We acknowledge Thomas Burglin, Tore Samuelsson and Lars Arvestad for reading this manuscript and giving useful suggestions. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2014. 08.008. References Akerborg, O., Sennblad, B., Arvestad, L., Lagergren, J., 2009. Simultaneous bayesian gene tree reconstruction and reconciliation analysis. Proc. Natl. Acad. Sci. USA 106, 5714–5719. http://dx.doi.org/10.1073/pnas.0806251106. Altschul, S., Gish, W., Miller, W., Myers, E., Lipman, D., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410, http://dx.doi.org/10.1016/S00222836(05)80360-2. Anthis, N., Haling, J., Oxley, C., Memo, M., Wegener, K., Lim, C., Ginsberg, M., Campbell, I., 2009. Beta integrin tyrosine phosphorylation is a conserved mechanism for regulating Talin-induced integrin activation. J. Biol. Chem. 284, 36700–36710. http://dx.doi.org/10.1074/jbc.M109.061275. Ashton, G., McLean, W., South, A., Oyama, N., Smith, F., Al-Suwaid, R., Al-Ismaily, A., Atherton, D., Harwood, C., Leigh, I., Moss, C., Didona, B., Zambruno, G., Patrizi, A., Eady, R., McGrath, J., 2004. Recurrent mutations in Kindlin-1, a novel keratinocyte focal contact protein, in the autosomal recessive skin fragility and photosensitivity disorder, Kindler syndrome. J. Invest. Dermatol. 122, 78– 83. http://dx.doi.org/10.1046/j.0022-202X.2003.22136.x. Banno, A., Goult, B., Lee, H., Bate, N., Critchley, D., Ginsberg, M., 2012. Subcellular localization of Talin is regulated by inter-domain interactions. J. Biol. Chem. 287, 13799–13812. http://dx.doi.org/10.1074/jbc.M112.341214. Barsukov, I., Prescot, A., Bate, N., Patel, B., Floyd, D., Bhanji, N., Bagshaw, C., Letinic, K., Di Paolo, G., De Camilli, P., Roberts, G., Critchley, D., 2003.

203

Phosphatidylinositol phosphate kinase type 1gamma and beta1-integrin cytoplasmic domain bind to the same region in the Talin FERM domain. J. Biol. Chem. 278, 31202–31209. http://dx.doi.org/10.1074/jbc.M303 850200. Bensenor, L., Barlan, K., Rice, S., Fehon, R., Gelfand, V., 2010. Microtubule-mediated transport of the tumor-suppressor protein merlin and its mutants. Proc. Natl. Acad. Sci. USA 107, 7311–7316. http://dx.doi.org/10.1073/pnas.09073 89107. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T., Higgins, D., Thompson, J., 2003. Multiple sequence alignment with the clustal series of programs. Nucl. Acids Res. 31, 3497–3500. Chishti, A., Kim, A., Marfatia, S., Lutchman, M., Hanspal, M., Jindal, H., Liu, S., Low, P., Rouleau, G., Mohandas, N., Chasis, J., Conboy, J., Gascard, P., Takakuwa, Y., Huang, S., Benz Jr., E., Bretscher, A., Fehon, R., Gusella, J., Ramesh, V., Solomon, F., Marchesi, V., Tsukita, S., Tsukita, S., Hoover, K., et al., 1998. The FERM domain: a unique module involved in the linkage of cytoplasmic proteins to the membrane. Trends Biochem. Sci. 23, 281–282. de Pereda, J., Wegener, K., Santelli, E., Bate, N., Ginsberg, M., Critchley, D., Campbell, I., Liddington, R., 2005. Structural basis for phosphatidylinositol phosphate kinase type igamma binding to Talin at focal adhesions. J. Biol. Chem. 280, 8381–8386. http://dx.doi.org/10.1074/jbc.M413180200. Dunty, J., Gabarra-Niecko, V., King, M., Ceccarelli, D., Eck, M., Schaller, M., 2004. FERM domain interaction promotes FAK signaling. Mol. Cell Biol. 24, 5353– 5368. http://dx.doi.org/10.1128/MCB.24.12.5353-5368.2004. Gao, J., Khan, A., Shimokawa, T., Zhan, J., Stromblad, S., Fang, W., Zhang, H., 2013. A feedback regulation between Kindlin-2 and GLI1 in prostate cancer cells. FEBS Lett. 587, 631–638. http://dx.doi.org/10.1016/j.febslet.2012.12.028. Garcia-Alvarez, B., de Pereda, J., Calderwood, D., Ulmer, T., Critchley, D., Campbell, I., Ginsberg, M., Liddington, R., 2003. Structural determinants of integrin recognition by Talin. Mol. Cell. 11, 49–58. Goksoy, E., Ma, Y., Wang, X., Kong, X., Perera, D., Plow, E., Qin, J., 2008. Structural basis for the autoinhibition of Talin in regulating integrin activation. Mol. Cell. 31, 124–133. http://dx.doi.org/10.1016/j.molcel.2008.06.011. Gough, J., Karplus, K., Hughey, R., Chothia, C., 2001. Assignment of homology to genome sequences using a library of hidden markov models that represent all proteins of known structure. J. Mol. Biol. 313, 903–919. http://dx.doi.org/ 10.1006/jmbi.2001.5080. Guy Tanentzapf, N.H.B., 2006. An interaction between integrin and the Talin ferm domain mediates integrin activation but not linkage to the cytoskeleton. Nat. Cell Biol. 1, 601–606. http://dx.doi.org/10.1038/ncb1411, . Hamada, K., Shimizu, T., Matsui, T., Tsukita, S., Hakoshima, T., 2000. Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain. EMBO J. 19, 4449–4462. http://dx.doi.org/10.1093/emboj/19.17.4449. Han, B., Nunomura, W., Takakuwa, Y., Mohandas, N., Jap, B., 2000. Protein 4.1r core domain structure and insights into regulation of cytoskeletal organization. Nat. Struct. Biol. 7, 871–875. http://dx.doi.org/10.1038/82819. Harburger, D., Bouaouina, M., Calderwood, D., 2009. Kindlin-1 and -2 directly bind the c-terminal region of beta integrin cytoplasmic tails and exert integrinspecific activation effects. J. Biol. Chem. 284, 11485–11497. http://dx.doi.org/ 10.1074/jbc.M809233200. Has, C., Castiglia, D., del Rio, M., Diez, M., Piccinni, E., Kiritsi, D., Kohlhase, J., Itin, P., Martin, L., Fischer, J., Zambruno, G., Bruckner-Tuderman, L., 2011. Kindler syndrome: extension of FERMT1 mutational spectrum and natural history. Hum. Mutat. 32, 1204–1212. http://dx.doi.org/10.1002/humu.21576. Hedges, S., Dudley, J., Kumar, S., 2006. Timetree: a public knowledge-base of divergence times among organisms. Bioinformatics 22, 2971–2972. http:// dx.doi.org/10.1093/bioinformatics/btl505. Huttenlocher, A., Horwitz, A., 2011. Integrins in cell migration. Cold Spring. Harb. Perspect. Biol. 3, a005074. http://dx.doi.org/10.1101/cshperspect.a005074. Kang, B., Cooper, D., Devedjiev, Y., Derewenda, U., Derewenda, Z., 2002. The structure of the FERM domain of merlin, the neurofibromatosis type 2 gene product. Acta Crystallogr. D Biol. Crystallogr. 58, 381–391. Khan, A., Janke, A., Shimokawa, T., Zhang, H., 2011. Phylogenetic analysis of Kindlins suggests subfunctionalization of an ancestral unduplicated Kindlin into three paralogs in vertebrates. Evol. Bioinform. Online 7, 7–19. http://dx.doi.org/ 10.4137/EBO.S6179. Kindler, T., 1954. Congenital poikiloderma with traumatic bulla formation and progressive cutaneous atrophy. Brit. J. Dermatol. 66, 104–111. Lai-Cheong, J., Parsons, M., McGrath, J., 2010. The role of Kindlins in cell biology and relevance to human disease. Int. J. Biochem. Cell Biol. 42, 595–603. http:// dx.doi.org/10.1016/j.biocel.2009.10.015. Lichtarge, O., Bourne, H., Cohen, F., 1996. An evolutionary trace method defines binding surfaces common to protein families. J. Mol. Biol. 257, 342–358. http:// dx.doi.org/10.1006/jmbi.1996.0167. Liu, J., Fukuda, K., Xu, Z., Ma, Y., Hirbawi, J., Mao, X., Wu, C., Plow, E., Qin, J., 2011. Structural basis of phosphoinositide binding to Kindlin-2 protein pleckstrin homology domain in regulating integrin activation. J. Biol. Chem. 286, 43334– 43342. http://dx.doi.org/10.1074/jbc.M111.295352. Liu, Y., Zhu, Y., Ye, S., Zhang, R., 2012. Crystal structure of Kindlin-2 PH domain reveals a conformational transition for its membrane anchoring and regulation of integrin activation. Protein Cell 3, 434–440. http://dx.doi.org/10.1007/ s13238-012-2046-1. Lock, J., Wehrle-Haller, B., Stromblad, S., 2008. Cell–matrix adhesion complexes: master control machinery of cell migration. Semin. Cancer Biol. 18, 65–76. http://dx.doi.org/10.1016/j.semcancer.2007.10.001.

204

R.H. Ali, A.A. Khan / Molecular Phylogenetics and Evolution 80 (2014) 193–204

Lua, R., Lichtarge, O., 2010. PyETV: a PyMOL evolutionary trace viewer to analyze functional site predictions in protein complexes. Bioinformatics 26, 2981–2982. http://dx.doi.org/10.1093/bioinformatics/btq566. MacCollin, M., Ramesh, V., Jacoby, L., Louis, D., Rubio, M., Pulaski, K., Trofatter, J., Short, M., Bove, C., Eldridge, R., et al., 1994. Mutational analysis of patients with neurofibromatosis 2. Am. J. Hum. Genet. 55, 314–320. Ma, Y., Qin, J., Wu, C., Plow, E., 2008. Kindlin-2 (mig-2): a co-activator of beta3 integrins. J. Cell Biol. 181, 439–446. http://dx.doi.org/10.1083/jcb.200710196. Marchler-Bauer, A., Bryant, S., 2004. CD-search: protein domain annotations on the fly. Nucl. Acids Res. 32, W327–31. http://dx.doi.org/10.1093/nar/gkh454. Montanez, E., Ussar, S., Schifferer, M., Bosl, M., Zent, R., Moser, M., Fassler, R., 2008. Kindlin-2 controls bidirectional signaling of integrins. Genes Dev. 22, 1325– 1330. http://dx.doi.org/10.1101/gad.469408. Rogalski, T., Mullen, G., Gilbert, M., Williams, B., Moerman, D., 2000. The UNC-112 gene in caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane. J. Cell Biol. 150, 253–264. Shi, X., Ma, Y., Tu, Y., Chen, K., Wu, S., Fukuda, K., Qin, J., Plow, E., Wu, C., 2007. The MIG-2/integrin interaction strengthens cell–matrix adhesion and modulates cell motility. J. Biol. Chem. 282, 20455–20466. http://dx.doi.org/10.1074/ jbc.M611680200. Shimizu, T., Seto, A., Maita, N., Hamada, K., Tsukita, S., Tsukita, S., Hakoshima, T., 2002. Structural basis for neurofibromatosis type 2. Crystal structure of the merlin FERM domain. J. Biol. Chem. 277, 10332–10336. http://dx.doi.org/ 10.1074/jbc.M109979200. Siegel, D., Ashton, G., Penagos, H., Lee, J., Feiler, H., Wilhelmsen, K., South, A., Smith, F., Prescott, A., Wessagowit, V., Oyama, N., Akiyama, M., Al Aboud, D., Al Aboud, K., Al Githami, A., Al Hawsawi, K., Al-Ismaily, A., Al-Suwaid, R., Atherton, D., Caputo, R., Fine, J., Frieden, I., Fuchs, E., Haber, R., Harada, T., Kitajima, Y., Mallory, S., Ogawa, H., Sahin, S., Shimizu, H., Suga, Y., Tadini, G., Tsuchiya, K., Wiebe, C., Wojnarowska, F., Zaghloul, A., Hamada, T., Mallipeddi, R., Eady, R., McLean, W., McGrath, J., Epstein, E., 2003. Loss of Kindlin-1, a human homolog of the caenorhabditis elegans actin-extracellular-matrix linker protein UNC-

112, causes Kindler syndrome. Am. J. Hum. Genet. 73, 174–187. http:// dx.doi.org/10.1086/376609. Sjostrand, J., Sennblad, B., Arvestad, L., Lagergren, J., 2012. DLRS: gene tree evolution in light of a species tree. Bioinformatics 28, 2994–2995. http://dx.doi.org/ 10.1093/bioinformatics/bts548. Svensson, L., Howarth, K., McDowall, A., Patzak, I., Evans, R., Ussar, S., Moser, M., Metin, A., Fried, M., Tomlinson, I., Hogg, N., 2009. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation. Nat. Med. 15, 306–312. http://dx.doi.org/10.1038/nm.1931. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. http://dx.doi.org/10.1093/molbev/msr121. Ussar, S., Wang, H., Linder, S., Fassler, R., Moser, M., 2006. The Kindlins: subcellular localization and expression during murine development. Exp. Cell Res. 312, 3142–3151. http://dx.doi.org/10.1016/j.yexcr.2006.06.030. Wang, H., Lim, D., Rudd, C., 2010. Immunopathologies linked to integrin signalling. Semin. Immunopathol. 32, 173–182. http://dx.doi.org/10.1007/s00281-0100202-3. Wehrle-Haller, B., 2012. Structure and function of focal adhesions. Curr. Opin. Cell Biol. 24, 116–124. http://dx.doi.org/10.1016/j.ceb.2011.11.001. Wehrle-Haller, B., Imhof, B., 2003. Actin, microtubules and focal adhesion dynamics during cell migration. Int. J. Biochem. Cell Biol. 35, 39–50. Ye, F., Petrich, B., 2011. Kindlin: helper, co-activator, or booster of Talin in integrin activation? Curr. Opin. Hematol. 18, 356–360. http://dx.doi.org/10.1097/ MOH.0b013e3283497f09. Yogesha, S., Sharff, A., Giovannini, M., Bricogne, G., Izard, T., 2011. Unfurling of the band 4.1, ezrin, radixin, moesin (FERM) domain of the merlin tumor suppressor. Protein Sci. 20, 2113–2120. http://dx.doi.org/10.1002/pro.751. Zhan, J., Zhu, X., Guo, Y., Wang, Y., Wang, Y., Qiang, G., Niu, M., Hu, J., Du, J., Li, Z., Cui, J., Ma, B., Fang, W., Zhang, H., 2012. Opposite role of Kindlin-1 and Kindlin-2 in lung cancers. PLoS One 7, e50313. http://dx.doi.org/10.1371/journal.pone. 0050313.