Force Directed 'Mechanointeractome' of Talin-Integrin

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Force Directed ‘Mechanointeractome’ of Talin-Integrin Soham Chakraborty, Souradeep Banerjee, Manasven Raina, and Shubhasis Haldar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00442 • Publication Date (Web): 08 Aug 2019 Downloaded from pubs.acs.org on August 9, 2019

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Biochemistry

Force Directed ‘Mechanointeractome’ of Talin-Integrin Soham Chakraborty#, Souradeep Banerjee#, Manasven Raina, Shubhasis Haldar* Department of Biological Sciences, Ashoka University Sonepat, Haryana 131029

# Contributed equally to this work *To whom correspondence may be addressed. Email- [email protected]

ACS Paragon Plus Environment

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Abstract: Mechanotransduction from the extracellular matrix into the cell is majorly supervised by a transmembrane receptor- integrin and a cytosolic protein- talin. Integrin binds ligands in the extracellular side, whereas talin couples integrin receptors to the actin cytoskeleton and later acts as a ‘force-buffer’. Talin and Integrin together form a mechanosensitive signalling hub that regulates crucial cellular processes and pathways including cell signalling and formation of focal adhesion complexes, which help cells to sense their mechano-environment and transduce signal accordingly. Although both proteins function in tandem, most literature focuses on them individually. Here we provide a focused review on the talin-integrin mechano-interactome network in light of its role in the process of mechanotransduction and its connection to diseases. While working under force, these proteins drive numerous biomolecular interactions and form adhesion complexes, which in turn control many physiological processes such as cell migration; thus, they are invariably associated with several diseases from leukocyte adhesion deficiency to cancer. Forming insights into their role in the occurrence of these pathological disorders might lead us to establish treatment methods and therapeutic techniques.

Key words: Mechanotransduction, Mechanical force, Integrin, Talin, Focal adhesion, Cancer


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Biochemistry

Introduction: Cells are capable of sensing the mechanical milieu surrounding them through mechanotransduction – a comprehensive process that translates mechanical cues into biochemical signals, allowing cells to cope with their physical environments.1 This ability of cells to respond to their physical surroundings is indispensable and crucial to several operations varying from developmental processes like morphogenesis to physiological processes like blood pressure.2 Fibroblasts, endothelial, and muscle cells are a few examples of excellent mechanosensitive cells with the capability of detecting significantly low amounts of mechanical force.3 Several cellular processes such as cell adhesion4, migration5, division6 and cellular locomotion7 are directly regulated by force. As a majority of cells and their functions depend upon mechano-signalling pathways, errors (like mutations) in cellular mechanotransducers can cause various human diseases like muscular dystrophies, deafness, cardiomyopathy, arteriosclerosis, cancer, lung dysfunction, and immune disorders.2,8–12 The capability of sensing mechanical cues and responding accordingly is majorly regulated by transmembrane proteins connecting the extracellular matrix (ECM) to cytosolic proteins, which remain connected to the cytoskeleton. Among these different proteins, talin and integrin are two major supervisors of force specific cellular mechanisms. While integrin, being a transmembrane receptor, interacts with ECM ligands (such as, fibronectin, collagen, laminin) and transmits the experienced tension within the cells; talin experiences force majorly from the actin cytoskeleton.13 Due to talin’s association with integrin, the force experienced by talin is also relayed to the extracellular region through integrin, leading to regulation of extracellular interactions. As a linchpin partner, talin plays a crucial role in activating integrins by the ‘inside-out’ mechanism and concurrently linking them to the actin cytoskeleton.14 The talin-integrin mechanical linkages are controlled by mechanical strain, regulating recruitment of other cytoplasmic partners such as vinculin, actin, kindlin and α-synemin to architect integrin based focal adhesions (FAs).15 These supramolecular complexes help cells sense their mechano-environment and transduce signal accordingly. During cell migration, the actomyosin system generated mechanical tension is streamed towards the ECM by these integrin-talin centered dynamic entities which act as a ‘molecular clutch’.16 As a part of these clutch systems, integrins activate FAK (focal adhesion kinase) and Src kinase, which further induce RhoA signalling during cell migration.17 Abnormalities in any of these FA proteins and in the related mechanosignalling hamper cellular mechanosensitivity, leading to fatal diseases such as cancer metastasis, collapse of primary immune response, leukocyte adhesion deficiency (LAD)18 and failure in developmental migration and morphogenesis.2 In this review, we will focus on how the integrin-talin mechanosensitive signalling hub (MSH) plays a decisive role in force transduction and eventually in regulation of mechanosignalling pathways. Being one of the most prominent force cushions in many cells, any malfunction in this hub might lead to the onset of several fatal diseases such as leukocyte adhesion deficiency, renal fibrosis, glioblastoma multiforme and prostate cancer. Thus, it is necessary to review our current understanding of mechanosensitivity and the recent progresses made in force-based studies of talin and integrin in light of their biophysical principles. We will also discuss key aspects of their structural and functional regulation in diverse mechanical responses of cells and finally explain how their dysregulation impacts diseased phenotypes including cancer progression. Structural details of talin and integrin in relation to mechanointeractome: Vertebrates express two closely related talin isoforms. Talin1 are ubiquitously expressed proteins, whereas talin2 are mainly expressed in skeletal muscle, cardiac muscle cells and brain cells.19–21 They show different expression patterns and share 74% amino acid sequence homology.11 Talin1 appears to


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be more extensively studied and is believed to have more prominent function than talin2 in cell migration and embryogenesis.22 A knock-out of talin1 in mice, developed progressive myopathy and decreased muscle force generation23,24. Conditional knock out of talin1 in platelets fails to activate integrin receptors in response to platelet agonists.25 Talin contains two main domains- N terminal FERM (four-point-one, ezrin, radixin, moesin) domain and C terminal rod domain. This C-terminal end remains connected with the N-terminal portion through an unstructured linker (9 kDa), which when fully extended increases the length of the protein by 20 nm.26 Talin FERM domain consisted of usual F1-F3 subdomains, where F1 is preceded by an uncharacterized region F0 subdomain. This makes talin an exceptional FERM protein.27 Instead of adopting a ‘cloverleaf’ conformation like usual FERM proteins, talin head domain forms an extended conformation.28 Talin rod domain is divided into 13 helical subdomains (R1-R13)29 containing a total of 61 alpha helices followed by a 62nd dimerizing alpha helix (dimerization domain, DD) required for the homo-dimerization of talin and binding with actin cytoskeleton.12,30 Talin alternates between two conformations, an inactive autoinhibited conformation in the cytoplasm and an active extended form near the plasma membrane. It adopts the autoinhibited form due to an intramolecular interaction between F3 and R9 subdomains which can be converted subsequently into the active state.31–33 RIAM interacts with R2-R3 subdomains of autoinhibited talin dimer in a Rap1 dependent manner which eventually converts it into an active extended state (Fig 1).33,34 Along with that, Gα13 (Guanine nucleotide binding protein alpha 13) switch region 2 (SR 2) interacts with talin to relieve its autoinhibited conformation and induce integrin activation.35

Figure 1: Conversion of autoinhibited talin to active extended state: Talin dimer forms a “donut shaped” autoinhibited conformation with their FERM domains in the centre. Autoinhibited conformation is formed by an intramolecular interaction between F3 and R9 subdomains. RIAM interacts with R2-R3 subdomains of autoinhibited talin and recruit them near plasma membrane in a Rap1-GTP dependent manner. In plasma membrane the autoinhibited talin dimer is converted into active extended state in which F3 subdomain binds to integrin β cytoplasmic tail, whereas F0, F1, F2 interacts with membrane phospholipid PIP2.33,34


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Biochemistry

Integrin is a heterodimeric protein which is made up of multidomain α and β subunits (Fig 2a).14,36,37 18α and 8β subunits combine to form 24 different heterodimeric isoforms with varying ligand binding ability, resulting in diversified functions in different tissues.14,38 Each of the integrin subtypes are made up of an extracellular region, a transmembrane portion and a flexible cytosolic tail.14,39 Extracellular regions of both the subunits consist of multiple domains. The α subunit is made up of a seven bladed βpropeller domain, followed by one thigh and two calf domains (Calf 1 and Calf 2) each with a βsandwich fold. The β half constitutes of a hybrid domain, PSI (plexin-semiphorin-integrin), EGF1-4 (Epidermal growth factor) and β tail domains. In β3 integrin, the PSI domain gets split up into two parts, that remain connected with a long-distance disulfide bridge between Cys13 and Cys435 residues.14,39,40 Following the PSI domain, there are four cysteine rich EGF (1-4) domains.14,36,40 The EGF4 being the last of its type is connected with the β-tail which attaches the subunit to the membrane. A hinged region known as the ‘genu’ or ‘knee’, placed in between the thigh and calf-1 of α subunit and at the junction of the EGF1 and 2 of β subunits causes flexible bending during conversion between the thermodynamic ‘bent-closed’ and ‘extended-open’ conformations (Fig 2a).14,36,39 The βI domain possesses a MIDAS (metal ion dependent adhesion site) which binds the Mg2+ ion and regulates ligand binding. The MIDAS on its adjacent sites has an ADMIDAS (adjacent to MIDAS) site and a synergy site/synergistic metal binding site (Sy-MBS) (Fig 2c). 14,38,41,42 Both the ADMIDAS and Sy-MBS sites generally bind to Ca2+, however the inhibitory effect of Ca2+ in ADMIDAS can be countered when replaced by Mn2+ .14,41

Figure 2: Structural details of integrin and talin: (a) Schematic tertiary structure of talin28–30,43–47 and integrin, integrin36,48,49 β-cytoplasmic tail’s proximal NPxY motif interacts with talin’s R358 and W359 residues in F3 subdomain. (b) The LD-motif of DLC1 binding with R8 subdomain.50 (c) The metal ion bound MIDAS (Mg2+), ADMIDAS (Ca2+) and Sy-MBS (Ca2+) housed in βI domain of integrin.48,49 (The structures were later modified to enable a proper view of every domain using CHIMERA51 and the unstructured and undefined domains were prepared using their amino acid sequences through NCBIBLAST52 and MODELLER.53)


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Mechanointeractome of talin and integrin: The integrin-talin complex interacts with a repertoire of proteins in both extracellular and intracellular regions of the cell (Fig 3). Talin binds with a plethora of cytosolic signalling proteins through its multiple rod subdomains and interacts with the cytosolic tail of integrins through its FERM domain. In contrast, integrins bind majorly with the ECM components and counter cell receptors through their ectodomain and multiple regulators through their cytoplasmic tail. This make the talin-integrin complex a key regulator of extracellular and intracellular signalling. The multiple subdomains of talin communicate with a variety of interactors, including structural and signalling proteins which are involved in the regulation of their own and others’ conformational and signalling activity. It’s major structural partners are integrin, vinculin, actin, α-synemin, whereas PIP2 (Phosphatidylinositol 4,5-bisphosphate), DLC1(Deleted in Liver Cancer 1), RIAM (Rap1-GTPinteracting Adaptor Molecule), Rap1(Ras-related protein 1), layilin, TIAM (T-lymphoma invasion and metastasis-inducing), KANK 1-454,55 and moesin are the signalling partners (Fig 3c).11,12 The binding of interactors with talin is strictly regulated by the conformational changes in its own subdomains, that occur due to the transmission of force through them, which in turn allows the binding and unbinding of ligands.

Figure 3: Talin-integrin force cascade and interactome: (a) transmission of the mechanical signals from the extracellular matrix to actin cytoskeleton through talin integrin force-cascade. (b, c) interactome of integrin and talin with the signalling (black) and structural (red) partners. Signalling partners are involved in various biochemical signalling for cell proliferation and migration; structural partners are mainly different cytoskeletal proteins which are involved in cell shape and structure maintenance.

Table1: Ligands interaction with specific subdomains of talin and their functional activities Ligands Integrin56 Vinculin34,57

PIP259

Talin subdomains F3 and R11 R1-R3; R6-R8, R10-R11, R13 F2-F3 (ABS1), R4-R8 (ABS2), R13-DD (ABS3) F1-F3

Rap160

F0

Actin58

Functions Cell adhesion, migration and signal transduction Formation and maturation of focal adhesion Maintaining cell shape and motility Activate integrin co-operatively, activation Cell spreading and integrin activation


platelet

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Biochemistry

RIAM61 DLC162,63

F3, R2-R3, R8, R11 R8

α-synemin64

R8

KANK1-454

R7

FAK65

F3

Layilin66

F3

Gα1335 TIAM 167 PIP15Kγ9068

F3 F3 F3

Moesin69,70

R11, R13

Unfolds F3 to induce integrin activation Acts as a tumor suppressor, decreases myosin activation and migration. Integrates mechanical stress and maintain structural integrity in eukaryotic cells Blocks talin-actomyosin interaction, regulates cell migration and polarity Regulates adhesion assembly, cell motility and adhesion turnover Assists in cell adhesion along with hyaluronan in ECM Removes talin’s autoinhibited state Activates Rac1 and induces cell spreading Synthesizes PIP2 in talin induced integrin activation Regulates cell migration signalling in cellular protrusion

Talin interacts with a diverse array of proteins through its FERM domain. The binding of FERM domain to integrin results in integrin activation and enhances its ligand binding capability through ‘inside-out signalling’. The talin autoinhibited conformation is removed during integrin and actin binding followed by its recruitment to focal adhesion.31,32,71,72 Talin head subdomain F0 binds to the Rap1 protein,27,73 whose effector RIAM bind to R2-R3 subdomains with higher affinity than R8 and R11 subdomains.29,74 Among other activators, Gα13 and PIP2 (both of which interact with F3) dislocate R9 to induce steric unmasking of talin domains.35,75 Additionally, KANK2 interacts with the R7 subdomain to induce talin activation54 and subsequent integrin activation near the plasma membrane. Activated talin links with actin cytoskeleton through its F2-F3 subdomain making an actin binding site 1(ABS1).76,77 Another interacting subdomains for actin binding is R4-R8, which makes up the ABS2.76,78 Interestingly, during retrograde motion, actin cytoskeleton pulls talin by interacting with R13 and the adjacent dimerization domain (ABS3). This is understood to be the primary actin binding domain for initial force transmission.30,79–81 ABS3 was renamed as I/LWEQ domain and is also found in Huntington interacting protein 1 (Hip1) and its related paralogue.82 Interaction between partner proteins and talin subdomains is majorly controlled by the α helices present in the adjacent subdomains. For example, actin binding to 58th-62nd α helices of ABS3 are opposed by the adjacent 57th helix of R13.30,78,80 Additionally, integrin binding to the 50th α helix of IBS2 is negatively regulated by the immediate next 51st helix in R11.83 The binding of these interactors is tightly regulated in terms of their individual role in the formation of focal adhesions. One of the important partners of talin is DLC1, a GTPase-activator and tumor suppressor protein whose mutations lead to a variety of cancerous phenotypes.84 DLC1 interacts with talin R8 subdomain by an α-helical leucine–aspartic acid (LD) motif (Fig 2b). 62,85 Apart from DLC1, paxillin and RIAM also interact with the R8 subdomain of talin through LD motif.50,86 Interactions of talin subdomains with partner proteins are not limited to the LD motif. Several other motifs of different proteins can also interact with different subdomains of talin. For example, integrin β subunit’s NPxY motif associates with F3 subdomain,87 α-synemin’s SNTIII motif interacts with R8 subdomain,64 KANK protein’s KNmotif bind to the R7 subdomain of talin. However, KANK also contains an LD motif to promote talin activation.54,55


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Integrin on the other hand, being a transmembrane protein, binds to several signalling and structural ligands on both outside and inside of the cell membrane (Fig 3c). The binding of ligands to integrin is regulated by the heterodimeric nature of integrins and divalent metal ions situated in the I domain. The heterodimeric nature of integrin receptor ensures that the different combinations of α and β subunits can selectively specify their ligands (Table 2).41 Although, Mg2+ in the MIDAS directly binds to the carboxylate oxygen of extracellular ligands, this interaction is additionally tuned by the Sy-MBS and ADMIDAS bound Ca2+.14,41 This emphasizes the importance of metal cations in βI domain for regulating the interaction with extracellular ligands. βI domain directed interactions are sometimes assisted by αI domain possessed Ca2+, in αIIbβ3, α1β1 integrin subtypes.88 Majority of the integrin extracellular ligands are enriched with acidic amino acid sequences in their active sites. During the interaction, the basic amino acid binds with the β propeller’s cleft of α subunit and the acidic residue binds with the MIDAS of βI domain, strengthening the binding of extracellular ligands at the junction of the two subunits’ ectodomain.14

Table2: Ligand interactions with specific integrin subtype, binding motifs and functions Ligand

Integrin subtypes

Binding motif

Function

Laminin89

α3β1, α6β1, α6β4, α7β1, α1β1, α2β1, α10β1

N- and C- terminal of laminin

Cell survival; random or directed cellular migration and spreading

αVβ3

RGD

α4β1, α4β7, α9β1, αDβ2 αIIbβ3, αVβ3, αVβ6, αVβ1, α5β1

LDV/IDSP

Leukocyte adhesion

RGD

Cell attachment and adhesion

α8β1, α4β1, α4β7

LDV

αIIbβ3, α8β1, αVβ5 α10β1, α2β1, α1β1, α11β1

RGD

αXβ2

LDV

I-CAM94

αXβ2, αMβ2, αDβ2, αLβ2

LDV

Immune response of T-cells and cell attachment

E-cadherin95

αEβ7

LDV

Thrombospondin96

α3β1, α2β1, α4β1

LDV

Lymphocyte adhesion around mucosal epithelia. Anti-tumor cytotoxic response of tumor infiltrating T-lymphocytes. Cell-ECM adhesion in different cancer

V-CAM90 Fibronectin91

Vitronectin92 Collagen93

GER/ GFOGER


Endothelial cell attachment to ECM, regulate tumor progression. Cell proliferation, cell adhesion

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Biochemistry

α9β1, α6β1, αVβ3, αIIbβ3

RGD

types, formation of myotendinous junction

α4β7, α4β1, α9β1

LDV

α8β1, α5β1, αVβ1, αVβ6, αVβ3, αVβ5

RGD

Cell adhesion, migration, immune response, mesenchymal stem cell differentiation

BSP98

αVβ3, αVβ5

RGD

Cell proliferation, migration, adhesion

MFG-E8 99

αVβ3, αVβ5

RGD

vWF100

αVβ3, αIIbβ3

RGD

Tenascin101

αVβ3, α8β1, αVβ6

RGD

α9β1

IDG

Phagocytosis of apoptotic- epithelial, endothelial cells Platelet-platelet adhesion, plateletendothelial cell adhesion Cell proliferation, apoptosis regulation, epithelial-mesenchymal like cell transition

Fibrillin102

αVβ3

RGD

Cell spreading, cell attachment

Fibrinogen103

αMβ2, αXβ2

LDV

αIIbβ3, αVβ3

RGD

Platelet-ECM binding to maintain homeostasis

Factor X104

αMβ2

LDV

MAdCaM90

α4β7, α4β1

LDV/LDTS

Cell adhesion, degranulation Leukocyte adhesion

PeCAM105

αVβ3

RGD

Leukocyte adhesion with endothelium

iC3b106

αMβ2, αXβ2

LDV

Phagocytosis

Osteopontin97

phagocytosis,

Other than these extracellular ligands, integrins bind to different cytosolic partners by their flexible cytosolic tail. Among these, talin is crucial as it activates integrins and eventually acts as a vital mediator for connecting the cytoskeletal system to the membrane. In addition to talin, kindlin directly associates with integrins and further recruits PINCH-parvin, paxillin and Arp2/3 multiprotein complex to regulate cell migration and overall cell stiffening via RhoA signalling.13 On the contrary, integrins-ligand interactions are destabilized by inactivators such as moesin, filamin A, DOK1, ICAP1, SHARPIN (SHANK-associated RH domain-interacting protein) and MDGI (Mammary-derived growth inhibitor) by interacting with integrin β cytoplasmic tail (Fig 3b). Moesin, filamin A, DOK1, ICAP1 compete with talin and kindlin recruitment, whereas SHARPIN and MDGI connect with integrin α subunit to maintain their low affinity state.13,107–113 The structural changes of integrin during its interaction with ligands majorly take place in the transmembrane (TM) and extracellular regions. Crystal structure of αIIbβ3 integrin subtype shows that TM domain separation occurs during active state, whereas in αVβ3 those TM domains remain separated in its inactive form. During these interactions, the ectodomain changes its conformation from thermodynamic inactive to a rare active state.36,114 Integrin β subunits’ ectodomain leg, comprising EGF and β tail domains, displays conformational fluctuations by either getting separated or coming closer towards the α subunit during its active state.13,39,115 Unlike the other domains of integrin, the cytosolic domain does not show much of a structural modification individually but acts along with the TM domain. Even in absence of such individual conformational changes, the cytosolic domain is a hotspot for interaction of different cytosolic adaptor proteins such as talin and kindlin. During talin attachment


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with NPxY motif in integrin β cytoplasmic domain (βcd), the salt-bridge between the α/β cytosolic tail gets broken, which consequently separates the domains. In contrast, kindlin interacts with a distal NPxY motif due to binding specificity provided by TTV/STF (TTV in β1/ STF in β3) sequences between two canonical NPxY motifs.116,117 Paxillin interacts with α4 integrin through their LD3 and LD4 region.118 In an extended-open conformation, integrin binds to several extracellular ligands with the help of the metal cations present in the βI domain. This interaction between ligand and integrin is assisted by MIDAS, which binds to the ligand both directly and indirectly in a highly regulated high and low conformation transition. The octahedral configuration of MIDAS of α3βII integrins possesses the top axial co-ordination point specifically for water or the ligand carboxylate site. The conformational stability of MIDAS changes from a higher to lower state due to the bond which binds the ligand to the divalent cation. When the bonding takes place directly with the ligand’s Ser residue in DXSXS motif, the MIDAShigh conformation is obtained. This bond when replaced with an indirect bond connected through water, takes the MIDASlow conformation. This regulation is extremely important as even in a thermodynamically inactive conformation, integrin can bind to specific ligand with less half-life, when stabilized by MIDAShigh conformation.12,37,17,45 The crystal structure of α5β1 integrin bound to fibronectin reveals that the specificity and binding affinity of the interaction is not only enhanced by core RGD motif of a ligand, but also modulated by the Ca2+ of the Sy-MBS. Furthermore, the binding of extracellular ligands is very precisely regulated by the presence of a post translational modification (N-glycosylation) of a specific amino acid residue at the β integrin head residue.41 Thus, this low and high conformations of MIDAS, along with post-translational modification of an amino acid residue, majorly decide the stability of an integrin-ligand complex. Talin and integrin mediated mechanotransduction: Mechanical force induced conformational changes and concurrent unfolding of subdomains are the key properties of talin to perform as a mechanosensor. Mechanical unfolding of talin transduces the force along its rod domains and regulates the interactions with other proteins. Among various interactorsactin, RIAM, DLC1, KANK1, KANK2 interact with folded talin subdomains29,50,69 whereas vinculin requires an unfolded subdomain120 to interact, allowing adhesion reinforcement. Talin domains fold to their native conformation below a force of 3pN.88 Above 5 pN, talin unfolds and exposes eleven vinculin binding domains. Thus, vinculin binding is directly controlled by mechanical force, which could take place by any of these two ways: either internally by actin retrograde flow or by exertion of force from extracellular matrix (ECM). Vinculin binding to talin requires force induced unfolding of talin subdomains,8,34,120,121 for example, R2-R3 subdomains are not exposed in native conformation but become accessible when actomyosin systems stretch talin, which in turn enhances vinculin binding.43,44,122 Vinculin binding to talin is not only required for force transmission, but also causes talin unfolding which finally regulates cellular responses.12 This interaction in turn mediates actin binding to talin through ABS2, which adds more complexity to force transduction in the context of conformational arrangement. It is to be noted that mutation or deletion in both ABS2 and ABS3 can cause significant reduction in force sensing and eventually vinculin binding ability.12 ABS3 has been shown to promote most of the talin function, however, the role of ABS3 in cell adhesion formation is not prominent, and mutation or functional impairment of it can cause failure in adhesion assembly78. Different subdomains sense tension and function accordingly during the course of adhesion maturation.123,124 Initially, nascent linkage between talin and actin experiences lower tension, whereas mature ones sense higher tension mainly through ABS2.69 To summarize, talin in nascent adhesions versus mature adhesions shows different force sensitivity to change their ligand profiles. It not only works as a mechanotransducing machinery, but also plays a role as “shock absorber” to withstand the impact of fluctuating mechanical changes.8


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Biochemistry

The application of force makes talin act as a combined mechanical regulator with a specific force range for each subdomain. Among all subdomains, R3 and R8 are exceptional with regard to their structural features as both subdomains contain a cluster of four threonine in the hydrophobic core of their helix8,29,44, which makes them mechanically unstable and so they unfold above 5 pN force. R3 has been confirmed as an initial mechanosensor during force transmission through talin, as it not only unfolds under lowest force, but also undergoes rapid unfolding and refolding.8,34 Additionally, RIAM, another adhesion partner of talin, interacts with folded R3 and eventually unfolds it into a helix string.29,69 This structural transition exposes two cryptic VBSs, enhancing the binding of vinculin that eventually leads to removal of RIAM in a mutually exclusive way.29 This mechanical event is reflected in cellular physiology, where an integrin-talin-RIAM complex at the tip of a lamellipodial and filopodial protrusion (‘sticky finger’) is converted into integrin-talin-vinculin linkage to form a mature focal adhesion (FA).125 Low mechanical stability of the R3 subdomain makes it suitable for acting as a mechanosensor during adhesion maturation. Mechanically stabilized talin R3 subdomain was found to affect fibroblast substrate rigidity sensing and YAP (Yes-associated protein) signalling, whereas its destabilization can lead to decrease in cell migration by effectively changing traction force generation. Importantly, the destabilized R3 also changes size, shape and protein composition of adhesion sites that ultimately affects ECM mechanosensing.26 Despite the structural homology between R3 and R8, the arrangement of R8 makes it a special subdomain in terms of force transmission. Normally, R8 is packed within two helices of R7 subdomain and remains “safe” from force. The protection provided by R7 is important for R8 to act as a “binding hotspot”. This domain topology suggests that R8 can unfold under ~5 pN force, only after R7 is stretched at 15 pN. This cooperative unfolding strategy is reflected in a single unfolding step size of ~80nm during stretching of R7-R8 subdomain at 15-20 pN. However, R8 subdomain can act as a novel mechano-chemical switch, which plays a critical role in adhesion maturation during the course of cell migration. In early adhesions, force unfolds R8 subdomain and disrupts DLC-1 localization in adhesion sites, reducing its negative regulation on RhoA signalling. This induces force generation in maturing adhesions and drives cell migration. As the adhesions matures, the force on individual talin decreases with the increasing number of protein partners in adhesion site that helps DLC-1 to interact with R8 subdomain. This efficiently controls the rate of cell migration by abrogating RhoA signalling pathway. Thus, reduction in force transduction allows R8 to shift into folded DLC-1 binding conformation.126 In contrast, R9 subdomain remains stable at force > 15pN even when vinculin is bound to talin, suggesting that it is a very stable domain in presence of tensile loads and thus is able to finely adjust focal adhesion dynamics.8 The structural information obtained from single molecule studies has helped in deducing the role of talin as a mechano-chemical regulator. Single molecule super-resolution imaging has shown the dynamic fluctuation of talin end to end at adhesion sites. In vivo FRET assays have shown that talin length varies within a range of 60-400 nm at adhesion sites due to actomyosin retrograde motion, which is counter evidence for the mechanical studies performed.10 Another prominent part of this mechanochemical regulation is R7 subdomain, which recruits the microtubules to the stable adhesion site.55 Microtubules are not stably associated with adhesion sites but their recruitment and stabilization to adhesions requires a cortical microtubule stabilization complex (CMSC), which is a multiprotein complex consisting of KANK, microtubule plus end binding protein, CLASP (cytoplasmic linker associated proteins) and multiple cortical adaptor proteins.17,127 CMSC accumulates near the adhesions and stabilizes microtubules in the cell cortex.128 The presence of CMSC in adhesion complexes depends on linkage between LD motif in KANK protein and R7 in talin rod domain, in a lean border along focal adhesion outer edge.54 This mechanically regulated microtubule recruitment to the adhesion site is important for focal adhesion dynamics (adhesion assembly and disassembly). The tension generated during cell-cell and cell-ECM adhesion or separation transmits the mechanical signals through an integrin-talin mechanical cascade to the cell for further responses. To initiate this,


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integrin is converted from its inactive thermodynamically stable ‘bent-closed’ conformation to the active unstable ‘extended-open’ state through a transient ‘extended-closed’ structure.13,14,39 This activation takes place by two mechanisms- ‘inside-out’ and ‘outside-in’ (Fig 4a, 4b). During ‘outsidein’ activation of integrin, extracellular ligands (fibronectin, collagen, laminin etc.) or agonists (chemokines, cytokine, ADP etc.) induce the integrin activation and successive association of talin and kindlin.129 While, ‘inside-out’ activation occurs when talin associates with integrin β cytoplasmic tail to induce a tilt angle change of α/β transmembrane domain, which will further separate their transmembrane domain and ectodomain.13,130 On the contrary, in case of ‘inside-out’ activation of the α5β1 and α4β1 integrin, the cellular concentration of talin owing to its low affinity towards integrin, is not enough to exert 1-3 pN force required to overcome the thermodynamic barrier to activate integrin.13,131 However, according to a talin induced integrin activation model, talin stabilizes a rare ‘extended-open’ integrin by exerting a minor force to integrin-ligand complex, instead of singlehandedly disrupting their transmembrane salt-bridge.13 Although, with the help of PIP2, talin can break the transmembrane salt bridge, as shown for αIIbβ3.132 Eventually this favours the conformation to shift towards an ‘extended-open’ state, which then proceed for the ‘outside-in’ activation and signalling. Due to this inability of talin (or adaptors) in inducing integrin activation, it was concluded that tensional force is a more potent regulator than physiological concentration of talin.131 Additionally, force can directly decrease the free energy, whereas adaptor concentration can only logarithmically reduce it.131 All of these regulations cause the integrin to shuffle between its three conformations by ‘inside out’ and ‘outside in’ mechanisms in a reciprocated manner.


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Biochemistry

Figure 4: (a) ‘Inside-out’ activation: 1) the inactive bent-closed conformation of integrin is unable to interact with talin or any extracellular matrix ligand. 2) The autoinhibited conformation of talin dimer gets activated by Rap1-RIAM complex which then binds to the inactive bent closed (BC) conformation at integrin βcd. 3) F0, F1, F2 subdomains of active extended talin dimer interact with PIP2 and F3 interacts with integrin. Both of these two interactions are important for converting integrins into its extended open (EO) conformation. On the other hand, the C-terminal domain of talin associates with actin filament, which possibly provides the required 1-3 pN of force. Now these activated integrins are capable of binding to ECM ligands (such as collagen, fibronectin etc.).13,33 (b) ‘Outside-in’ activation: The BC conformation of integrin remains inactive and is unable to bind with extracellular matrix proteins. During thermodynamic equilibrium condition, conformational fluctuation occurs between the inactive BC and rare active EO state, where the EO is stabilized by the minimum amount of required force, provided by talin. This causes the EO to bind with an immobilized extracellular ligand as well as the actomyosin complex simultaneously through talin, thus transducing the signals from outside to inside.13 Integrins are known to experience a wide range of forces within 1-40 pN during ligand interaction, as revealed by different force-based techniques. Measurement of single molecule force with molecular


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tension sensors (MTSs) has shown that most integrins experience 1-5 pN force during force transmission.133,134 Additionally, DNA based MTSs reveal integrin can exert up to 15 pN force.135,136 Furthermore, studies with DNA based tension probes imply that a single integrin molecule can transmit up to ~ 40 pN during adhesions or even higher in its maturation.137 However, it was observed that in this ensemble, the cytosolic domain of integrin experiences about 1.5-6 pN of force. External forces disrupt the protein-protein interaction as a result of slip-bond formation, where bonds lifetimes are substantially shortened. Application of mechanical loads of 10-30 pN can induce both bond strengthening and extended bond lifetimes resulting in the formation of ‘catch bond’.13 This can be exemplified by integrin α5β1-fibronectin (FN) interaction in the extracellular region. In the relaxed state, α5β1 interacts with RGD (Arg-Gly-Asp) peptide of FN but during tensional loads, synergy sites in FNIII9 are also engaged to form a ‘catch bond’.138,139 Indeed, application of 5 pN of force to α5β1fibronectin results in bond lifetimes of 1s on average, which could be lengthened when force loading was increased to 25 pN. To inspect whether bond lifetimes can be prolonged even after force removal, single integrin α5β1-fibronectin bonds were first loaded with 25 pN and subsequently unloaded to 5 pN for measuring bond lifetimes, which led to the observation that mean lifetime was considerably extended to 30s. This event is called ‘cyclic mechanical reinforcement’, which is different from a ‘catch bond’. Catch bonds signify that bonds are stable and strengthened under force, but disrupted during release of tension. In contrast, this ‘cyclic mechanical reinforcement’ proposes more effective regulation with cyclic force where bonds lasted for a longer time even after tension removal.140 Shifts in mechanical force and fluctuation between these ‘catch bonds’ and ‘slip bonds’ of integrin-ligand interaction, effectively determine the organization of adhesion sites. Another plausible idea for stabilizing integrin-based adhesion is parallel integrin clustering, which significantly increase the mechanical linkages to initiate robust mechanotransduction. It not only induces the lateral redistribution of force through the bonds of integrin assembly, but also increases integrin-ligand binding sites and duration of force transmission.141 On soft ECMs of ~1.5 kPa tensional loads, integrins form clusters with an intermolecular distance of ~200 nm142; but higher tensions (~150 kPa) of stiff ECMs dictate integrins to form denser clusters with shortened distance of ~60 nm, in order to design stable adhesions.143 The formation of integrin assembly must be accomplished by kindlin dimerization and Factin crosslinking to withstand higher force. Finally, catch bond formation and integrin clustering considerably extends adhesion lifetime leading to the formation of a stable adhesion. Integrin-ligand ‘catch bond’ formation and subsequent force transduction play a vital role in regulating the overall cellular mechanics and gene expressions.17 Both of these changes require integrin-adhesion strengthening and a downstream RhoA signalling cascade. Importantly, adhesion strengthening is intricately connected to cellular stiffness via mutual crosstalk of various integrins with RhoA-guanine nucleotide exchange factors (GEFs). In early adhesion strengthening, tensional loads on integrin activates key cytoskeletal and signalling molecules such as FAK, Src kinases, Arp2/3 and vinculin to reconstruct the actin cytoskeletal system, which mediates increased force transduction through integrin. Furthermore, amplification of force transduction induces activation of RhoA GEFs such as, LARG and GEF-H1144 and eventual myosin-II activation, which ultimately leads to cell stiffening.13,17 As a concluding remark, RhoA pathways are critical for matrix rigidity sensing and the cell can regulate the dynamics of this signalling in order to respond to ECM stiffness and to maintain their own geometry for differentiation and proliferation. Talin-Integrin centred focal adhesion complex: Focal adhesions are large and dynamic multiprotein complexes that bridge extracellular matrix to the actin cytoskeleton via integrin transmembrane receptors. These complexes act like a mechanosensitive signalling hub, which control its own dynamics in a force dependent manner. Dynamic remodelling of


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Biochemistry

these adhesion sites is an important influencer of cell migration and spreading. During migration, integrins are activated to form small clusters near the cellular protrusions. These nascent complexes either get disassembled or integrated into more pronounced structures by the convergence of protrusive F-actin scaffold accompanied by PIP lipids which recruit and activate talin, vinculin, FAK, kindlin and ILK to form focal adhesion complexes.15,145 These focal complexes mature further to form larger adhesive structures, such as, focal adhesions (FAs) or podosomes in contractile and activated cells respectively. In rapidly migrating cells, the focal adhesion complexes act as mechanical linkages. The dynamic association between the ECM bound integrin receptors and force generating actomyosin cytoskeleton is recognized as the “molecular clutch”. This interaction between integrin and actomyosin complex is mechanically coupled by talin with assistance from vinculin, kindlin, actinin; together these constitutes the “molecular clutch”. These clutch molecules individually manipulate the extent of actin retrograde motion, generated due to actin polymerization and actomyosin contraction by myosin II, which pulls the adhesion complex (Fig 5).17,146 Each interaction in different clutch components works in a regulatory manner to integrate the mechanical response of the entire molecular clutch. Eventually, different components of the molecular clutch make up a 3D-heterogenous focal adhesion complex.

Figure 5: Model of talin centred “molecular clutch” as part of a mature focal adhesion: In focal adhesion, integrin-based adhesions are strengthened by the accumulation and binding of adaptor proteins like talin, vinculin, kindlin and scaffolding F-actin. Membrane lipid PIP2 are also engaged to activate integrin. Integrin β subunit linked talin undergoes mechanical stretching which further recruits vinculin. The binding of vinculin in turn, induces actin binding to ABS2. Although ABS3 acts as initial


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actin binding domain for force transmission, ABS2 enhances the force transmission and strengthen the adhesions by increasing the force redistribution. Vinculin binds to paxillin which in turn interacts with kindlin. Alternatively, kindlin interacts with integrin β subunit. Talin-integrin driven “Molecular clutch” plays a critical role in controlling force transmission through focal adhesion. The dynamic clutches control the adhesions turnover through sensing the actomyosin dependent contraction and cellular traction force.16,17,146 The focal adhesions effectively respond to a regime of forces applied by ECMs of different rigidities. Force transduction through these dynamic entities is dependent on the mechanical response of talin as its centrepiece. On a stiff matrix, the rapid loading of mechanical tension via molecular clutches enhances talin unfolding, allowing vinculin to bind. This interaction along with the actin cytoskeleton triggers focal adhesion growth by elevated force transmission through the clutch. The tension induced reinforcement mechanism of integrin-ligand ‘catch-bond’ formation assisted chiefly by talin and vinculin, occurs on a timescale of seconds or longer. On soft matrix, the slow force loading rates (and thus low force transmission) are not enough to boost up the force transmission by talin centered clutch reinforcement16,146 (Fig 6). Thus, duration and magnitude of force determines structural transitions and ligand profiles of talin, giving an insight into its spatio-temporal outlook. The matrix sensing and force transmission across talin additionally controls signalling circuits, which accordingly reflect the biochemical outputs of cells. Though focal adhesion mediated mechanotransduction is majorly regulated by the talin and integrin together, adhesions are differently decorated due to positioning of other proteins- vinculin, paxillin, FAK, Src kinase. Vertical layering of vinculin during force transmission is not only controlled by talin but also by an interaction with paxillin.147,148 Furthermore, the mature adhesion sites congregated on fibronectin exhibit horizontal positioning with β3 integrin in adhesion centre and KANK at the focal adhesion belt.17 Force transmission through integrin induces FAK and Src activation. This helps in cytoskeletal rearrangements and concurrent growth of adhesion site, leading to cellular stiffness through LARG and GEF-H1. Hence, the mechanosensitivity of the whole cell depends on integrated circuits constructed by these molecules with synchronization between a particular time and space.


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Biochemistry

Figure 6: Mechanotransmission through dynamic molecular clutch: The molecular clutch shows mechanosensitivity through its force sensing capabilities. (a) On soft matrix, slow force loading rate cannot induce talin unfolding and subsequent vinculin binding. This leads to slip bond (bond dissociation) formation in integrin-ligand interaction under low tension. The bond disruption ultimately diminishes the force transmission and resultant disassembly of adhesion sites. (b) On stiff matrix, high force loading rate is transmitted through clutches faster than integrin-ECM ligand interaction lifetime, which leads to talin unfolding and vinculin binding. This results in talin centred adhesion reinforcement and “catch bond” formation between integrin and its ligands. Finally, this leads to high force transmission through the adhesion system.16,17,117,146 Diseases linked to mechanointeractome: Recent research in mechanobiology shows that a diverse group of diseases are linked with abnormal mechanotransduction. Glaucoma, a leading case of irreversible blindness, is caused by trabecular meshwork mechanics149. Similarly, cardiomyopathy150,151, immune disorder152–154, cancer155–160, Williams syndrome161, autism162 and schizophrenia163 are also directly linked with mechanotransduction. The diversity of systems affected by this phenomenon range from a muscle to even a single hair cell.2 Cell-ECM interaction, a key regulator in mechanotransduction, is majorly controlled by the talinintegrin MSH. Integrin related mechanotransduction is disrupted in metastasis and aging.17 Removal of α8β1 integrin hampers hair cell differentiation and stereocillia maturation.164 The indirect role of


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integrin is evidenced in the pathology of rheumatoid arthritis.165 Integrin α9β1 interacts with tenascinC and osteopontin, which in turn induce the expression of matrix metalloproteases such as MMP1, MMP3, MMP13 and IL-6 cytokines. These in-turn contribute to the pathogenesis of rheumatoid arthritis.165 Mutations of integrin types are found to be linked with several diseases which are listed below.

Table3: Diseases with their characteristics, involved proteins and their causes Disease Renal fibrosis166

Disease character

Proteins Causes involved Progressive detrimental connective tissue α3β1 integrin Mutation in α3 integrin deposition on kidney parenchyma

Focal segmental Scarring of kidney glomerulus, failing to α6β4 integrin Mutation in β4 integrin glomerulosclerosis16 filter and cleanse blood 7

Prostate Cancer168

Myelodysplastic syndromes11 Angiogenesis169

Ulcerative colitis170

Glanzmann’s thrombasthenia171

Cancer in the prostate gland leading to abnormal reproductive and excretory behaviour Blood cells in bone marrow remain immature and do not function properly

α3 and α6 Laminin receptor α3 and integrin α6 are not expressed on subunit cell surface. Talin Under expression of talin

Formation of new blood vessels, helps in cancer progressions. Cancer cells fulfil their requirement of oxygen and nutrients by this process Innermost lining of intestine or colon is inflamed and becomes ulcerous, facilitates the chance of colon cancer

αVβ5 and Genetic aberration in αVβ3 αVβ5 and αVβ3 integrins integrin α4β7 integrin and β1 integrin subunit

Platelets become defective and coagulation αIIbβ3 is impaired integrin

Leukocyte adhesion Neutrophil dysfunctions; characterized by αLβ2 deficiency (LAD)18 malfunctioned immune system integrin

Psoriasis172 Melanoma metastasis173

Skin inflammation and red patches on skin.

αLβ2 integrin Cancer develops in melanin producing skin αVβ3 cells and spreads through lymph nodes to integrin distant sites in the body. Mainly occurs in areas, which are directly exposed to sunlight


Recruitment of leukocytes in gut, binding of α4β7 integrin to overexpressed MdCAM1 Fibrinogen binding to platelet specific αIIbβ3 integrin causes platelet activity. Deficiencies in αIIbβ3 leads to platelet abnormalities. Defect in β2 integrin.

activation and migration of T cells by αLβ2 integrin αVβ3 integrin induce the formation of activated MMP2 and protease processing. This helps in

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Biochemistry

cell migration and eventually metastasis Glioblastoma Multiforme 174,175

Osteoporosis176

Dilated cardiomyopathy177

Atherosclerosis178

Tumor initiates in star shaped glial cells (astrocytes) and rapidly spreads in the brain. Symptoms are vomiting, blurred vision, headache Bones become weak and brittle. Bone mass is reduced

Talin, αVβ3, Overexpression of αVβ3, αVβ5 αVβ5 integrins integrin Osteoclast α1β1 and αVβ3 integrin

Polymorphisms in osteoclast integrins leading to abnormalities in binding with collagen

Left ventricle of heart becomes dilated, β1 integrin weakens and is unable to pump blood subunit, properly. Talin1 and Talin2 The artery walls become narrowed due to Talin1. deposition of fats and cholesterol which Talin2, metarestricts the blood flow. vinculin and vinculin

Loss of talin1 and talin2 leading to instability of β1 integrin in cardiomyocyte Down-regulation of talin leading to loosening of cell-ECM interaction.

Talin and integrin have composite roles in several diseases, where both of them are responsible for the abnormal phenotype. In myelodysplastic syndromes, lower levels of talin1 contribute to diminish αIIbβ3 integrin signalling, which ultimately leads to haemorrhagic diathesis.179 Similarly, in dilated cardiomyopathy, loss of all talin isoforms leads to a complete reduction of β1 subunit of integrin, which causes myocardial fibrosis.180 The loss of talin or its knockdown is prominent in the pathology of leukocyte adhesion deficiency (LAD) where the resultant abrogation of talin, kindlin and integrin makes the platelets defective11,181 and the leukocytes are unable to attach with the endothelium of the inflammation site. Further they are unable to interact with antigen presenting cells, thus creating a suppressed immune response. Notably, the indirect mechanosensitivity of talin is also evident in platelet function. Talin knocked out (Talin-/-) platelets show defective fibrin clot retraction even in presence of the integrin activating Mn2+ ion. However, clot retraction in platelets containing mutant talin (L325R), in which talin holds all of its binding partners but is only defective in activating integrin, can be rescued by exogenous Mn2+ ion. Furthermore, this rescue can again be blocked by treatment with CytochalasinD, indicating a dual aspect of talin mechanosensitivity required for fibrin clot retraction by platelets.182 Importance of integrin and talin in cell migration, proliferation and invasion, nominates them as major mechanosensing proteins in cancer biology. The Cancer Genome Atlas (TCGA) project reveals that talin and integrin are mutated or amplified up to 20% and 60% in all tumor types respectively, highlighting their relevance.183 Talin levels are substantially upregulated in metastatic prostate cancer than its primary tumor, suggesting a possible role in cell migration and invasion. Furthermore, talin is crucial for tensional homeostasis in deadly brain tumor glioblastoma multiforme (GBM), where suppression of the protein results in loss of stiffness sensing ability. Another role of talin in cancer is maturation and stabilisation of invadopodia. It is required for maturation of invadopodia and concurrent


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recruitment of sodium/hydrogen exchanger 1 (NHE-1) to the invadopodia.184 NHE-1 acidifies periinvadopodia microenvironment, activating proteases and subsequent degradation of the ECM for cell migration.185–187 In oral squamous cell carcinoma, talin overexpression has also been shown to correlate with invasiveness.188 Deletion of talin in prostate cancer cells leads to decreased metastatic ability which makes them a remarkable supporter of anoikis (programmed cell death when cells get separated from their ECM) resistance. This makes talin a therapeutic target for anoikis resistance in the context of tumor microenvironment.189 However, upregulation of talin1 promotes anoikis resistance through FAK/AKT, GSK3β signalling.190 Thus, the role of talin in cancer progression is not only limited to its own influence on focal adhesion formation but also depends on its downstream signalling cascade. In spite of its role as master regulator in metastasis, the mechanical property of talin in tumorigenesis is not properly elucidated and is still highly debated. The altered expression of talin partners which mediate its recruitment to integrin tail could eventually result in cancerous phenotypes. Along with talin, integrin recruits various scaffolding and signalling molecules to activate different biochemical pathways involved in cancer progression. Depending on the composition of surrounding microenvironment, integrin has a dual nature- either to induce cell death (integrin mediated death, IMD) or cell survival.191 Recent studies suggest that although integrins are overexpressed in tumours, integrins α5β1 block oncogenic transformation.192 Deletion of β1 integrin can enhance tumorigenesis or cell proliferation in prostate cancer. In contrast, the conditional deletion of the same β isoform in mouse mammary tumor results in a substantial 30-days delay in tumor induction in mammary epithelial cells, indicating a role in depletion of metastatic potential.193 Integrin α6β4 is overexpressed in basal-like breast cancers and this correlates with its invasiveness. A mutual crosstalk between laminin receptor α6β4 and p-cadherin indicates the intricate role of integrin signalling pathway during invasion of breast cancer cells.194 Integrin α11β1, a stromal cell-specific receptor for fibrillar collagens is overexpressed in carcinoma-associated fibroblasts (CAFs). They induce collagen reorganization thus regulating stromal stiffness. Additionally, α11β1 activates crucial signalling pathways for tumor growth in nonsmall cell lung carcinoma (NSCLC) to enhance tumorigenicity.195 Furthermore, Integrin subtype α1β1 plays a critical role in colorectal cancer, where cell invasion is regulated by integrin association with talin and paxillin.196 β1 integrin present in the filopodial shaft is responsible for activating quiescent tumor leading to micro-metastasis at distant sites.197,198 Tumor progressions are typically correlated with increasing tissue stiffness due to a desmoplastic reaction: an event of excessive collagen deposition. Integrin dependent collagen depositions promote tumor cell proliferation and chemoresistance.191 A common example is PDAC, one of the most dangerous of its types because of its insensitivity to chemotherapy and immunotherapy. This occurs due to a thick desmoplastic stroma throughout the tumor that maintains an immunosuppressive tumor microenvironment and impedes drug delivery.199–201 In breast myoepithelial cells, differential bond dynamics of αvβ6 and α5β1 integrins have been involved in rigidity sensing of both malignant and healthy tissues respectively.202 In glioblastoma cancer stem cells (CSCs), α6 integrins are only coexpressed with integrin β1. Repression of α6 diminishes the CSC numbers, suggesting a role of integrin in CSC maintenance in primary tumor.203 Transcriptional upregulation of β1-integrin by ZNF304 (a zinc-finger transcription factor) followed by activation of a Src–FAK–paxillin, promotes anoikis resistance in ovarian cancer. Subsequent silencing of ZNF304 gene inhibits tumor growth in mouse.204 Significant upregulation of talin and downregulation of integrin is common in malignant prostate epithelial cells due to the hypoxic condition (1% O2) of tumor microenvironment, which leads to tumor metastasis and progression. Exposure of prostate tumor epithelial cells to hypoxia reveals a pronounced talin overexpression. This largely affects the integrin signalling network and subsequent recruitment of focal adhesion proteins thereby inducing adaptation of metastatic cells under hypoxia.205 Gene Ontology (GO) profiling predicts upregulation of talin1 is correlated with cellular mechanisms such as ion transport and membrane depolarization in hepatocellular carcinoma (HCC) progression.206 However,


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Biochemistry

another study indicates the plausible role of both talin1 and talin2 in HCC tumorigenesis.207 Core 1 β1,3-galactosyltransferase (C1GALT1) is recurrently upregulated in HCC and subsequently modifies O-glycans on integrin β1 thus regulating its activity. This predicts a role of C1GALT1 in the promotion of HCC invasion through integrin β1.208 During HCC, cell migration and invasion is reduced via FAK/ Src-Rho GTPase signalling with regulation from integrin α9 as a tumor suppressor.209 In prostate cancer, talin phosphorylation at S425 activates β1-integrin which is effectively inhibited using nonphosphorylated mutant talin1 or silenced p53-activated Cdk5, as it is responsible for that modification. Usage of this mutant or knockdown of talin, prevents its colonization within bone.210 Without this modification, talin remains in its autoinhibited form which cannot bind to integrin and fails to activate downstream signalling cascade for cell survival.33,210 Jointly both of these proteins in a mutual and composite fashion, could be a significant therapeutic target. Future directions: Studies in the recent past have inferred the biochemical and biophysical aspects of how the force sensing capabilities of talin and integrin, being a part of focal adhesion, play an important role in cellular functions such as cell adhesion and migration.26,69 Although these studies speculated that mechanical force transmitted through talin and integrin is one of the decisive factors for mechanotransduction,11,13,69 our current knowledge is still not enough to elucidate how mechanical force finely orchestrates the overall interactome which reflects the cellular physiology. To add another level of complexity, binding to several cytoplasmic proteins (such as, KANK2) diminishes the force transmission through the integrin-actomyosin linkage.54 Force plays a critical role in mechanotransmission and mechanosensing during cell adhesion, migration26 and invasion in the pathological state, indicating that a better understanding of force mediated adhesion formation and turnover is required. Furthermore, how the integrin-talin ‘force cushion’ directs focal adhesion architecture must be deciphered. Talin and integrin are both complementary and indispensable to one another,12,13 thus it would be better to characterize them as the ‘integrin-talin’ complex, rather than just individual proteins. How this complex induces force redistribution along with remodelling its interactome during adhesion dynamics, is a question that should be answered with more clarification. This revelation will put the spotlight on how the mechanosensitive signalling hub transduces and distributes force along with regulating cell movement. In force directed temporal regulation, if talin subdomains remain unfolded due to absence of immediate ligand interactions, the subdomains become completely stretched into random coils even in low forces and impede further ligand binding. This issue creates a ‘dual aspect’ of force transduction, which may be responsible for adhesion disassembly under high tension. Furthermore, disordered motifs can disclose a new interactome profile of talin because ligands interact to distorted linear polypeptides too.69 When undergoing mechanical stretching, talin regulates its ligand profiles and a few of these ligands bind to unfolded subdomains of talin. How they stabilize those unfolded subdomains has not been properly elucidated yet. Also, talin may experience different magnitudes of force depending on their cellular localization that might have an impact on migration and invasion in pathological conditions. Despite of a growing body of knowledge about the in vivo importance of mechanotransduction and how its dysregulation leads to altered organism physiology at the tissue level during diseased condition still remains unexplored. Recently it has been shown that focal adhesion can be stabilized even in the presence of mutant or modified talin rod subdomains.81 This calls for further debate on which is more critical for adhesion formation and its outlook- mechanotransduction or force transmission. The FA outlook depends not only on the ‘talin-integrin’ hub, but also on other FA components which change at different time scales under different force magnitudes. The driving nature of force mediated molecular mechanisms


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underlying focal adhesion formation is not known so far. The simultaneous force sensing capabilities with uninterrupted interactome dynamics, is very challenging to convey due to technical limitations. To decode these difficulties, a cutting-edge duplex mechanistic approach is needed, which can monitor binding of ligands to stretched proteins. A combination of fluorescence along with force spectroscopy could be useful, where talin-integrin complex will be stretched under force and interactome partner binding will be monitored through changes in fluorescence intensity. Conclusion: Integrin and talin, by virtue of being mechanosensitive proteins form a crucial force cascade through which mechanotransduction is regulated; while talin acts as a force buffer and mechanochemical regulator, integrins are key in forming adhesion sites and in transmitting signals, into and out of the cell. A key aspect of this process is the clustering of several proteins which leads up to the formation of focal adhesions. This transduces the force through the molecular clutch and influences important processes like cell migration and adhesion. Because of this system’s key role in many cellular processes and pathways, it is not surprising that they play larger roles in cellular pathologies. Indeed, the recent TCGA report remarkably highlights the mammoth role of both these proteins in cancer: Talin is found to be mutated or amplified in up to 20% and integrins in up to 60% of all tumor types.183 A plausible conclusion to consider at the end of this review is to draw an increased attention towards the role of the integrin-talin complex as a therapeutic target for cancer and other cellular abnormalities. Acknowledgement: We thank Ashoka University for support and funding. S.H thanks DBT Ramalingaswami fellowship for funding. We acknowledge Mr. Vaibhav Wagh for Figure 2.

Author Contribution: S.C., S.B., contributed equally to the work. Conflict of Interest: The authors declare no conflict of interest.


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Biochemistry

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Biochemistry

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Force Directed 'Mechanointeractome' of Talin-Integrin

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