Serial Number Freehand 951
Trolled for some more serial numbers. They are in blocks, that's for sure. Here are those seen so far:00920013xx01842 - Israeli Star of David and Y Stamp02400 - Israeli Marked (Israeli?
Serial Number Freehand 951 Free
No marks) - Markings on Right of Slide 'P.B. 104 Made in Italy'38177 - Markings on Right of slide 'P.B. 104 Made in Italy'C1352C3124D005xxEC0365 - Early Egyptian EagleEC04xxEC0458 - Early Egyptian EagleEC362X - Later Egyptian CrestV01140.
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The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs. Cell motility is central to the development and homeostasis of multicellular organisms, and defining the mechanisms involved will inform strategies to modulate aberrant cell migration and promote tissue regeneration.
Cell migration involves the cyclical attachment and detachment of the integrin family of adhesion molecules to extracellular matrix (ECM), as well as the generation of force required to translocate the cell body. Such events occur in focal adhesions (FA), dynamic macromolecular complexes in which integrins are linked via cytoplasmic adhesion plaque proteins to the actomyosin contractile machinery. Two plaque proteins that are key to this link are the interacting actin-binding proteins talin and vinculin. Cells depleted of talin cannot maintain cell spreading, while cells without vinculin have smaller more dynamic FAs, and are compromised in coupling plaque proteins to F-actin.Structurally, talin consists of an atypical N-terminal FERM-domain (talin head) that binds integrins, PIP2 and F-actin (actin-binding site 1; ABS1) linked to a C-terminal flexible rod consisting of 13 alpha-helical bundles (R1-R13) terminating in a dimerization motif.
The rod contains binding sites for the Rap1-interactor RIAM, vinculin and integrins plus two additional regions that bind F-actin (ABS2 and ABS3). Binding of the talin head to integrins regulates their affinity for ECM, while talin binding to actin is thought to form the primary link to the force-transmitting machinery. Vinculin, which binds talin via its globular N-terminal head and F-actin via its C-terminal tail, acts as a molecular clutch, reinforcing the link between talin and actomyosin,. Moreover, vinculin binding to talin maintains integrins in an active conformation, stabilizing the entire FA structure containing a large number of signalling components. However, expression of constitutively active vinculin compromises cell polarity and directional cell migration. Thus, co-ordinated cell migration requires that the activity of talin and vinculin are precisely controlled, but the mechanisms that regulate assembly of the talin/vinculin complex and its interaction with the cellular force machinery remain to be explored.
( a) Cartoon of talin constructs expressed as N-terminally tagged GFP-fusion proteins. The talin FERM domain is linked to the flexible talin rod which consists of 13 helical bundles (R1-R13) terminating in a dimerization helix (DD). Constructs in which the R4-R10 (talΔR4-R10) and R1-R10 (talΔR1-R10) domains have been deleted are shown.
Colours indicate binding sites for actin (orange) and vinculin (red). ( b) Talin1 and talin2 knockout (TKO) cells cells transfected with talinFL, 24 h after plating on fibronectin; arrows indicate non-transfected cells, which do not spread. ( c) TKO cells expressing indicated GFP-talin fusion constructs were plated on fibronectin and stained for F-actin. Magnified regions are from the area framed in red. Note the colocalisation of talΔR1-R10 or talΔR4-R10 with F-actin at the cell edge. Scale bars, 10 μm. ( d) Quantification of FA size in TKO cells expressing indicated constructs.
Note that talΔR1-R10 or talΔR4-R10 have smaller FAs compared with cells expressing talinFL ( n70 cells, from three independent experiments, error bars are ±s.e.m.,.= P65 cells, from three independent experiments, error bars are±s.e.m.,.= P. The activity of talin and vinculin is regulated by conformational changes at several levels.
In both cases, the N-terminal head domains interact with the C-terminal regions of the proteins resulting in an autoinhibited state. Despite evidence that activation involves either chemical signals (binding to activating proteins or lipids) and/or physical force, the detailed mechanisms underlying activation have not been elucidated. In addition, structural and biochemical studies on talin show that the vinculin binding sites (VBSs) within the helical bundles that make up the talin rod are cryptic, and single molecule experiments, indicate that force-induced unfolding of the bundles is required to unmask the VBSs.
Thus, it is hypothesised that force-induced conformational changes in talin lead to the recruitment of vinculin and the stabilization of FAs. However, to what extent such mechanisms operate in cells is unclear.Structure–function studies on talin have been hampered by the fact that (i) most cell types express two structurally and functionally related talin isoforms, (ii) knockout or knockdown of talin1 leads to upregulation of talin2 and (iii) knockdown leaves residual talin, complicating the interpretation of results.Here, we use newly derived talin1 and talin2 knockout (TKO) cells that grow in suspension, and only adhere to ECM, spread and assemble FAs following expression of functional talin constructs. By reconstituting TKO cells with structure-based talin point and deletion mutants, we define the role of specific talin and vinculin domains in engaging the actomyosin machinery, the assembly and stability of FAs, and the establishment of cell polarity.
We demonstrate that vinculin binding within domains R2R3 acts to ‘unlock’ ABS2 of talin, and that this is regulated either by prior activation of vinculin, or by the application of force across talin provided by actin-binding to ABS3. These findings demonstrate how talin, vinculin and actin interact with one another to form the major mechanosensory module of the FA. Talin regulates FA size and cell polarityTo investigate how the 11 potential VBSs in the talin rod contribute to FA formation, we deleted rod domains containing either 4 (talΔR4-R10) or 9 (talΔR1-R10) of the VBSs (; ). These constructs were tagged with GFP and expressed in TKO cells, which lack endogenous talin and do not adhere to ECM and do not spread (; ). Expression of GFP-tagged full-length talin (talinFL) rescued integrin activation , cell adhesion, spreading and FA formation , and cells displayed both small peripheral adhesions and mature FAs connected to prominent actin stress fibres. In contrast, although the talin rod deletion mutants rescued integrin activation and spreading, the cells lacked large FAs (; ).
TalΔR1-R10 cells displayed mostly small peripheral adhesions, and while talΔR4-R10 cells assembled slightly larger structures , both generally lacked actin stress fibres, although adhesions were connected to thin actin filaments (; ). Moreover, cells expressing these mutants were largely unpolarized , and cell motility was reduced compared with talinFL cells ; cell polarization is a prerequisite for directional migration.Vinculin, which binds talin and actin, is important for full engagement of adhesion complexes with the force machinery. Thus, the small peripheral adhesions exhibited by talΔR1-R10 and talΔR4-R10 cells might result from reduced vinculin-binding. Indeed, we observed a twofold reduction in the vinculin/talin ratio in talΔR4-R10 cells, and a fivefold reduction in talΔR1-R10 cells compared with talinFL cells. We conclude that both talin R1-R3 and R4-R10 contain functional VBSs that recruit vinculin to FAs.
The presence of vinculin in talin ΔR1-R10 adhesions likely indicates that the two VBSs in R11-R13 are also functional, as proposed by others. Talin domains R1-R3 contain key vinculin binding sitesInhibition of actomyosin-mediated tension with Rho Kinase (ROCK) or myosin inhibitors normally results in FA disassembly. By binding to talin, constitutively active full-length vinculin (vinT12) stabilises FAs which become insensitive to such drugs. To investigate which talin rod domains are key to this effect, talinFL or the talin deletion mutants were co-expressed with either full-length vinculin (vinFL) or vinT12, and the cells treated with Y-27632.
As the talin deletion mutants do not support FA maturation in TKO cells, we used NIH3T3 cells for these experiments (endogenous talin supports FA formation which become GFP-talin positive; ). As expected, Y-27632 reduced the size of GFP-talinFL positive FAs while vinT12 stabilized FAs. The intensity of GFP-talinFL in FAs also decreased more than in cells co-expressing vinT12. Interestingly, while vinT12 also stabilized talΔR4-R10 in FAs to a significant degree, it had no effect on talΔR1-R10 ( and ), suggesting that binding of vinculin to the R1-R3 region of the talin rod is particularly important for adhesion stability. ( a) NIH3T3 cells expressing talinFL, talΔR1-R10 or talΔR4-R10 with vinFL (control) or vinT12 were treated with DMEM containing 50 μM Y-27632 or an equivalent volume of water for 30 min before fixation. The effect on adhesions was analysed assessing the signal from the expressed GFP-talin fusion constructs.
FAs in control cells expressing talinFL are dramatically reduced in size following treatment with Y-27632. Note that talinFL and talΔR4-R10, but not talΔR1-R10, in FAs are stabilised by co-expression of vinT12 ( n=15 cells, representative of three independent experiments, error bars are±s.e.m.,.= P. Vinculin-induced FA stabilization goes alongside reduced talin turnover. To investigate the turnover of talinFL and the deletion mutants, we used talin constructs tagged with photoactivatable-GFP (PAGFP) and assessed fluorescence loss after photoactivation (FLAP; for details see and ). The results showed that the rates of turnover of talΔR4-R10 and talΔR1-R10 were significantly faster than talinFL (;; ). Co-expressing vinT12 significantly decreased turnover of talinFL, and to some extent talΔR4-R10, but not the talΔR1-R10 mutant (; ). These results were confirmed using FRAP.
To establish that the inhibitory effect of vinT12 on talin dynamics was due to a direct interaction, we used a vinT12 A50I mutant with reduced talin binding. This construct had no effect on talinFL turnover as assessed by FLAP ( and ).
These results clearly indicate that binding of active vinculin to the R1-R3 region of the talin rod plays an important role in stabilizing FAs. ΔR2R3 suppresses FA dynamics independently of vinculinFour out of the five VBSs in talin R1-R3 are in R2R3. We therefore expected that TKO cells expressing a talΔR2R3 construct would have smaller and less stable FAs.
To our surprise, they were even larger than FAs in talinFL cells, although the cells were more rounded. All FAs in talΔR2R3 cells were linked to prominent actin stress fibres , and strikingly, the FAs remained stable when treated with Y-27632 ( and ). Comparable results were seen in NIH3T3 cells. Co-expression of vinT12 with talΔR2R3 caused no further increase in FA stability. Moreover, FLAP experiments showed that talΔR2R3 turnover was greatly diminished compared with talinFL ( and ).
As expected, FAs in talΔR2R3 cells had a significantly reduced vinculin to talin ratio compared with control cells ( and ). Furthermore, talΔR2R3 exhibited reduced turnover even when expressed in vinculin null cells. Clearly, the stabilizing effect induced by deleting talin R2R3 is vinculin independent. ( a) Cartoon of the talΔR2R3 construct. ( b, c) TKO cells expressing talΔR2R3 have more prominent actin stress fibres associated with larger FAs than those in cells expressing talinFL; cells expressing talΔR2R3 are more circular than talinFL cells ( n=70–90 cells, from three independent experiments, error bars are±s.e.m.,.= P70, from three independent experiments, error bars are±s.e.m.,.= P. A role for talin ABS2 in FA dynamicsOne possibility is that deletion of R2R3 may activate the adjacent actin-binding site (ABS2) originally mapped to residues 951–1327 (ref. ) (roughly equivalent to R4-R6).
However, R7R8 has also now been shown to bind F-actin. To map ABS2 more precisely, we expressed several new talin rod fragments designed according to domain boundaries. Talin R4-R8 co-sedimented with F-actin to about the same extent as ABS1 in the talin head and ABS3 in the C-terminal R13 rod domain.
Interestingly, R1-R3 and R9-R12 also co-sedimented with F-actin, though to a lesser degree, indicating that like filamin, talin interacts with actin at several sites distributed along the length of the molecule. ( a, b) Recombinant talin polypeptides were incubated with F-actin, the actin pelleted, and supernatants (S) and pellets (P) analysed by SDS–PAGE. ( a) Talin FERM domain F0-F3 (ABS1) followed by talin rod domains R1-R3, R4-R8 (ABS2), R9-R12 and R13-DD (ABS3). Asterisks show the domains containing the known ABSs. ( b) Actin co-sedimentation assays show that actin binding to ABS2 is reduced when either domain R3 or R9 is present; numbers show percent band density ( c) Actin co-sedimentation studies using the 4 sub-domains that make up ABS2; Note that R4 and R7-R8 bind actin.
Serial Number Freehand 951 3
( d) Scheme outlining point mutations in the talin rod that reduce actin binding to ABS2. ( e) Expression of talin ABS2 mutant (FL ABS2mut) in TKO cells rescued cell spreading, but resulted in rounder cells with smaller FAs compared with talinFL cells (see also; scale bar, 10 μm). ( f) FLAP experiments in TKO cells show that the turnover of talinFL ABS2mut is increased compared with talinFL (FL control). Note that co-expression with vinT12 reduced the turnover of talinFL ABS2mut to a lesser extent than talinFL. The almost linear decay of the talinFL in presence of vinT12 (FL VinT12) prevented appropriate fitting, hence accurate t½ FLAP could not be determined; N/D=not determined ( n=28–56 FAs, from three independent experiments, error bars are±s.e.m.,.= P. To explore the possibility that domains flanking ABS2 might influence its activity, we designed ABS2 constructs containing the adjacent R3 and R9 domains. Interestingly, inclusion of these domains either together (R3-R9) or individually (R4-R9 or R3-R8) actually reduced actin binding to ABS2.
These results indicate that domains flanking ABS2 suppress its activity, and suggest that conformational changes in these domains have the potential to activate ABS2.To identify the major actin binding determinants in R4-R8, we examined the ability of individual domains to bind F-actin. Both R4 and the R7R8 double domain bound F-actin (the latter is consistent with previous results ). In contrast, R5 and R6 bound only weakly if at all to F-actin, and previous studies have shown that R7 alone does not bind actin. The results indicate that R4 and R8 are the key determinants of ABS2, and perhaps bind cooperatively to F-actin. Both domains have anomalously high pI values (9.5 and 7.8, respectively) compared with an average pI of 5.4 for the talin rod, and will be positively charged at physiological pH as required for binding to the negatively charged surface of F-actin. High pI values are also observed for ABS1 and ABS3.Analysis of the distribution of conserved positively charged and hydrophobic amino acids in the R4 and R8 domain structures highlights a number of residues that may contribute to actin binding. By serially mutating these residues , a R4-R8 construct with reduced ability to bind F-actin ( ∼60% inhibition) was generated (B.Goult and M.Schwartz personal communication).
A GFP-tagged talinFL ABS2mut containing these mutations rescued cell spreading in TKO cells, but the FAs were small.