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.

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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 ( n>70 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 polarity To 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 sites Inhibition 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 vinculin Four 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 dynamics One 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. ( 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 smaller and the number of actin stress fibres markedly reduced compared with talinFL cells (; for quantification ). Furthermore, FLAP experiments showed that turnover of talinFL ABS2mut was significantly faster than talinFL and similar results were obtained by deleting the R8 domain ( and, and ).

Both talinFL and talinFL ABS2mut were stabilized by co-expression of vinT12; however, it had a less pronounced effect on talinFL ABS2mut. Thus, ABS2 is an important factor determining talin turnover in FAs. ABS2-actin and vinculin-actin regulate talin dynamics To test whether the stabilizing effects of talΔR2R3 are dependent on ABS2, we expressed a talΔR2R3 ABS2mut mutant in TKO cells. The construct rescued cell spreading, but the cells had much smaller FAs and lacked the prominent F-actin stress fibres seen with the talΔR2R3 construct (). Furthermore, using FLAP, we found that the talΔR2R3 ABS2mut had a significantly faster turnover than talΔR2R3 ( and ) demonstrating that the stabilizing effects of talΔR2R3 are indeed partly mediated through ABS2.

( a) The talin ABS2 point mutations were introduced into talΔR2R3 and this construct (talΔR2R3 ABS2mut) was expressed in TKO cells. Scale bar, 10 μm. TalΔR2R3 ABS2mut cells had a similar circularity to talΔR2R3 cells, but lacked prominent actin stress fibres and had smaller FAs than talΔR2R3 cells ( n>70 cells, from three independent experiments, error bars are±s.e.m., ***= P. An additional factor influencing FA dynamics, migration and traction force is the ability of vinculin to bind actin via its C-terminal tail. To establish whether this is also a factor regulating talinFL turnover, we used a vinT12 I997A mutant with reduced actin-binding and bundling activity.

The vinT12 I997A construct still produced a significant reduction in the turnover of talinFL, but the reduction was not as great as with vinT12 ( and ). Altogether, these experiments strongly suggest that actin binding to talin ABS2 contributes to FA stabilization, and that this is regulated by vinculin binding to R2R3. However, the ability of vinculin to bind directly to both talin and F-actin also plays a significant role. Talin-actin link is required for traction force generation The link between the adhesion plaque and actomyosin is essential to generate the forces required for cell migration. To investigate the role of talin in this process, we used TKO cells expressing various talin mutants, and traction force microscopy to examine force transmission to substrate.

The largest traction stresses detected were at the cell periphery, where the majority of adhesion complexes are localized (). Highest traction stresses were observed in talinFL and talΔR2R3 cells, while cells expressing talin ABS2 deletion or point mutants exhibited significantly reduced force transmission (). Quantification showed that cells expressing talΔR2R3 were able to exert similar forces on the ECM to cells expressing talinFL (). In contrast, cells expressing talΔR4-R10 or talΔR1-R10, which lack ABS2 and several VBSs, showed a 45 and 55% reduction respectively in the force transmitted to ECM (). Similarly, TKO cells expressing talinFL ABS2mut displayed a 50% weaker force exertion than cells expressing talinFL (). These data clearly demonstrate that ABS2 plays a key role in actomyosin-mediated force transmission.

( a) Representative images from traction force microscopy (TFM) experiments. Colour spectrum indicates stress magnitude (Pa), with areas of low traction in blue and high traction in red.

( b) Quantification of total force (nN) from TFM experiments shows that TKO cells expressing constructs lacking ABS2 (talΔR1-R10 and talΔR4-R10) exert less force than cells expressing talinFL ( n=26–46 cells, error bars are±s.e.m., *= P. Talin ABS3 is required for FA initiation and cell spreading While the above experiments demonstrate a key role for ABS2 in FA maturation and engagement with the actomyosin machinery, the role of ABS3 has remained unclear. One hypothesis, largely derived from in vitro experiments, is that ABS3 might support the initial force transduction that is required to unravel the R2R3 helical bundles, and promote vinculin binding. If this were the case, one would expect that abolishing actin binding to ABS3 would result in a failure to form stable adhesions.

To test this hypothesis, we used a talinFL KVK/DDD mutation that reduces actin binding to ABS3 by >70%, without affecting dimerization which is essential for actin binding. Interestingly, while the GFP-tagged talinFL KVK/DDD mutant rescued TKO cell spreading, ∼45% of cells lacked FAs (). Moreover, in the majority of cells, GFP-talinFL KVK/DDD showed a diffuse cytoplasmic distribution with no clear localization to actin filaments, unlike constructs with an intact ABS3. These experiments suggest that F-actin binding to ABS3 supports the force required for the initial events leading to FA formation. ( a) While expression of talinFL KVK/DDD in TKO cells rescues cell spreading,70 cells, ***= P70 cells, ***= P. However, about half of talinFL KVK/DDD cells contained some FAs ().

These cells exerted weaker traction forces, migrated slower and contained less vinculin in FAs compared with cells expressing talinFL (). However, the fact that they had clearly visible FAs suggested that FA initiation via ABS3 can be bypassed. We therefore tested whether activated vinculin, which in turn activates ABS2, and can itself bind directly to F-actin, might bypass ABS3. Indeed, we found that co-expression of vinT12 with the talinFL KVK/DDD mutant was able to fully rescue FA formation, unlike co-expression of vinT12 A50I (). Together, our data demonstrate that ABS3 plays a key role in the initiation of FA assembly, but that its role can be bypassed by pathways that activate vinculin and allow the association between vinculin and talin.

The development of FAs involves (i) formation of small, dot-like nascent adhesions independent of actomyosin-mediated tension and (ii) their tension-dependent maturation into FAs. Talin is seen early in adhesion complex assembly, binds and activates integrins, and regulates the recruitment of other proteins including vinculin, paxillin and FAK to FAs.

Using TKO cells, we demonstrate that talin is indeed essential for integrin activation, cell adhesion and spreading, and is at the core of adhesion complex assembly, the regulation of adhesion strength and engagement with actin. TKO cells expressing a talin ABS3 mutant (talinFL KVK/DDD) exhibit a marked reduction in assembly of adhesion clusters suggesting that in the absence of ABS3-mediated coupling to actomyosin, the force-dependent conformational changes in talin required to expose cryptic VBSs and to activate ABS2 are inoperative. As a result, adhesion complexes that do form are unstable. ABS3 may also be involved in the correct localization of talin, bringing it into position to bind and activate integrins and initiate adhesion complex assembly. Such a model is suggested by our finding that the talinFL KVK/DDD mutant showed little enrichment in actin-rich lamellipodia () compared with constructs with an intact ABS3 (). However, the role of talin ABS3 in adhesion assembly can be bypassed by constitutively active vinculin, suggesting that pathways that activate endogenous vinculin may also drive complex assembly. Consistent with this, the muscle-detachment phenotype caused by expressing a talin ABS3 mutant during Drosophila embryogenesis was partially rescued by vinculin.

To date, the role of ABS2 in the central part of the talin rod has been ignored. Here we establish that talin ABS2 (R4-R8) is essential for stabilization of FA complexes, and for the generation of maximal traction force ( and ). Our biochemical experiments indicate that domains flanking ABS2 suppress its activity (), suggesting that talin must undergo a conformational change that relieves this inhibitory effect. Since the talinFL KVK/DDD mutation inhibits FA assembly (), it seems likely that activation of ABS2 is initially regulated by engagement of ABS3 with actomyosin. However, the fact that the talinFL KVK/DDD phenotype is rescued by vinT12 suggests additional regulation of ABS2 by vinculin binding to R3 ().

Indeed, our experiments with talin rod deletion mutants show that the VBSs in the R1-R3 domains are particularly important for FA stabilization, and magnetic tweezer experiments demonstrate that (i) force unmasks the VBSs in R2R3 (ref. ) and (ii) that R3 is the initial mechanosensing domain unfolding at ∼5 pN (refs, ). This conclusion is consistent with structural data which shows that while R2 is stabilized by R1 (ref. ), R3 is the least stable of the three domains because of the presence of four threonine residues buried within its hydrophobic core. Based on these results, we postulate that the initial association between talin ABS3 and actin provides sufficient force to overcome the 5 pN threshold to unfold the talin R3 helical bundle. This allows vinculin to bind to R3, relieving the inhibitory effect of R3 on ABS2 which then binds F-actin, reinforcing the link with the actomyosin machinery, leading to adhesion maturation. When the initial force acting on talin ABS3 is insufficient to unfold the R3 domain, binding of vinculin is suppressed, ABS2 is not activated and the complex disassembles, as seen in short-lived nascent adhesions.

Ratiometric imaging with talin deletion mutants and vinculin ( and ) shows that the R2R3, R4-R10 and the C-terminal R11-R13 talin rod domains can all bind vinculin in vivo, confirming in vitro data of multiple VBSs along the talin rod. Data demonstrating that activated vinculin stabilizes talin mutants containing talin R2R3 and R4-R10 domains in FAs, and the FA structure itself () indicates that vinculin drives adhesion assembly through binding to VBSs in these domains. Vinculin-mediated adhesion stabilization depends in part on the actin binding activity of vinculin itself since maximal stability was not achieved using a constitutively active form of vinculin containing a mutation in its own actin binding site ().

These observations, together with the previously reported force mediated recruitment of vinculin to FAs,,, and the increased ratio of vinculin/talin during FA maturation, suggest that a gradual increase of force induces vinculin to bind to different sites in talin, thus strengthening the connectivity between the adhesion complex and the cytoskeleton. This additional mode of regulation through vinculin, whose own activity can be regulated by actomyosin-mediated forces, adds a regulatory module which appears key for the cell to respond to changes in the mechanical environment. We propose the following two-step model for talin activation and adhesion maturation which is key for co-ordinated force transduction and polarized cell migration (). Initially, talin, the bulk of which exists in an autoinhibited form in the cytosol,, is recruited to the plasma membrane, the actin-rich lamellipodium and adhesion sites through pathways involving actin binding to ABS3, or its interaction with RIAM, integrins, PIP 2, exosomes and FAK. Whether kindlin, which appears to precede talin in nascent adhesions, is involved in recruiting talin is unclear. At the leading edge, the talin head-rod auto-inhibitory interaction is disrupted possibly by PIP2 (ref. ), and talin synergises with kindlins to activate integrins leading to the formation of nascent adhesions.

Subsequently, actomyosin-mediated tension across talin, bound to integrins at one end and actin via ABS3 at the other, leads to unfolding of the R3 helical bundle relieving inhibition of ABS2 and exposing the two high affinity VBSs in R3, which can bind vinculin. Vinculin may prevent refolding of R3 locking ABS2 into an active state. The combined link of talin ABS2 and ABS3 to F-actin and the ability of vinculin to cross-link talin to actin stabilises talin in the adhesion complex. This hypothesis is supported by the recently published findings that talin engaged with F-actin at FAs is under tension, and is stretched and positioned by this F-actin linkage, thus orchestrating the molecular architecture of the FA. Further increases in myosin-II mediated force generation may subsequently activate additional VBSs localized along the talin rod leading to recruitment of more vinculin molecules, resulting in the maximal stabilization of the overall FA structure.

The fact that vinT12 can bypass the role of talin ABS3 in FA assembly, suggests that pathways that activate vinculin (for example, via PIP2 (refs, )) may also drive FA assembly. FAs then act as signalling hubs controlling many aspects of cell behaviour including polarity and migration.

( a) Talin in which the vinculin and actin binding sites in R2R3 and R4-R8 (ABS2), respectively are cryptic. Inactive vinculin in green.

( b, c) Two possible mechanisms for linking talin with the actomyosin machinery; ( b) force-dependent pathway: actin binding to ABS3 leads to force exertion across talin, stretching R2R3 and unmasking its previously cryptic vinculin binding sites. A combination of force and vinculin binding to R2R3 also relieves the inhibitory effect of R3 on ABS2, allowing actin binding. ( c) Vinculin driven pathway: binding of activated vinculin to talin R2R3 domains unlocks ABS2, allowing actin to bind to talin independent of ABS3. ( d) Full engagement with actin occurs through actin binding of ABS2 and ABS3, with vinculin stabilizing the link between talin and actin. Cell lines and transfections NIH3T3 fibroblasts and vinculin −/− mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS, 1% L-glutamine and 1% non-essential amino acids. For the generation of talin1 and talin2 knock out cells, cells were isolated from the collecting ducts of 5–6-weeks-old talin1 flox/flox mice/ talin2 null mice as described by Husted et al. And immortalized with pSV40 plasmid.

Loci for talin1 gene in collecting duct cells were deleted with adenovirus expressing Cre recombinase. Multiple clones of the double null cells were prepared by performing serial dilution cloning and verified similar functions in different clones. Gene deletion was verified by both PCR and immunoblotting for talin 1 and talin 2 (). Talin null cells were cultured in DMEM F-12 supplemented with 10% FCS, 1% L-glutamine, 15 μM HEPES and 1% Antimycotic solution (Sigma).

All cells were cultured at 37 °C with 5% CO 2. Transient transfections were performed using Lipofectamine and Plus reagents (Life Technologies) as per the manufacturer’s instructions. Antibodies, reagents and plasmids Bovine fibronectin was purchased from Sigma and used at 10 μg ml −1 diluted in PBS (Sigma). Y-27632 dihydrochloride (Tocris Bioscience) was dissolved in water and used at 50 μM for 30 min. Mouse anti-vinculin antibody (clone hVin1) (Sigma, UK) was diluted (1:500) in 1% Bovine Serum Albumin (BSA) (cat: V9131, Sigma, UK).

Dylight 594-conjugated AffiniPure Donkey Anti-Mouse IgG (cat: 715-585-150, Jackson ImmunoResearch, USA) was used as a secondary antibody, diluted in 1% BSA (1:500). Site-directed mutagenesis was performed using the QuikChange Lightning Multi Site-Directed Mutagenesis kit (Agilent, USA). For western blotting, the primary antibodies used were mouse anti-talin (8d4) (cat: T3287, Sigma) and mouse anti-GFP (cat: 11 814 460 001, Roche), diluted 1:500 and 1:250, respectively, in 2% milk (PBS 0.1% Tween). The secondary antibody was goat anti-mouse IgG conjugated to horseradish peroxidase (cat: A9917, Sigma), diluted 1:5,000 in 2% milk (PBS 0.1% Tween). Bands were detected using enhanced luminol-based chemiluminescent substrate (Promega). Western blots Cells were transfected with the desired talin constructs, incubated for 24 h before lysis with Laemmli buffer.

Samples were run on an SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel, 90 v, 15 min followed by 120 v, 1 h. Proteins were transferred to a nitrocellulose membrane at 250 mAmps, 2 h.

The membrane was blocked for 1 h with 5% milk (PBS 0.1% Tween) and probed with the indicated primary antibodies. Uncropped scans of the blots are shown in. Immunofluorescence and microscopy Cells transfected with GFP- and/or mCh-tagged proteins were incubated overnight on glass bottom dishes (MatTek), fixed with 4% paraformaldehyde and permeabilised with 0.5% Triton X-100 (Sigma). Samples were incubated with the primary antibody for 60 min, and then washed thrice with PBS.

Secondary antibody staining followed the same procedure. Fixed samples were imaged using a Delta Vision RT microscope (Applied Precision) equipped with a 60 × /1.42 Plan Apo oil immersion objective (Zeiss).

Images were acquired with a CoolSnap HQ camera (Photometrics). Image analysis and ratio imaging Image analysis was carried out using Fiji-ImageJ (version 1.48d) software. For all constructs, we analysed cells with low to intermediate expression levels of talin. Expression levels were determined by fluorescence intensities measured across a large number of cells/FAs in cells exposed to the same amounts of fluorescent light (exposure time below 500 ms).

Adhesion size measurements were performed using the particle analysis function of imageJ after careful background subtraction. For cell circularity measurements, a freehand region of interest was drawn and circularity was measured. Cells were classified as being either ‘circular’ or ‘elongated’ as has been described previously, where elongated cells have a circularity ⩾0.56 (ref. For ratio imaging experiments, all images were acquired using the same exposure times.

Ratio images were created using custom software; briefly, background noise was removed by thresholding, followed by division of the vinculin immunofluorescence image by the GFP-talin image. For ratio quantification, GFP-talin and vinculin immunofluorescence images were background subtracted, a region of interest was drawn around individual, peripheral adhesions (7 per cell) and the integrated density was measured for both channels. A ratio value was produced for each adhesion by dividing the values from vinculin images by talin images. Fluorescence loss after photoactivation (FLAP) Cells co-expressing the PAGFP-tagged protein of interest and the appropriate mCherry-tagged marker were plated onto fibronectin-coated glass bottom dishes. Both plasmids where expressed in a 1:1 ratio (cells were transfected with 1 μg of each) and only cells of healthy appearance with low to intermediate levels of the mCherry plasmid were selected for FLAP experiments.

One hour before imaging, DMEM was replaced with Ham’s F-12 medium supplemented with 25 mM HEPES buffer, 2% FCS, 1% penicillin/streptomycin and 1% L-glutamine. Cells were allowed to equilibrate in a pre-warmed environment at 37 °C with 5% CO 2. Experiments were performed on a spinning disk confocal microscope (CSU10, Tokogawa) supplied by Intelligent Imaging Innovations, Inc. (3i), equipped with a 60 × /1.42 Plan Apo oil immersion objective (Zeiss).

To photoactivate, a 405-nm laser was pulsed at full power for 5 ms at user-defined regions of interest. Fluorescence images were captured in 10 s intervals for a total period of 5 min using 488-nm and 561-nm laser lines used at full power with an appropriate exposure time. Approximately 4–5 FAs within a similar region of the cell were selected. To obtain intensity values over time, photoactivated fluorescence (PAF) images in the 488-nm channel were processed using a custom MATLAB (The Mathworks) script. Briefly, a user-defined threshold is used to detect FAs and create a mask.

The mean intensity within each mask is measured and recorded. A second user-defined region is used to obtain background values, which are subtracted from the adhesion intensity values.

Background-subtracted mean intensity values are then normalized to the first post-bleach intensities. Normalized intensity values were then fitted using Prism 6 (GraphPad Software) to single-phase exponential decay curves. The equation coefficients were extracted and transformed to generate F M and t 1/2 loss of PAF. Fluorescence recovery after photobleaching (FRAP) Cells were transfected with GFP-tagged FA proteins of interest and FRAP experiments were performed using a Delta Vision RT microscope equipped with a 100 × /1.40 UPlan Apo oil immersion objective (Zeiss). FRAP laser beam diameter size was 1 μm and was held for 0.075 s per region of interest to bleach.

Images were recorded every 10 s for 5 min following previously published methods. Normalized fluorescence intensities were obtained using Softworx FRAP analysis software. Data were imported into MATLAB and fitted with single exponential curve fits according to published FRAP models. A custom MATLAB script was used to extract the coefficients of the curve fits and the half-time ( t 1/2) of recovery and mobile fraction (MF) were calculated.

Traction force microscopy Fibronectin (FN) coated polyacrylamide (PAA) gels (6% of gel diluted from 30% Protogel in PBS, 37.5:1 fixed ratio of arylamide/bis-acrylamide; EC-890, National Diagnostics) containing 1:100 dilution of 0.2 μm FluoSpheres carboxylate-modified microspheres (F8805, blue fluorescent (365/415), Life Technologies) were prepared using previously published methods with modifications. The stiffness (8.760±0.209 kPa) of 6% PAA gels were measured with a CellHesion Atomic Force Microscope (Nanowizard, CellHesion 200; JPK Instruments, Berlin, Germany) with tip-less cantilevers (NP-O10, Bruker AFM Probes) modified by attaching 10 μm diameter polystyrene beads (PPS-10.0, Kisker). Transfected cells were plated on gels and allowed to spread overnight. Cell spreading deformed the surfaces of the gels thus changing the position of the embedded beads.

Fluorescence images of the embedded beads were captured on a Delta Vision microscope system (Applied Precision, Washington, USA) using an oil immersion × 60 (NA=1.42) objective. After taking bead images, 0.05% Triton X100 was added to detach the cells and thus release forces mediated.

Phosphatidylserine exposed on the surface of apoptotic mammalian cells is considered an “eat-me” signal that attracts phagocytes. The generality of using phosphatidylserine as a clearance signal for apoptotic cells in animals and the regulation of this event remain uncertain.

Using ectopically expressed mouse MFG-E8, a secreted phosphatidylserine-binding protein, we detected specific exposure of phosphatidylserine on the surface of apoptotic cells in Caenorhabditis elegans. Masking the surface phosphatidylserine inhibits apoptotic cell engulfment. CED-7, an ATP-binding cassette (ABC) transporter, is necessary for the efficient exposure of phosphatidylserine on apoptotic somatic cells, and for the recognition of these cells by phagocytic receptor CED-1. Alternatively, phosphatidylserine exposure on apoptotic germ cells is not CED-7 dependent, but instead requires phospho lipid scramblase PLSC-1, a homologue of mammalian phospholipid scramblases. Moreover, deleting plsc-1 results in the accumulation of apoptotic germ cells but not apoptotic somatic cells. These observations suggest that phosphatidylserine might be recognized by CED-1 and act as a conserved eat-me signal from nematodes to mammals. Furthermore, the two different biochemical activities used in somatic cells (ABC transporter) and germ cells (phospholipid scramblase) suggest an increased complexity in the regulation of phosphatidylserine presentation in response to apoptotic signals in different tissues and during different developmental stages.

INTRODUCTION In metazoans, cells undergoing apoptosis are recognized and internalized by living cells via phagocytosis, and they are degraded inside phagocytes. This process removes unwanted cells before they release potentially harmful contents, and it is important for immunological tolerance, suppression of inflammatory responses, and tissue remodeling ( ). Phagocytes use cell surface receptors to recognize surface features of apoptotic cells.

Several changes have been detected on the surface of mammalian cells undergoing apoptosis, including the exposure of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, and changes in cell surface carbohydrates and ionic charges ( ). PS is a component of cellular membranes, making up 2–10% of total phospholipids (for review, see ). In living cells, PS is almost exclusively localized to the inner leaflet of plasma membrane, at least partially due to an ATP-dependent aminophospholipid translocase activity that selectively returns PS and phosphatidylethanolamine (PE) from the outer to the inner leaflet (for review, see ). The exact identity of the protein(s) responsible for this activity in vivo remains to be revealed. During the early stage of apoptosis, PS is detected on the outer leaflet, as observed in mammals, Drosophila, Xenopus, and chick (; ), suggesting a process of transbilayer redistribution. Not much is known about the exact mechanism that triggers the exposure of PS in response to apoptotic stimuli in animals. Phospholipid scramblases (PLSCs), by catalyzing the random, bidirectional “flip-flop” of phospholipids across the membrane bilayer, could potentially counter the aminophospholipid translocase activity ( ).

Four homologous phospholipid scramblases have been identified in humans, however, the in vivo roles of these scramblases in the redistribution of PS have not been established ( ). In addition, ABCA1, the prototype of the A subclass of mammalian ATP-binding cassette (ABC) transporter 1, has been implicated in the translocation of PS from inner to the outer leaflet ( ). ABCA1 facilitates the engulfment of apoptotic cells ( ). The inactivation of aminophospholipid translocase and the activation of phospholipid scramblases or ABC transporters together might lead to the exposure of PS on the surface of apoptotic cells. However, it is unclear whether and how apoptotic cells of a particular cell type choose to activate either the ABC transporter or the phospholipid scramblase or both to promote PS exposure. PS on the surface of apoptotic mammalian cells could act as one of the “eat-me” signals and be recognized both directly by certain phagocytic receptors that have PS-binding activity, or indirectly by others that associate with “bridging” molecules that bind to PS (for review, see ). One such bridging molecule is mammalian milk fat globule- EGF-factor 8 (MFG-E8) ( ).

This glycoprotein, when secreted from macrophages, which are professional phagocytes, interacts with apoptotic cells via its high-affinity, Ca 2+-independent PS binding activity and with macrophages via its association with integrin α vβ 5, a prominent phagocytic receptor ( ). The apoptotic cell–MFG-E8–integrin α vβ 5 interaction results in the activation of Rac1 GTPase-mediated cytoskeleton reorganization and phagocytosis ( ). One hundred and thirty-one of the 1090 somatic cells developed in the nematode Caenorhabditis elegans hermaphrodite undergo apoptosis, among those 113 die during embryogenesis (; ).

In addition, >300 germ cells undergo apoptosis during germline development in adult hermaphrodite gonads ( ). In living animals, apoptotic cells, or “cell corpses,” can be distinguished from living cells by using Nomarski differential interference contrast (DIC) microscopy by their highly refractile, button-like appearance (; ). Elegans, cell corpses are swiftly engulfed and digested by their neighboring cells (for review, see ). Genetic screens have identified eight genes acting within two parallel and partially redundant pathways to regulate the engulfment of apoptotic cells, cell corpse-engulfment defective ( ced) -1, -6, -7, and dyn-1 in one pathway, and ced-2, -5, -10, and -12 in the other pathway (;; ). Ced-10 may also convey certain activities for the ced-1 pathway ( ). Mutations of each of these genes result in the accumulation of cell corpses in the body.

The protein products of ced-6 (PTB-containing adaptor), dyn-1 (dynamin), ced-2 (CrkII homologue), ced-5 (Dock180 homologue), ced-10 (Rac GTPase), and ced-12 (ELMO1 homologue), all act exclusively inside engulfing cells to control engulfment (;;;; ). CED-1, a single-pass transmembrane protein, in contrast, is localized to the surface of engulfing cells and acts as a phagocytic receptor to recognize cell corpses and initiate their engulfment ( ). The exact ligand(s) that CED-1 recognizes on the surface of cell corpses is not known; however, the function of CED-7, a homologue of mouse ABCA1, is essential for the recognition of cell corpses by CED-1 ( ). Among the eight known engulfment genes, ced-7 is the only gene that plays a role in dying cells ( ). It is hypothesized that CED-7 promotes the exposure of a particular signaling molecule to the surface of apoptotic cells to attract CED-1 ( ). In the past, it was unknown whether PS is exposed on the surface of apoptotic cells in C.

Elegans, and if so, whether PS plays any role in cell corpse engulfment. In addition, the identity of the eat-me signal presented by CED-7 remains elusive. In this study, we investigated these issues. Sadistic Intent Resurrection Of The Ancient Black Earth Rar more. We established that PS acts as an eat-me signal and a CED-1 ligand in C. Elegans, and we further identified the roles of two potential PS exposure activities during C. Elegans development.

Elegans Strains C. Elegans strains were grown at 20°C as described previously ( ). The N2 Bristol strain was used as the reference wild-type strain.

Mutations are described by, except where noted otherwise: LGI, ced-1(e1735), ced-12( n3261), plsc-2(tm820) (this study), plsc-3(ok1313) (this study); LGIII, ced-6( n2095), ced-7( n1996); LGIV, ced-5(n1812), ced-10( n1993); LGV, unc-76(e911), plsc-1(ok1178) (this study). Plsc-1(ok1178) and plsc-3(ok1313) were provided by the C. Elegans Gene Knockout Consortium in strains RB1149 and RB1249, respectively. Plsc-1(ok1178) was outcrossed twice with the wild-type strain. Plsc-2(tm820) was provided by Shohei Mitani (Tokyo Women's College, Tokyo, Japan). The strain UL809, which carries the reporter P plsc-1 NLS-gfp on an extrachromosomal array ( ), was provided by Ian Hope (University of Leeds, Leeds, United Kingdom). Transgenic lines were generated by microinjection ( ).

Plasmids were coinjected with p76-18B ( ) into unc-76(e911) mutants, and non-Unc progeny were identified as transgenic animals. The enIs7[P ced-1 ced-1::gfp] and enIs18[P lim-7 mfg-e8::gfp] transgenes were both integrated on chromosome X. Double mutants between plsc-1(ok1178) and ced-1(e1735) and between plsc-1(ok1178) and plsc-2(tm820) were generated by standard genetic crosses, and the presence of homozygous ok1178 and tm820 alleles was verified by polymerase chain reaction (PCR) (Supplemental Figure S2). Because plsc-2 and plsc-3 are both on chromosome I, instead of generating plsc-1(ok1178); plsc-2(tm820) plsc-3(ok1313) triple mutant strain, we chose to inactivate plsc-3 by using RNAi in the plsc-1(ok1178); plsc-2(tm820) double mutant strain. Plasmid Construction mfg-e8 cDNA was PCR amplified from a Mus musculus breast cDNA library by using primers designed based on M. Musculus mgf-e8 mRNA (GenBank accession no.

The splice variant that lacks the Pro/Thr-rich domain (amino acids 111–146) ( mfg-e8Δ P/T), also known as mfg-e8-S ( ), was first isolated. To construct a full-length mfg-e8 clone, the IMAGE clone 6333582 (GenBank accession no. ) was digested using StuI and BamHI, a 440-base pair (bp) fragment containing the 111-bp Pro/Thr domain was inserted to replace the StuI–BamHI fragment in the mfg-e8Δ P/T clone. To construct mfg-e8Δ C2, nucleotides 1–918 were PCR amplified from mfg-e8 cDNA. The three mfg-e8 isoforms were fused with green fluorescent protein (GFP) at their C termini and placed behind the following promoters: P ced-1, which is expressed in all types of engulfing cells for somatic and germ cell corpses ( ); P dyn-1, which is ubiquitously expressed in embryos ( ); and P lim-7, which is specifically expressed in gonadal sheath cells ( ). RNA Interference (RNAi) The RNAi feeding constructs for dyn-1, plsc-1, plsc-2, and plsc-3 were obtained from a C.

Elegans RNAi library ( ), and animals of particular genotypes were treated with RNAi as described previously ( ). To score germ cell corpses, mid-L4–stage animals were transferred to RNAi plates, and then they were transferred to a new set of RNAi plates after 24 h. The numbers of germ cell corpses per gonad arm were scored under DIC microscope 48 h later. The progeny of these adults as L1 larvae hatched within an hour were used to score the number of somatic cell corpses in the head. Irradiation and Time-Lapse Recording of Duration of Germ Cell Corpses Time-lapse analysis was performed on a DeltaVision system with a Olympus IX70 microscope (Applied Precision) by using the following protocol (Yu and Zhou, unpublished data). Adult hermaphrodites 24 h post-L4 stage were irradiated with a 137Cs source (Gammacell 1000, Atomic Energy of Canada Ltd., Kanata, ON, Canada; 8.33 Gy/min) at 180 Gy. Two hours after irradiation, samples were transferred onto NGM plates containing 1 mM Aldicarb; after 20 min, samples were mounted on a slide immersed in 0.5 mM Aldicarb solution, and then they were observed under 60× DIC optics at 20°C.

Thirty serial z-sections, at 1.0 μm/section, of the gonadal region were recorded every 3 min. Recording began at 3 h after irradiation, and the period of duration of cell corpses that show up during the first 2 h were scored, because in the germline of wild-type hermaphrodites, the number of cell corpses reaches its peak 4 h after irradiation ( ).

Animals were closely monitored for signs of viability during recording. Fluorescence Microscopy Animals were anesthetized in 30 mM sodium azide and placed on a glass slide on a 4% Agar pad. DIC and GFP images from serial z-sections were used to score the number of cell corpses and GFP-labeled cell corpses. Olympus IX70-Applied Precision DeltaVision microscope equipped with CoolSNAP digital camera (Photometrics, Tucson, AZ) and Softworx software (Applied Precision) was used to acquire serial Z-stacks of fluorescence images at 0.5-μm intervals and to deconvolve these images of embryos, newly hatched L1 larvae, and hermaphrodite gonads. Hermaphrodite gonads were sometimes analyzed using an Axioplan 2 compound microscope (Carl Zeiss, Thornwood, NY) with Nomarski DIC accessories, AxioCam camera, and AxioVision version 4.3 imaging software. ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify GFP signal intensity in the gonads.

The “Freehand” tool was used to trace GFP(+) circles around germ cell corpses and on the surface of adjacent living germ cells, the intensity of GFP signal was measured in the chosen areas, and relative ratio of GFP signal intensity was calculated. Phosphatidylserine Is Detected on the Surface of Apoptotic Germ Cells Mouse MFG-E8 has an N-terminal signal sequence but no putative membrane-spanning region, and it was reported to be a secreted protein (A) ( ). We generated a construct that allows the expression of a mfg-e8::gfp fusion cDNA (GFP was fused to the C terminus of MFG-E8) under the control of P lim-7, a promoter specifically active in gonadal sheath cells ( ) that wrap around the germline and are responsible for the engulfment and degradation of germ cell corpses that die in the adult hermaphrodite gonad ( ).

We anticipated that MFG-E8::GFP, once secreted from gonadal sheath cells, would label the surface of nearby germ cell corpses if PS was present on the surface of these cells (A). PS is specifically exposed on the surface of germ cell corpses. (A) a, domain structures of mouse MFG-E8 and two deletion constructs. SS, signal sequence; E1 and E2, epidermal growth factor (EGF) domains; C1 and C2, Factor VIII-homologous domains. In wild-type animals, MFG-E8::GFP was specifically detected on the surface of dying germ cells but not on living germ cells (B). In the gonad of ced-1 null mutant adult hermaphrodites, where a large number of unengulfed germ cell corpses reside, ∼65% of germ cell corpses identified by DIC microscopy were labeled with MFG- E8::GFP on the surface (, C and G). Similar to wild type, no GFP signal was detected on the surface of living germ cells (C).

Consistently, no MFG-E8::GFP circle was observed on the surface of any germ cells in ced-3(n717) mutant animals, where programmed cell death was abolished due to a loss-of-function mutation in the C. Elegans caspase CED-3 (G) ( ). We further observed a strong GFP signal in coelomocytes, scavenger cells that nonspecifically internalize fluid from the pseudocoelom (body cavity) via endocytosis and micropinocytosis ( ) (D). Because P lim-7 is not active in coelomocytes (Supplemental Figure S1), this observation suggests that MFG-E8::GFP is secreted from gonadal sheath cells as expected and subsequently internalized by coelomocytes from the extracellular space. Previous studies indicate that C1 and C2, the two factor-VIII homologous domains in MFG-E8 (A), are essential for the PS binding activity ( ). We found that a truncated form of MFG-E8 lacking the C2 domain (MFG-E8ΔC2::GFP) expressed under the control of P lim-7 failed to label germ cell corpses (E). Meanwhile, strong MFG-E8ΔC2::GFP signal was detected in coelomocytes near the gonad (E), indicating that the production and secretion of this truncated protein were normal.

In addition, MFG-E8ΔP/T::GFP, another truncated form lacking the proline/threonine-rich (P/T) domain (A) and reported to display decreased PS binding activity in vitro ( ), was detected on merely 20% of germ cell corpses (G). Together, these results indicate that MFG-E8 is recruited to the surface of apoptotic cells through its specific binding to PS. To further confirm the above-mentioned results using a different PS detector, we stained dissected adult hermaphrodite gonads with FITC-conjugated Annexin V, a known PS-binding protein (see Materials and Methods) ( ).

Annexin V was detected specifically on the surface of germ cell corpses but not on that of living germ cells in gonads dissected from ced-1 mutants (F), indicating that PS is indeed specifically presented on the surface of apoptotic cells. Together, our observations indicate that MFG-E8::GFP is a specific marker for detecting PS exposed on the surface of apoptotic germ cells in living C. PS Is Specifically Presented on the Surface of Apoptotic Somatic Cells During embryogenesis, the execution of apoptosis in somatic cells, like that in germ cells, requires the functions of cell death genes ced-4 and ced-3 ( ). However, whether the downstream events regulated by the caspase CED-3 are identical remains unclear.

To examine whether apoptotic somatic cells expose PS on their surfaces, we expressed mfg-e8::gfp under the control of P dyn-1, the promoter of dyn-1 that is ubiquitously expressed in embryos ( ). In this scenario, we anticipated that the secreted MGF-E8::GFP would label the outer surface of any cells that exposed PS.

In wild-type embryos, GFP was detected on the surface of transiently present cell corpses; in contrast, no GFP signal was detected on the surface of living somatic cells (A). In ced-1 null mutant embryos, GFP was detected on the surface of cell corpses inside embryos as well as on unengulfed cell corpses that are subsequently extruded to the embryonic cavity (B). In ced-1 L1 larvae expressing P dyn-1 mfg-e8::gfp, 92% of the persistent somatic cell corpses are labeled with a GFP ring; however, in ced-1 L1 larvae expressing MFG-E8ΔC2::GFP, only 5.9% somatic cell corpses are labeled with GFP (A). Together, these results indicate that, like germ cell corpses, somatic cell corpses specifically expose PS on their outer surfaces.

The Association of MFG-E8 with the Surface of Apoptotic Cells Interferes with Their Engulfment In the gonad of wild-type hermaphrodites expressing P lim-7 mfg-e8::gfp, we detected a 2.4-fold increase in the number of germ cell corpses over the wild-type animals not expressing any transgene or expressing P lim-7 mfg-e8Δ C2::gfp (G). To further determine whether the ectopic expression of MFG-E8 affected cell corpse removal, we monitored the duration of germ cell corpses induced by γ-ray irradiation, which occur rather synchronously, by using time-lapse recording (see Materials and Methods). Four hours after irradiation, >2-fold increase in the numbers of germ cell corpses were observed in wild-type animals both expressing and not expressing P lim-7 mfg-e8::gfp compared with their corresponding nonirradiated controls (A), indicating that irradiation was effective in inducing apoptosis.

Moreover, a significantly larger number of irradiation-induced cell corpses were observed in animals expressing than those not expressing P lim-7 mfg-e8::gfp (A), suggesting that the overexpressed MFG-E8 may affect the removal of these cell corpses or the irradiation-induced apoptosis. In adult hermaphrodites not carrying any transgene, all germ cells undergoing apoptosis disappear within 1 h of its first appearance (B); however, in animals expressing P lim-7 mfg-e8::gfp, 44% of germ cell corpses analyzed persisted for a significantly longer period (B). These results indicate that MFG-E8 delays the removal of cell corpses. The overexpression of MFG-E8::GFP in the gonad reduces the efficiency for the removal of germ cell corpses. (A) Significantly more germ cell corpses were observed in the gonad of adult hermaphrodites overexpressing MFG-E8::GFP in the absence or presence.

In wild-type animals, somatic embryonic apoptotic cells are swiftly engulfed and degraded during embryogenesis; thus, no cell corpse is detected in the head of newly hatched L1 larvae (A). We thus scored the number of persistent cell corpses in the head of newly hatched L1 larvae to measure the degree of engulfment defect during embryogenesis. We observed that the expression of MFG-E8::GFP, but not that of MFG-E8ΔC2::GFP in wild-type embryos, resulted in a weak yet still significant (p. CED-7, a Member of the ABC Transporter Family, Is Involved in Exposure of PS on the Surface of Apoptotic Somatic Cells but Not Germ Cells To examine whether the functions of any of the engulfment ced genes are required for the exposure of PS on the surface of somatic apoptotic cells, the P dyn-1 mfg-e8::gfp transgene was expressed in mutant alleles of each of the seven genes, and the number of cell corpses as well as that of MFG-E8::GFP rings around cell corpses was scored in the heads of newly hatched L1 larvae (see Materials and Methods). Any persistent cell corpses observed in the head of newly hatched L1 larvae should have been generated during embryogenesis (; ). Ced-1(e1735), ced-5(n1812), and ced-7(n1996) are null mutants of each corresponding gene, whereas ced-6(n2095), ced-10(n1993), and ced-12(n3261) represent strong loss-of-function mutants of the corresponding genes ( ).

We found that in ced-1, ced-5, ced-6, ced-10, and ced-12 mutants, at least 85% of somatic cell corpses were labeled with MFG-E8::GFP circles (, B–E). However, in ced-7 mutants, only on average 57% of cell corpses were labeled with MFG-E8::GFP circles (, B and F). These results indicate that ced-7, unlike ced-1, ced-5, ced-6, ced-10, or ced-12, is required for the efficient association of MFG-E8 with cell corpses.

A likely interpretation is that CED-7 contributes to the presentation of PS on the surface of apoptotic cells. We also quantified whether mutations in engulfment genes in the ced-1 pathway affect the association of MFG-E8::GFP on the surface of persistent germ cell corpses. Contrary to what we observed with somatic cell corpses, the percentage of germ cell corpses labeled with MFG-E8::GFP in ced-7 mutant hermaphrodites seemed similar to that obtained from ced-1 or ced-6 mutant hermaphrodites or hermaphrodites that lost dyn-1 function due to RNAi treatment () (see Materials and Methods) ( ), suggesting that inactivating ced-7 or other known ced-1 pathway components does not affect the exposure of PS on the surface of germ cell corpses. PS and Perhaps an Additional Ligand(s) Both Contribute to the Recognition of Cell Corpses by Phagocytic Receptor CED-1 The phagocytic receptor CED-1 recognizes cell corpses and clusters in a high concentration on the side of an engulfing cell in contact with a cell corpse. A CED-1::GFP reporter, which retains full CED-1 activity, forms bright green circles around transiently existing apoptotic cells in wild-type embryos, and primarily partial circles around persistent cell corpses in engulfment mutant embryos and larvae ( ). Previously, we reported that in ced-7 mutants, the clustering of ced-1 around cell corpses was severely defective. In this study, we scored the number of CED-1::GFP circles around persistent cell corpses using the DeltaVision deconvolution microscope (see Materials and Methods).

In ced-5, ced-6, ced-10, and ced-12 mutant L1 larvae, we identified a larger percentage of persistent somatic cell corpses labeled with CED-1::GFP circles or partial circles than previously reported (∼60 vs. ∼30%) (;, B–E). This increase is primarily due to the increased sensitivity in detecting fluorescence signals by the DeltaVision deconvolution microscope compared with the fluorescence microscope we used previously. Strikingly, in ced-7 mutant L1 larvae, merely 8.5% of cell corpses were labeled with CED-1::GFP circles or partial circles (, B and F), consistent with our previous observation ( ) and confirming that the function of ced-7 is essential for CED-1 to recognize and cluster around cell corpses. Thus, in ced-7 mutants, the efficiency of PS exposure on somatic cell corpses and the efficiency of CED-1's recognition of cell corpses both decrease. The loss of ced-7 function seems to affect CED-1's recognition of somatic cell corpses to a much larger extent than affecting the exposure of PS. Compared with ced-6 mutants that are defective in the engulfment events downstream of CED-1's recognition of cell corpses ( ), in ced-7 L1 larvae, the percentage of cell corpses presenting PS is decreased from 91 to 57%, yet the percentage of cell corpses recognized by CED-1 is dramatically decreased from 64 to 8.5% (B).

A comparison with ced-5, ced-10, or ced-12 mutants that are defective in cytoskeleton reorganization during engulfment generates a similar conclusion (B). These differential effects suggest that PS exposure alone is necessary but might not be sufficient for cell corpses to be recognized by CED-1. Among multiple possibilities, perhaps an additional signaling molecule is exposed on the surface of apoptotic cells in a CED-7-dependent manner and acts together with PS to attract CED-1 (see Discussion).

A Null Mutation in ced-7 Does Not Affect CED-1's Recognition of Germ Cell Corpses To determine the correlation between PS exposure and CED-1 clustering in the adult hermaphrodite gonad, we examined whether CED-1 clusters around germ cell corpses in engulfment mutants. We used a CED-1ΔC::GFP reporter, in which the intracellular domain of CED-1 was replaced with GFP, for this assay. CED-1ΔC::GFP on the surface of engulfing cells retains the ability to cluster around cell corpses yet loses the ability to initiate engulfment and thus failed to rescue ced-1 mutant phenotypes ( ).

Interestingly, unlike in the soma in which a ced-7 null mutation blocks CED-1 clustering, similar percentages of persistent germ cell corpses are labeled with CED-1ΔC::GFP rings in ced-1, ced-6, and ced-7 mutants and dyn-1(RNAi) animals (). This observation correlates well with the normal exposure of PS on the surface of germ cell corpses observed in ced-1, ced-6, and ced-7 mutants and dyn-1(RNAi) animals (), and further suggests that somatic and germ cell corpses may use different mechanisms to regulate the exposure of PS and/or another unidentified eat-me signal(s). Also observed the clustering of CED-1 around germ cell corpses in ced-7 mutants. Mutations of Phospholipid Scramblase Homologues PLSC-1, PLSC-2, and PLSC-3 Do Not Cause Obvious Defect in the Engulfment of Somatic Cell Corpses Human phospholipid scramblase PLSCR1 was reported to induce the random movement of phospholipids between plasma membrane leaflets in response to the increase of intracellular Ca 2+ and perhaps other signals (; ). This activity could potentially promote the exposure of phospholipids that are usually kept in the inner leaflet of the plasma membrane, such as PS and PE, to the outer surface.

Elegans genome, we identified four predicted polypeptides, C04E12.7, T22H2.5, ZK1053.5, and F11A6.2, that display extensive homology to PLSCR1 throughout their entire length (A). The genes encoding C04E12.7, T22H2.5, ZK1053.5, and F11A6.2 are named plsc-1, plsc-2, plsc-3, and plsc-4, respectively (). F11A6.2 lacks a part of the scramblase domain at the N-terminal region and is not studied in this report. We analyzed whether PLSC-1, -2, and -3, which possess the intact scramblase domain, are required for the exposure of PS and for cell corpse engulfment by using deletion alleles of each gene (see Materials and Methods). Elegans homologues of human PLSCR1 contain persistent germ cell corpses. (A) The alignment of human PLSCR1 with its three C. Elegans homologues. Kimpex Arrow Skis Installation Artists.

The amino acid residues identical or similar among at least three proteins are shaded in black or gray. In plsc-1(ok1178) deletion mutants, we detected a 1240-base pair deletion that removed exons 1 and 2 and intron 1 of the predicted plsc-1 gene as well as 444 base pairs 5′ of the translational start site (B and Supplemental Figure S2). Such a deletion eliminates N-terminal 2/3 of the open reading frame and the 5′ promoter sequence for plsc-1 transcription, and it should result in a null allele. In the plsc-3(ok1313) allele, we detected a 1171-base pair deletion that removed the entire coding region of plsc-3 as well as 67 base pairs upstream of the ATG start codon and that should create a null allele (B and Supplemental Figure S2). The plsc-2(tm820) allele bears a 571-base pair deletion that removes most of exon 1 of plsc-2 and 230 base pairs upstream of the ATG start codon (), again likely to result in a null allele (B and Supplemental Figure S2). We further confirmed that in each of the homozygous mutant strains, the wild-type allele of the corresponding genes was absent from the genome (Supplemental Figure S2), excluding the possibility of the presence of duplicated wild-type alleles elsewhere in the genome. All three mutant alleles are homozygous viable and lack obvious developmental defects.

In the head of L1 larvae, we did not observe persistent somatic cell corpses in any single mutant background, or in animals subjected to RNAi treatment of each of the three genes (C). In plsc-1(ok1178) or plsc-2(tm820) single mutant embryos, or in plsc-1(ok1178); plsc-2(tm820); plsc-3(RNAi) triple mutant embryos, the numbers of somatic cell corpses scored at four different stages of embryogenesis are similar to that observed in wild-type embryos (p >0.05, Student's t tests) (D). These results seem to indicate a lack of obvious defect in the engulfment of somatic cell corpses or in the execution of apoptosis. We further analyzed the embryonic expression pattern of plsc-1. A GFP reporter containing a nuclear localization signal (NLS) fused to its N terminus and expressed under the control of a 6.4-kb plsc-1 promoter (P plsc-1NLS- gfp) ( ) was reported to express predominantly in neurons and body wall muscles in larvae and adults ().

Using this reporter, we observed that in embryos, plsc-1 was predominantly expressed in body wall muscle precursor cells and certain yet-to-be identified cells that could be of neuronal lineages (A). We further observed that throughout embryogenesis, none of the cell corpses expressed any GFP signal. In particular, in embryos 310–330 min post-first cleavage, 100% of the 17 cell corpses are GFP − (, A and B).

These observations, together with the above-mentioned observation indicating that the deletion of plsc-1 does not affect the removal of somatic cell corpses, suggest that plsc-1 is unlikely to be involved in the exposure of the eat-me signal in somatic apoptotic cells. The Inactivation of PLSC-1, PLSC-2, and PLSC-3 Affects the Removal of Germ Cell Corpses to Different Extents In the gonad of adult hermaphrodites of plsc-1, -2, and -3 single mutants, in contrast, we observed significantly larger numbers of germ cell corpses than in wild-type animals (p. PLSC-1 Promotes the Exposure of PS on the Surface of Apoptotic Germ Cells We observed two closely related defects regarding the exposure of PS on the surface of germ cells corpses in homozygous plsc-1(ok1178) mutants carrying the P lim-7 mfg-e8::gfp reporter construct. First, the MFG-E8::GFP signal is significantly weaker on the surface of germ cell corpses than in wild-type animals. The reduction of GFP signal intensity was observed in the plsc-1(ok1178) single mutants (data not shown) as well as in ced-1(e1735); plsc-1(ok1178) double mutants (, A and B). The overall GFP signal intensity in the gonad remains normal (A); in addition, bright GFP signals were observed in coelomocytes near the gonad (Supplemental Figure S3), indicating that the deletion of plsc-1 does not affect the production or the secretion of MFG-E8::GFP from gonadal sheath cells.

Second, in plsc-1 mutants, the number of germ cell corpses labeled with MFG-E8::GFP is significantly lower than in wild-type animals (C). Similarly, the number of persistent germ cell corpses labeled with MFG-E8::GFP in the ced-1; plsc-1 double mutants is significantly lower than that in ced-1 mutants (, A and C).

Together, the reductions of GFP signal intensity on the surface of germ cell corpses and the percentage of germ cell corpses labeled with MFG-E8::GFP strongly suggest that PLSC-1 plays an important role in promoting the presentation of PS on the surface of germ cell corpses. The plsc-1(ok1178) mutants expressing MFG-E8::GFP display a further enhanced Ced phenotype—containing more germ cell corpses (20.2) than plsc-1(ok1178) mutants alone (12.5) or wild-type animals expressing MFG-E8::GFP (13.2) (E and C), suggesting an additive effect of two independent forces that impair the function of PS as a signal to attract engulfing cells: decreased efficiency of PS exposure in plsc-1 mutant background, and further sequestration of PS from phagocytic receptor(s) by MFG-E8. Due to the frequently occurring germline-specific repression of the expression of transgenes in the extrachromosomal array ( ), P plsc-1NLS- gfp is not a suitable reporter for examining whether plsc-1 is expressed in the germline. The expression pattern of plsc-1 in the germline remains to be determined.

The Exposure of PS Is a Conserved Feature of Apoptotic Cells from Nematodes to Human The cuticle that covers larvae and adults and the chitin-based eggshell, which are impermeable to most staining reagents without fixation, made it difficult to determine the surface features of apoptotic cells in living C. In this study, we detected PS exposure on the surface of apoptotic cells by using ectopically expressed, secreted MFG-E8::GFP in living animals, and by using Annexin V staining in surgically dissected gonads. In ced-1 mutants that contain many unengulfed cell corpses, strong PS accumulation was detected on the surface of >90% of somatic cell corpses and ∼60% of germ cell corpses, and during embryogenesis, larval development, and adulthood, suggesting that the exposure of PS is a common feature for apoptotic cell, regardless of tissue types or developmental stages.

Recently, using Annexin V staining, also reported PS exposure on the surface of apoptotic germ cells in C. PS May Act As an Eat-Me Signal and a Ligand for CED-1 in C. Elegans Our research provided several lines of evidence to suggest that the exposure of PS may facilitate the engulfment of apoptotic cells in C. First, the ectopic expression of mouse MFG-E8::GFP but not its PS binding-deficient form (ΔC2) in gonadal sheath cells results in the accumulation of persistent germ cell corpses. MFG-E8 is likely to delay cell corpse engulfment, because germ cell corpses last significantly longer than in animals not expressing MFG-E8. No obvious defects in germline morphology or fertility that could be associated with excessive death of germ cells were observed. In addition, during the time-lapse recording experiment, we observed that in both animals expressing or not expressing MFG-E8, similar numbers of germ cells underwent apoptosis in a fixed period after irradiation, suggesting MFG-E8 did not induce excessive apoptosis in response to irradiation.

These observations suggest that MFG-E8 is unlikely to cause a significant amount of excessive germ cell death. Rather, the binding of MFG-E8 to cell-surface PS may sequester PS and/or another adjacent signaling molecule from phagocytic receptor(s) or bridging molecules. A similar yet much weaker engulfment-inhibitory effect was detected when MFG-E8::GFP was ubiquitously expressed in embryos and larvae. P dyn-1, the somatic cell-specific promoter, is rather weak (Zhou, unpublished observation).

In addition, as a secreted protein, a significant amount of MFG-E8::GFP was observed in embryonic cavity, unavailable to associate with apoptotic cells (data not shown). These observations might explain why the effect of MFG-E8 in embryos is weak. Second, defects in PS exposure, observed in somatic cell corpses in ced-7 mutants and in germ cell corpses in plsc-1 mutants, are closely associated with defects in cell corpse engulfment, suggesting that the exposure of PS is necessary to attract engulfing cells. Thus, the exposure of PS is not only a conserved feature of apoptotic cells in metazoans but also plays a conserved role in the removal of apoptotic cells from nematodes to mammals. CED-1 was proposed to recognize PS as a ligand ( ).

We report that in ced-7 mutants, both PS exposure and CED-1's recognition of somatic cell corpses are impaired. In addition, masking PS and/or an unknown signaling molecule via MFG-E8 does not seem to further increase the number of germ cell corpses in ced-1 mutants (G and ), suggesting that MFG-E8 elicits its effect over engulfment through CED-1. These observations thus provide further support to the above hypothesis. Previously, PSR-1, a C. Elegans homologue of mammalian phosphatidylserine receptor, was implicated in recognizing PS on the surface of apoptotic cells and activating the CED-5 pathway for engulfment ( ). Both CED-1 and PSR-1 may recognize PS as a ligand and each activate a separate signaling pathway. Psr-1 deletion mutants seem to display a rather weak and transient engulfment defect ( ), suggesting that among the two receptors, CED-1 may play a major role in recognizing apoptotic cells.

In mammals, MFG-E8 acts to link apoptotic cell and phagocytes by its affinity for PS and for α vβ 5 integrin, a phagocytic receptor ( ). Elegans genome does not contain any known homologue of MFG-E8. In addition, none of the three known C. Elegans integrin subunits are involved in cell corpse engulfment (; ).

Our observation that the ectopically expressed MFG-E8 impairs instead of promoting engulfment suggests that MFG-E8 is unlikely to act as a bridging molecule for a phagocytic receptor in C. There May Be Two Alternative, Tissue-specific Mechanisms That Regulate PS Exposure ABCA1, the closest mammalian homologue of CED-7, promotes the translocation of PS from the inner to the outer leaflet of the plasma membrane ( ). In addition, mammalian ABCA7 enhances phagocytosis of apoptotic cells by macrophages, and it was proposed to be involved in the translocation of phospholipids across the lipid bilayer ( ). However, the specific phospholipid-flipping function of ABCA1 or ABCA7 in apoptotic cells is yet to be demonstrated (; ). Our identification of CED-7's function in promoting PS exposure on the surface of apoptotic cells provides this missing piece of the puzzle. Interestingly, CED-7's ability to promote PS exposure and to facilitate CED-1's recognition of cell corpses seems to be limited to somatic cells, suggesting a novel tissue-specific regulation.

Consistent with its function, CED-7 is wildly expressed in almost all cells in embryos and is localized to plasma membrane ( ). Although in vitro reconstitution assays and cell culture based assays indicate that mammalian PLSCR1 can mediate a Ca 2+-activated bidirectional transport of phospholipids between the two membrane leaflets, whether PLSCR1 functions as a phospholipid scramblase in vivo remains controversial (for review, see ). The multiple closely related phospholipid scramblases in mammals may act redundantly and prevent the disclosure of gene function by single gene deletion analysis. We identified PLSC-1, a C. Elegans homologue of PLSCR1 that contributed significantly and specifically to the exposure of PS in apoptotic germ cells but not somatic cells. In plsc-1 deletion mutant animals, the accumulation of germ cell corpses and the reduced PS exposure together strongly suggest the involvement of a putative phospholipid scramblase in the exposure of PS. Recently, also reported that C.

Elegans SCRM-1 (another name for PLSC-2) is involved in the exposure of PS in response to apoptotic signal. Mutants of plsc-2 and -3 display very minor Ced phenotype in the gonad. Furthermore, the plsc-1; plsc-2; plsc-3(RNAi) triple mutants only display very slight increase of the number of persistent germ cells, suggesting that plsc-2 and -3 might contribute to PS exposure either in a plsc-1–dependent manner or in a manner partially redundant to plsc-1 (A). Diagrams for the proposed mechanisms for the presentation and recognition of eat-me signals.

(A) CED-7 and PLSC-1 regulate the exposure of PS on the surface of apoptotic cells in the soma or the germline, respectively. Smaller size and gray color are. We did not observe defects in the removal of somatic cell corpses in any of the plsc-1, -2, -3 single mutants or the plsc-1; plsc-2; plsc-3(RNAi) triple mutants. Although it was reported that the scrm-1/plsc-2 mutant animals have minor delays in the removal of cell corpses in embryos ( ), because the Ced phenotype of ced-7 mutant embryos is much stronger than that reported for scrm-1 embryos, and because only ced-7 mutant animals display an obvious defect in the exposure of PS on apoptotic somatic cells (), it seems that CED-7 makes a more significant contribution to the exposure of PS on somatic cell corpses than any of the PLSC proteins. CED-7 and PLSC-1 may represent two alternative mechanisms that promote PS exposure in apoptotic cells of different tissue types (A).

Although germ cells and somatic cells in C. Elegans both undergo apoptosis via the ced-3– and ced-4–mediated pathway, certain aspects of cell death are different. In particular, living germ cell nuclei are included in one germline syncytium and share a common cytoplasm and a common plasma membrane; when a germ cell nucleus dies, it undergoes a rapid cellularization process and is separated from the syncytium ( ). Such a process does not occur to somatic apoptotic cells.

In addition, cell corpses in the germline and soma are engulfed by different types of engulfing cells, one exclusively by gonadal sheath cells and the other by several cell types, including hypodermal cells, intestinal cells, and muscle cells (for review, see ). The difference in the mechanism and dynamics of cell death and the types of engulfing cells may lead to the activation of different mechanisms that regulate PS exposure, hence the differential involvement of PLSC-1 and CED-7 in germ cell and somatic cell apoptosis. The existence of these alternative mechanisms may serve to increase the complexity for the regulation of eat-me signal exposure upon apoptotic stimuli during development. It would be of general interest to determine whether such differential mechanisms are also used in the mammalian system.

Why, then, in ced-7 mutant germline, are there still a large number of persistent germ cell corpses? Genetic mosaic analysis indicated that CED-7 functions are required in both germline and engulfing cells for efficient engulfment of germ cell corpses ( ). The defects observed in ced-7 mutant gonad might be primarily due to the loss of a major contribution of CED-7 activity from engulfing cells. The Evidence for a Novel Eat-Me Signal(s) A loss-of-function mutation in ced-7 results in a much stronger defect in the clustering of CED-1 around cell corpses than in the exposure of PS. Multiple explanations may exist.

First, the detection of PS on the surface of a cell corpse by CED-1 might be subject to a threshold. Second, CED-7 may play two distinct roles during engulfment: promoting the exposure of PS in apoptotic cells, and assisting CED-1 in recognizing eat-me signals on the surface of engulfing cells. Alternatively, a mutation in CED-7 may disrupt the presentation of a yet unknown eat-me signal recognized by CED-1 in addition to PS. Several molecules or structures have been implicated as eat-me signals besides PS.

These molecules include cell surface carbohydrates, oxidized lipids, intercellular adhesion molecule-3, cell surface calreticulin (for review, see ), and certain unidentified molecules sensitive to proteolytic cleavage ( ). Lysophosphatidylcholine secreted from apoptotic cells was also reported to attract phagocytes to the vicinity ( ).

It is possible that a novel eat-me signal(s) is exposed to the surface of C. Elegans cell corpses in a CED-7–dependent manner and acts cooperatively with PS as CED-1 ligands (B). Mitani, Ian Hope, A. Fire (Stanford University School of Medicine, Palo Alto, CA), the C.

Elegans Gene Knockout Consortium, and the Caenorhabditis Genetics Center for reagents; X. He for the DeltaVision; X. Edlund, and C. Chuang for technical assistance; S. Nagata for helpful suggestions; and A. Roger, and members of the Zhou laboratory for helpful comments. Previous and current support for Z.Z.

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