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Integrin expression and activity have been strongly correlated with developmental and pathological processes involving cell invasion through basement membranes. The role of integrins in mediating these invasions, however, remains unclear. Utilizing the genetically and visually accessible model of anchor cell (AC) invasion in C. elegans, we have recently shown that netrin signaling orients a specialized invasive cell membrane domain towards the basement membrane. Here, we demonstrate that the integrin heterodimer INA-1/PAT-3 plays a crucial role in AC invasion, in part, by targeting the netrin receptor UNC-40 (DCC) to the AC’s plasma membrane. Analyses of the invasive membrane components phosphatidylinositol 4,5-bisphosphate, the Rac GTPase MIG-2 and F-actin further indicate that INA-1/PAT-3 plays a broad role in promoting the plasma membrane association of these molecules. Taken together, these studies reveal a role for integrin in regulating the plasma membrane targeting and netrin-dependent orientation of a specialized invasive membrane domain.
Basement membranes (BMs) are thin, dense and highly cross-linked forms of extracellular matrix that provide the structural underpinning for all epithelia, endothelia and many mesenchymal cells in metazoans (Kalluri, 2003; Rowe and Weiss, 2008; Yurchenco et al., 2004). Despite its barrier properties, numerous cells during development and in normal physiological processes undergo highly regulated invasions through BMs to disperse, form tissues and mediate immune system responses (Hughes and Blau, 1990; Risau, 1997; Wang et al., 2006). Metastatic cancer cells are thought to exploit the same underlying regulatory networks to breach BMs during their spread to distant tissues (Friedl and Wolf, 2003). Efforts to elucidate the genetic pathways that control invasive behavior have been limited by the experimental inaccessibility of cell-matrix interactions in the complex and dynamic in vivo tissue environments where cell invasions occur (Even-Ram and Yamada, 2005; Hotary et al., 2006; Wang et al., 2006). As a result, the mechanisms that promote cell invasion through BMs during development and cancer remain poorly understood (Machesky, 2008; Rowe and Weiss, 2008).
Anchor cell (AC) invasion into the vulval epithelium in C. elegans is an in vivo model of invasive behavior that allows for genetic and single-cell visual analysis of invasion (Sherwood et al., 2005; Sherwood and Sternberg, 2003). During the mid L3 larval stage, a basally-derived invasive process from the AC crosses the gonadal and ventral epidermal BMs and then moves between the central 1° fated vulval precursor cells (VPCs) to mediate uterine-vulval attachment (Sharma-Kishore et al., 1999; Sherwood and Sternberg, 2003). Recent studies have shown that the invasive cell membrane of the AC is a specialized subcellular domain that is polarized towards the BM by the action of the unc-6 (netrin) pathway (Ziel et al., 2009). Approximately four hours prior to invasion, expression of the secreted guidance cue UNC-6 (netrin) from the ventral nerve cord targets its receptor UNC-40 (DCC) to the AC’s invasive membrane. There, netrin signaling localizes a number of actin regulators that promote invasion, including the Rac GTPases MIG-2 and CED-10, the Ena/VASP ortholog UNC-34 and the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Ziel et al., 2009). The proper orientation of these components at the basal membrane is required to generate robust protrusions that breach the BM in response to a later cue from the 1° VPCs that stimulates invasion. Although the molecular components of the invasive membrane are misoriented in unc-6 mutants, they still associate in a non-polarized manner with the AC’s plasma membrane, suggesting a distinct mechanism exists for regulating their targeting to the cell membrane.
Integrins are one of the major cell surface receptors used by metazoan cells to mediate direct cell-matrix interactions (Yurchenco et al., 2004). All integrins are heterodimers composed of a single α and β subunit. In vertebrates integrins have been implicated in regulating cell invasion during blastocyst implantation, angiogenesis and leukocyte trafficking (Hodivala-Dilke, 2008; Sixt et al., 2006; Staun-Ram and Shalev, 2005). Furthermore, the dysregulation of integrin expression and function has been associated with a number of metastatic cancers (Felding-Habermann, 2003; Hood and Cheresh, 2002). Mammals utilize 18 α and eight β subunits, which combine to form an array of different heterodimers (Hood and Cheresh, 2002). The complexity of the mammalian integrin receptor family, combined with the difficulty of in vivo analysis has hindered an understanding of the requirement and function of integrin receptors in mediating BM invasion (Felding-Habermann, 2003; Friedl and Wolf, 2003; Sixt et al., 2006). C. elegans possess only two predicted integrin receptors, composed of an α PAT-2 or α INA-1 subunit bound with the sole β subunit PAT-3 (Kramer, 2005), providing a simplified genetic landscape for examining integrin function.
We have conducted an RNAi screen to identify additional pathways that regulate invasion and report here that the C. elegans integrin heterodimer INA-1/PAT-3 is a crucial regulator of AC invasion. Cell biological and genetic analyses indicate that INA-1/PAT-3 functions within the AC to control the formation of invasive protrusions that breach the BM. Our analysis identifies a key role for integrin in regulating the membrane association of components of the invasive cell membrane, including the netrin receptor UNC-40 (DCC). This work demonstrates an essential role for integrin in controlling BM invasion and reveals an integrin-netrin pathway interaction that mediates the membrane targeting and polarization of the molecular constituents of the AC’s invasive membrane.
During the early L3 larval stage, the gonadal AC is separated from the underlying P6.p vulval precursor cell (VPC) by the juxtaposed gonadal and ventral epidermal basement membranes (BMs). At this time UNC-6 (netrin) secreted by the ventral nerve cord orients a specialized F-actin rich invasive cell membrane domain within the AC towards the BM (P6.p one-cell stage; Figure 1A). Approximately four hours later, the underlying 1° fated P6.p cell divides (P6.p two-cell stage) and produces an unidentified cue, which stimulates the formation of invasive protrusions that breach the BM and contact the P6.p granddaughters (P6.p four-cell stage; Figure 1A and 1B). Breaching the BM is dependent upon the transcription factor FOS-1A, which regulates the expression of genes in the AC that mediate BM removal (Hwang et al., 2007; Rimann and Hajnal, 2007; Sherwood et al., 2005). AC invasion is completed at the L3 molt when the basolateral portion of the AC moves between the P6.p great-granddaughters at the apex of the vulva (P6.p eight-cell stage; see Figure S1). AC invasion initiates uterine-vulval attachment and its timing and targeting are invariant in wild-type animals (Sherwood and Sternberg, 2003).
Ablation of the AC just prior to invasion disrupts uterine-vulval attachment and results in a protruded vulval (Pvl) phenotype (Kimble, 1981; Seydoux et al., 1993). To identify genes that regulate invasion, we examined genes whose knockdown by bacterial feeding of RNAi has been reported to produce a Pvl phenotype (Kamath et al., 2003) and found that RNAi targeting the α integrin subunit ina-1 resulted in an AC invasion defect (Table 1). Null mutations in ina-1 cause L1 larval lethality (Baum and Garriga, 1997). We therefore examined two hypomorphic mutations in ina-1, ina-1(gm39) and ina-1(gm144) that appear to affect different functions of ina-1 (Baum and Garriga, 1997). ina-1(gm39) mutants, which harbor a missense mutation within the putative ligand binding β propeller region of the protein, had a significant invasion defect, with 50% of ACs showing a lack of invasion at the P6.p four-cell stage and 22% failing to invade by the P6.p eight-cell stage (Figure S1; Table 1). In contrast, ina-1(gm144) animals, which contain a distinct missense lesion adjacent to the transmembrane domain (a region that might regulate integrin affinity for its ligand) had normal AC invasion despite this allele causing neuronal migration defects (Baum and Garriga, 1997) (Table 1). These results confirm a role for ina-1 in regulating AC invasion, and provide further evidence of separable ina-1 functions.
In both Drosophila and vertebrates, α and β integrin subunits require heterodimerization within the secretory apparatus to be transported to the cell surface (Leptin et al., 1989; Marlin et al., 1986). Consistent with a similar regulatory mechanism in C. elegans, high levels of full-length transgenes encoding the sole β integrin PAT-3::GFP or the α integrin INA-1::GFP alone showed internal localization within the AC, as well as in neighboring somatic gonad and vulval cells (Figure S2). Co-transformation of pat-3::GFP with genomic DNA encoding INA-1 resulted in PAT-3::GFP localization to the cell surface with increased levels at the AC’s invasive cell membrane (Figure 1D), indicating that INA-1 forms a heterodimer with PAT-3 in the AC. Co-transformation with genomic DNA encoding the other C. elegans α subunit, PAT-2, did not result in translocation of PAT-3::GFP to the cell surface (Figure 1E). Further analysis using a translational PAT-2::GFP fusion revealed that pat-2 was not expressed in the AC at the time of invasion (Figure S2), and RNAi-mediated depletion of pat-2 did not perturb AC invasion (Table 1). These results strongly suggest that the α subunit PAT-2 does not regulate AC invasion.
To further investigate a role for integrin signaling during AC invasion we used RNAi to target two major downstream effectors of integrin signaling, talin and pat-4 (ILK). RNAi depletion of each of these genes resulted in a defect in AC invasion (Table 1). Notably, RNAi targeting talin resulted in a stronger invasion defect than RNAi to pat-4 (ILK), consistent with the former being a more essential component of the integrin signaling pathway (Delon and Brown, 2007). Taken together, these results indicate that the integrin α subunit ina-1 and core components of the integrin pathway promote AC invasion.
To determine where integrin signaling functions during AC invasion, we first used a tissue-specific RNAi strain, fos-1a>rde-1, where only the uterine tissue (which includes the AC) is sensitive to RNAi (see Supplemental Methods). Treatment of fos-1a>rde-1 animals with RNAi targeting the sole β integrin pat-3 resulted in a defect in AC invasion (Table 1). These results indicate that PAT-3 functions within the uterine tissue during AC invasion, consistent with a direct role in the AC.
To determine if pat-3 functions within the AC we disrupted pat-3 function here utilizing a previously characterized dominant negative construct encoding the PAT-3 transmembrane and cytoplasmic domains connected to a heterologous hemagglutin (HA) extracellular domain (HA-βtail) (Lee et al., 2001). Autonomous expression of the β integrin cytoplasmic domain has been shown to act as a dominant negative inhibitor of endogenous integrin function (LaFlamme et al., 1994; Lukashev et al., 1994; Martin-Bermudo and Brown, 1999). We expressed the HA-βtail construct using the AC-specific zmp-1 promoter, which initiates expression before invasion at the late L2 stage and drives increasing levels in the AC up to the time of invasion (Figure S3). At the P6.p four-cell stage, when AC invasion normally occurs, >70% of transgenic HA-βtail animals failed to invade, and approximately 50% of ACs failed to invade by the P6.p eight-cell stage (Figure S3; Table 1). The stable chromosomal integration of zmp-1>HA-βtail (qyIs15 and qyIs48) led to nearly a complete block in invasion at the P6.p four-cell stage (Table 1). Confirming the specificity of this perturbation, AC invasion was normal in animals expressing a control construct (zmp-1>HA-βtailΔ), which lacks the cytoplasmic signaling domain of β integrin (Table 1; see Supplemental Methods). Furthermore, expression of HA-βtail under the control of the 1° VPC-specific promoter egl-17 did not affect AC invasion (Table 1). Finally, mosaic analysis using PAT-3::GFP expression to rescue the pat-3 null mutant pat-3(rh54) further indicated that pat-3 functions within the AC, but not the vulval cells to regulate invasion (Figure S4). Taken together, these results indicate that PAT-3 function is required within the AC to promote invasion.
ina-1 encodes an integrin most similar to vertebrate and Drosophila integrins that bind the BM component laminin (Baum and Garriga, 1997). To determine if INA-1/PAT-3 might mediate AC invasion by maintaining matrix attachment, we examined the interaction of the AC with the BM after perturbation of integrin signaling. We visualized the AC plasma membrane with the pleckstrin-homology (PH) domain from phospholipase C-δ fused to mCherry and driven by the AC-specific promoter cdh-3 (cdh-3>mCherry ::PLCδPH ) (Rescher et al., 2004). The BM was simultaneously observed with a functional laminin β subunit (LAM-1::GFP) (Kao et al., 2006). Similar to wild-type animals, ACs expressing HA-βtail were in direct contact with the BM at the P6.p one- and two-cell stages leading up to invasion, and remained attached to an intact BM after the time of normal invasion (≥ 20 animals each stage; Figure 1B and 1C; Movie S1 and Movie S2). Notably, however, at the normal time of invasion there was a significant 27% reduction in the width of AC contact with the BM in HA-βtail animals (from 7.2µm to 5.3µm; P < 0.05; Student’s t-test), consistent with a role in modulating BM adhesion. In ina-1(gm39) mutants approximately 10% of ACs were detached from the BM from the time of AC specification at the late L2 stage up to the time of invasion (10/74 P6.p one-cell stage and 6/81 P6.p two-cell stage), reflecting a possible earlier requirement in mediating BM association. The majority of ACs that failed to invade at the P6.p four-cell stage in ina-1 mutants, however, were attached to an intact BM (66/74 animals). The AC also remained attached to the BM in all ACs that failed to invade after RNAi-mediated depletion of ina-1, pat-3, pat-4 (ILK) and talin (Table 1). Taken together, these results suggest that INA-1/PAT-3 has an active role in promoting AC invasion that extends beyond mediating BM attachment.
AC invasion requires the generation of invasive processes that penetrate the BM in response to a chemotactic cue from the 1º VPCs (Sherwood and Sternberg, 2003). To examine whether INA-1/PAT-3 regulates this protrusive activity, we ablated all VPCs except the posterior most P8.p cell in wild-type and HA-βtail animals containing egl-17>CFP to visualize 1° VPC fate specification and the AC marker cdh-3>YFP. Under these conditions the descendants of P8.p adopt the 1° VPC fate and generate the chemotactic cue that stimulates invasion, thus providing an assay for the ability of the AC to generate processes towards displaced 1° VPCs (Sherwood and Sternberg, 2003). In wild-type animals, 80% of ACs initiated the extension of processes towards the isolated 1° fated P8.p cell descendants at the P8.p two-cell stage (16/20 animals), and at the P8.p four-cell stage all ACs responded with an invasive process that breached BM (14/14 animals; Figure 1F). In contrast, only 35% of ACs in HA-βtail animals initiated the extension of processes at the P8.p two-cell stage (6/17 animals), and less than 20% did so at the P8.p four-cell stage (3/16 animals), with most ACs failing to generate protrusions (Figure 1G). Furthermore, of the few processes observed, none breached the BM (3β animals). These results suggest that INA-1/PAT-3 mediates AC invasion, at least in part, by regulating the protrusive activity of the AC.
The proper formation of the AC’s invasive cell membrane is required for the extension of invasive processes (Ziel et al., 2009). To determine whether INA-1/PAT-3 regulates invasive cell membrane formation, we examined the localization of a full-length GFP-tagged transgene of the C. elegans Rac ortholog MIG-2, as well as AC-specific expression of the F-actin marker mCherry::moeABD and the PI(4,5)P2 reporter mCherry::PLCδPH. In wild-type animals MIG-2, F-actin and PI(4,5)P2 are enriched at the invasive cell membrane at the P6.p one-cell stage and their polarized localization increases through the time of invasion (Figure 2A, B, C, G). In HA-βtail animals MIG-2, F-actin and PI(4,5)P2 were polarized normally at the P6.p one-cell stage (Figure 2G). Notably, this was at the time when levels of the dominant negative HA-βtail were low in the AC. After this initial establishment of polarity, however, as levels of HA-βtail increased, MIG-2 and PI(4,5)P2 failed to further concentrate and there was a loss of F-actin polarity (Figure 2D, E, F, G). The polarity of these molecules was similarly decreased after RNAi knockdown of ina-1, pat-3 or talin, but unaffected by expression of the dominant negative control HA-βΔ, thus confirming the specificity of the dominant negative HA-βtail (Figure S5 and data not shown). These results suggest INA-1/PAT-3 is required for the maturation of the invasive membrane, and that failure to generate protrusions in HA-βtail animals was a result of perturbation in this specialized membrane domain. Importantly, PAR-3::GFP, which localizes to apical and lateral membranes of wild-type ACs, and AJM-1::GFP, which is found in apical spot junctions, both localized normally in HA-βtail animals (Figure 2H and 2I; data not shown). Thus, INA-1/PAT-3 specifically regulates the invasive cell membrane, but not the overall polarity of the AC.
UNC-6 (netrin) secretion from the ventral nerve cord directs its receptor UNC-40 (DCC) to the AC’s basal cell membrane where netrin signaling functions to orient additional components of the invasive membrane (Ziel et al., 2009). To determine whether INA-1/PAT-3 might regulate invasive membrane formation through the netrin pathway, we examined the localization of UNC-40::GFP in wild-type and HA-βtail animals. Similar to F-actin and actin regulators (Figure 2G), UNC-40::GFP initially localized normally to the invasive cell membrane in HA-βtail animals (Figure 3A and B). This nascent polarity, however, was subsequently lost by the P6.p four-cell stage (Figure 3C–G) and there was an apparent overall decrease in UNC-40::GFP membrane localization compared with wild-type ACs (Figure S6). The polarity of UNC-40::GFP was similarly perturbed after RNAi-mediated depletion of ina-1, pat-3 or talin, while polarity was normal in control HA-βΔ animals (Figure S5). Conversely, INA-1/PAT-3::GFP was localized normally in both unc-6(ev400) and unc-40(e271) mutant animals (20/20 animals for each; Figure S7). These results suggest that INA-1/PAT-3 acts upstream of the netrin pathway in the AC to regulate invasive membrane formation.
The hypomorphic mutant ina-1(gm39), which has a weaker AC invasion phenotype than HA-βtail animals (Table 1), allowed us next to determine the relationship between invasive membrane formation, UNC-40 localization and the ability of the AC to invade. Strikingly, ina-1(gm39) mutant ACs that had UNC-40, F-actin, MIG-2 and PI(4,5)P2 polarized normally were invading (Figure 4A, C and E; Figure S8). In contrast, ina-1(gm39) mutant ACs that had significant reductions in the polarized distribution and membrane localization of UNC-40 and other invasive membrane components did not invade (Figure 4B, D and E; Figure S8). The polarity of the invasive membrane components was normal in ina-1(gm144) mutants (Figure S5, data not shown), consistent with this allele not affecting an ina-1 function necessary for invasion. These results strongly support the notion that the key role of INA-1/PAT-3 in regulating AC invasion is promoting the formation of the invasive membrane. In addition, the positive correlation between the proper localization of UNC-40::GFP and invasion in ina-1(gm39) mutants, suggests that the integrin and netrin pathways function together to form the invasive membrane.
The effects after reduction or loss of INA-1/PAT-3 function on the invasive membrane differed noticeably from those observed after loss of UNC-6. While the components of the invasive membrane (actin regulators, PI(4,5)P2 , F-actin and UNC-40) are not properly polarized in unc-6 mutants, they still associate with the cell cortex (Ziel et al., 2009). In contrast, loss or reduction of INA-1/PAT-3 appeared to result in an overall decrease in the localization of these molecules to the cell membrane (see Figure 2D–F; Figure 3F; Figure S6). These observations led us to further compare invasive membrane formation after loss of netrin versus integrin activity.
The assembly of actin filaments is the target of a wide range of signaling networks (Disanza et al., 2005), and a dense cortical F-actin network is a key component of the AC’s invasive membrane. Thus, to more definitively assess the relative functions of integrin and netrin signaling on invasive cell membrane formation, we measured the integrated fluorescence intensity of the F-actin probe mCherry::moeABD in wild-type, as well as unc-6(ev400) and ina-1(gm39) mutants that failed to invade at the P6.p four-cell stage. A threshold value was set to measure the volume and amount of the dense F-actin network that composes the invasive membrane of wild-type ACs (see methods). Importantly, the AC-specific promoter cdh-3> used to drive mCherry::moeABD is not regulated by ina-1 or unc-6 and drove similar levels of mCherry::moeABD under all conditions (see Experimental Procedures). While the polarity of the dense F-actin network was perturbed in unc-6(ev400) mutants as previously reported (Figure 5A and B), the overall estimated volume and amount was similar to that observed in wild-type animals (Figure 5D). In ina-1(gm39) mutants, there was some mispolarization of the F-actin network (Figure 5C), consistent with reduction of netrin function. The most notable defect, however, was the greater than four-fold loss in the total volume and amount of the dense F-actin network (Figure 5C and 5D; movie S3–movie S5), suggesting that INA-1/PAT-3 regulates F-actin recruitment or polymerization at the cell membrane. These results indicate that integrin and netrin have distinct roles that act together to properly form the invasive cell membrane: INA-1/PAT-3 plays a broad role in promoting the membrane association of invasive membrane components (including the netrin receptor UNC-40), while netrin signaling specifically orients these molecules towards the BM. Further supporting this cell biological evidence of a synergistic function for these pathways in invasive membrane formation, we detected a strong synergistic genetic interaction between ina-1(gm39) and unc-40(e271) mutants. While 22% and 20% ACs in singly mutant ina-1(gm39) and unc-40(e271) animals failed to invade at the P6.p eight-cell stage, respectively, ACs in an ina-1(gm39); unc-40(e271) double-mutant strain displayed a more severe phenotype than the additive effects of these mutations alone with a >80% block in invasion (Table 1).
Cell invasion through BMs requires the integration of multiple signaling pathways that regulate distinct aspects of invasion. Thus, we examined the interaction of INA-1/PAT-3 with the FOS-1A pathway, a major regulator of BM removal during AC invasion (Hwang et al., 2007; Rimann and Hajnal, 2007; Sherwood et al., 2005). ACs in fos-1a mutants still form an invasive cell membrane and extend processes, however, these protrusions fail to break through the BM (Sherwood et al., 2005; Ziel et al., 2009). Treatment of fos-1(ar105) animals with ina-1(RNAi) enhanced the invasion defect (Table 1), indicating that ina-1 has functions distinct from the fos-1a pathway. Consistent with this notion, transcriptional reporters for FOS-1A and all of its known targets were expressed normally in HA-βtail animals (data not shown; Figure 6A, B). Conversely, expression of INA-1/PAT-3::GFP was normal after fos-1(RNAi) treatment (20/20 animals). Although these data indicate distinct activities during invasion, recently we have shown that the FOS-1A and netrin pathways converge at the invasive membrane to regulate BM removal (Ziel et al., 2009), suggesting FOS-1A and INA-1/PAT-3 functions might similarly intersect at this crucial subcellular domain.
A key downstream target(s) of FOS-1A function remains to be identified, as null mutations in known FOS-1A targets produce only mild defects in invasion (Sherwood et al., 2005). For example, loss of the FOS-1A target zmp-1, which encodes a matrix metalloproteinase, does not affect AC invasion, and its loss does not enhance the phenotype of HA-βtail animals (Table 1). Null mutations in the FOS-1A target hemicentin (him-4), a conserved extracellular matrix protein, however, do cause a moderate invasion defect (Table 1). A functional hemicentin::GFP fusion protein is deposited under the invasive cell membrane of wild-type ACs just prior to invasion, where it promotes BM removal (Figure 6C and E). Although hemicentin was expressed normally in the AC in HA-βtail animals (Figure 6B), it was not assembled under the AC’s invasive membrane in >95% of these animals (93/99 animals; FigureD and F). RNAi targeting ina-1, pat-3 or talin caused similar reductions in hemicentin deposition, while expression of the control construct HA-βΔ did not affect this process (Figure S9). Furthermore, loss of hemicentin did not enhance the invasion defect of HA-βtail animals (Table 1). These results indicate a clear dependency of hemicentin deposition and function on integrin activity and demonstrate that the FOS-1A and integrin pathways intersect at the invasive membrane to regulate BM removal.
Cell invasion through BMs play crucial roles in normal physiological processes and the dissemination of cancer. We show here that the C. elegans integrin heterodimer INA-1/PAT-3 is a key regulator of this process during AC invasion, functioning within the AC to generate the specialized invasive cell membrane. A key element of this function is promoting the activity of the netrin pathway within the AC, which ensures the correct polarization of the invasive front towards the BM.
Understanding the requirements and potential roles for vertebrate integrins during cell invasion through BMs in vivo has been clouded by loss-of-function phenotypes that are either lethal (Stephens et al., 1995), extremely complex at the cellular and tissue level (Brockbank et al., 2005) or absent because of apparent genetic redundancy (Bader et al., 1998). Compared with at least 24 known integrin heterodimers in vertebrates, only two are predicted in C. elegans, simplifying genetic analysis of integrin function (Kramer, 2005). Of the two predicted integrin heterodimers in C. elegans, only the INA-1/PAT-3 heterodimer was found to regulate AC invasion. ina-1, pat-3, talin and pat-4(ILK) RNAi treated animals and ina-1(gm39) hypomorphic mutants were defective in AC invasion. Furthermore, tissue-specific RNAi and tissue-specific expression of a dominant negative form of pat-3 (HA-βtail) in the AC or vulval cells, as well as mosaic analysis in the pat-3(rh54) null mutant, revealed that reduction of INA-1/PAT-3 function within the AC blocks invasion, while perturbation of INA-1/PAT-3 function in the vulval cells does not affect invasion. The reduction of INA-1/PAT-3 function slightly reduced the contact area of the AC with the BM, consistent with a function for this integrin heterodimer in modulating adhesion. However, the vast majority of ACs that failed to invade, however, were attached to an intact BM, suggesting that the role of INA-1/PAT-3 extends beyond regulating matrix attachment. Taken together, our results indicate an active, cell-autonomous requirement for the INA-1/PAT-3 integrin heterodimer in mediating BM invasion in vivo and support the idea that integrins might function generally in development and cancer to mediate passage through BMs.
The membrane structures that cells utilize to migrate through BMs in vivo are poorly understood (Machesky, 2008). The protrusive activity of the AC relies on the formation of a specialized invasive cell membrane containing the netrin receptor UNC-40 (DCC), a number of actin regulators, including the Rac GTPase MIG-2 and the phospholipid PI(4,5)P2, as well as F-actin (Ziel et al., 2009). The levels of HA-βtail within the AC mirrored perturbations in the invasive membrane: approximately five hours prior to invasion when expression of HA-βtail was low, the polarization of UNC-40, MIG-2, PI(4,5)P2 and F-actin to the invasive membrane was normal, but as expression levels of HA-βtail increased approaching the time of invasion, there was a corresponding decrease in the localization of these components to the invasive front. Reduction of ina-1, pat-3 and talin by RNAi similarly resulted in decreases in the polarity of these markers. These observations suggest that INA-1/PAT-3 has a direct role in regulating the formation of the invasive cell membrane.
INA-1 is most similar to laminin-binding integrins (Baum and Garriga, 1997), consistent with its mediating a direct interaction between the invading AC and its BM target. Furthermore, studies performed in vertebrate cell culture have shown that the cytoplasmic domain of integrins are capable of anchoring a large complex of proteins that regulate Rac localization to the cell membrane, PI(4,5)P2 generation at the plasma membrane and cell cortex associated recruitment and nucleation of F-actin (Delon and Brown, 2007; Wiesner et al., 2005). These studies support a close relationship between INA-1/PAT-3 function and its regulation of the cell membrane association of the Rac MIG-2, the phospholipid PI(4,5)P2 and F-actin. Moreover, the positive correlation between the proper localization of these components and the ability of the AC to invade in ina-1 hypomorphic mutants, offers strong evidence that INA-1/PAT3 controls invasion by promoting invasive membrane formation.
Our results showing a clear dependence of UNC-40 (DCC) membrane localization on INA-1/PAT-3 activity might have important implications to integrin-netrin pathway interactions in other contexts. Previous work in vertebrates has indicated that several laminin-binding integrins can act as direct receptors for Netrin-1(Yebra et al., 2003). It has been postulated that these integrins might act as a co-receptor with DCC in signaling to downstream effectors (Nikolopoulos and Giancotti, 2005), and thus could stabilize DCC localization through this interaction. While our data does not rule out this possibility, the different patterns of UNC-40 (DCC) localization after loss of UNC-6 versus loss of INA-1/PAT-3 indicate that INA-1/PAT-3 plays a broader role in regulating UNC-40 localization. Specifically, loss of UNC-6 resulted in mispolarization of UNC-40 along all cell membranes, while loss of INA-1/PAT-3 led to an overall decrease in membrane localization. These observations support the idea that INA-1/PAT-3 regulates the stabilization or trafficking of UNC-40 at the cell membrane.
We have also found that INA-1/PAT-3 mediates assembly of the extracellular matrix protein hemicentin under the invasive membrane of the AC. Hemicentin is transcribed in the AC and deposited extracellularly at the site of invasion where it promotes breaching of the BM (Sherwood et al., 2005). Expression of HA-βtail within the AC did not alter hemicentin transcription, but nearly abolished its assembly under the invasive membrane, as did RNAi treatments targeting INA-1/PAT-3 signaling. INA-1/PAT-3 might directly regulate the assembly of hemicentin, similar to known roles for vertebrate integrins in fibronectin matrix deposition (Wierzbicka-Patynowski and Schwarzbauer, 2003). Alternatively, INA-1/PAT-3 could influence hemicentin assembly indirectly through its role in invasive membrane formation. Consistent with the later hypothesis, in unc-6 mutants the AC’s invasive membrane is mispolarized and hemicentin deposition is decreased and misdirected to other surfaces (Ziel et al., 2009).
In vitro studies have shown that integrins recruit surface proteases towards matrix attachment sites to mediate matrix degradation (Wolf et al., 2003). The only identified protease expressed in the AC during invasion is the matrix metalloproteinase ZMP-1. A FLAG-tagged ZMP-1 protein is not strongly localized to the invasive membrane, and genetic deletion of zmp-1 does not perturb AC invasion (Sherwood et al., 2005). Furthermore, we did not detect a genetic interaction between zmp-1 and pat-3. ZMP-1 might function redundantly with other proteases. Alternatively, AC invasion may be predominantly driven by non-proteolytic remodeling of the BM as has been suggested by experimental observations during the trafficking of leukocytes and the activity of epithelial and carcinoma cell lines in vertebrates (Rabinovitz et al., 2001; Rowe and Weiss, 2008).
One of the challenges in understanding the diversity of integrin functions in vivo is elucidating the interactions with other signaling pathways that together mediate specific cellular functions (Wiesner et al., 2005). Our results reveal an interaction between the integrin and netrin pathways within the AC that is required for proper generation and organization of the invasive cell membrane. While both UNC-6 and INA-1/PAT-3 participate in formation of the AC’s invasive membrane, their roles in regulating this invasive front are distinct. In unc-6 mutants, UNC-40 (DCC), actin regulators, PI(4,5)P2 and F-actin are still associated with the AC’s plasma membrane, but they mispolarize along all surfaces of the AC (Ziel et al., 2009). In contrast, there was a reduction in the membrane targeting of these molecules after perturbation in INA-1/PAT-3 function, but not dramatic mislocalization to other membranes. Taken together, these observations suggest that INA-1/PAT-3 has a function in promoting the membrane association of components of the invasive cell membrane, including the netrin receptor UNC-40, while the UNC-6 (netrin) pathway acts to direct or confine these components towards the BM, consistent with a proposed scaffolding function for UNC-40 (DCC) (Gitai et al., 2003; Shekarabi et al., 2005). Strongly supporting cooperative functions for these pathways in regulating AC invasion, animals harboring mutations in both unc-40 and ina-1 showed a synergistic genetic interaction and displayed a more severe block of invasion than the additive defects of these mutants when present alone. A schematic diagram summarizing the role of INA-1/PAT-3 (integrin) and UNC-6 (netrin) pathway function on invasive membrane formation and its role in AC invasion is shown in Figure 6G.
Integrin and netrin pathways play key roles in a number of shared cellular processes involving cell invasion through BMs, including angiogenic vessel sprouting, leukocyte transmigration and metastatic cancer (Avraamides et al., 2008; Baker et al., 2006; Fitamant et al., 2008; Hodivala-Dilke, 2008; Ly et al., 2005; Nikolopoulos and Giancotti, 2005; Sixt et al., 2006; White and Muller, 2007). These shared functions indicate that the integrin and netrin pathway interaction revealed here might be a common mechanism to generate and polarize an invasive cellular response. The dramatically enhanced block in AC invasion after reduction of function in both pathways, further suggests that combined therapeutic targeting of integrin and netrin signaling may provide a powerful strategy to modulate cell-invasive behavior in development and human disease.
Details of tissue-specific expression of dominant negative pat-3, pat-3 and ina-1 expression studies, transgenic animal construction and P8.p isolation experiments are described in Supplemental Methods.
Strains were reared and viewed at 20ºC by standard methods (Brenner, 1974). Wild-type nematodes were strain N2. In the text and figures we designate linkage to a promoter using the (>) symbol and linkages that fuse open reading frames using the (::) annotation. The following transgenes and alleles were used for studies performed in this paper: qyEx4[zmp-1>HA-βtail], qyEx9[pat-3::GFP], qyEx35[zmp-1>HAJ3A], qyEx36[pat-3::GFP; genomic ina-1], qyEx33[pat-3::GFP; genomic ina-1], gmIs5[ina-1::GFP], qyEx41[pat-3::GFP; genomic ina-1], qyIs42[pat-3::GFP; genomic ina-1], qyIs43[pat-3::GFP; genomic pat-2], qyIs44[pat-3::GFP; genomic pat-2], qyIs25[cdh-3>mCherry:: PLCδPH ], syIs157[cdh-3>YFP), zuIs20(par-3::GFP), qyIs103[fos-1a>rde-1]; qyIs110[egl-17>HA-βtail], qyIs125[unc-62>rde-1]; LGI, muIs27[GFP::mig-2], unc-40(e271), rhIs2(pat-3::GFP); LGII, syIs77(zmp-1::YFP), qyIs17[zmp-1>mCherry], qyIs23[cdh-3>mCherry:: PLCδPH ], rrf-3(pk1426); LGIII, dpy-2(e1), pat-3(rh54), ncl-1(e1865), ina-1(gm144), ina-1(gm39), pha-1(e2123ts), unc-119(ed4), rhIs23[hemicentin::GFP], syIs129[hemicentin-ΔSP::GFP]; LGIV, qyIs10[lam-1::GFP], qyIs15 [zmp-1>HA-βtail], qyIs48 [zmp-1>HA-βtail], jcIs1[ajm-1::GFP]; LGV, rde-1(ne219), fos-1(ar105), qyIs50[cdh-3>mCherry::moeABD]; LGX, syIs123[fos-1a::YFP-TL], him-4(rh319), qyIs66[cdh-3>unc-40::GFP], qyIs7[lam-1::gfp], qyIs24[cdh-3>mCherry:: PLCδPH], syIs59[egl-17>CFP], unc-6(ev400).
A complete analysis of AC invasion defects in genes whose RNAi knockdown produce a Pvl phenotype will be described elsewhere. Double stranded RNA (dsRNA) targeting ina-1, pat-3, talin, pat-4,pat-2 and fos-1 used in this study was delivered by feeding to rrf-3 and fos-1(ar105) mutants as described (Sherwood et al., 2005). RNAi knockdown was confirmed by the presence of an embryonic Pat phenotype and defect in AC invasion; RNAi vectors were sequenced to verify the correct insert. Uterine-specific RNAi was conducted by expressing RDE-1 protein under the control of the fos-1a promoter (expressed at the mid L2 stage through the time of AC invasion) in rde-1(ne219) mutants. rde-1 is a necessary component of the RNAi pathway in C. elegans; expression of RDE-1 using tissue-specific promoters in an rde-1 mutant restores RNAi sensitivity only in the tissue expressing the RDE-1 protein (Qadota et al., 2007).
Transgenic L3 stage animals were synchronized and fixed for HA-βtail and HA-βΔ localization as previously described (Sherwood and Sternberg, 2003). Mouse Anti-HA monocolonal antibody clone HA-7 (Sigma) was used at 1:200 and FITC-conjugated Goat anti-mouse IgG secondary antibodies (Jackson Immunoresearch Laboratories) were used at 1:500. Quantification of fluorescence intensity in fixed and stained animals revealed a two-fold higher expression level of HA-βtail in strain qyIs15 versus qyIs48 (p < 0.05, one-tailed, unpaired t-test), consistent with the more severe invasion defect of qyIs15. Quantification of fluorescence intensity of GFP driven by the AC-specific regulatory element of zmp-1mK50–51 from the P6.p one-cell stage at the early L3 to the P6.p four-cell stage at mid L3 showed a 7-fold increase in expression levels of GFP over this time.
To perform a mosaic analysis of pat-3 function in the AC, 10ng of the plasmid encoding full-length PAT-3::GFP was injected into dpy-1(e1) pat-3(rh54) ncl-1(e1865); sDp3(III:f) animals. Rescued F1 progeny were identified by the Dpy phenotype and PAT-3::GFP. The presence of pat-3(rh54) was confirmed in rescued animals by the Pat phenotype. AC invasion was scored at the P6.p four-cell stage and the presence of the transgene in the AC determined by the expression of PAT-3::GFP. We were able to detect mosaic loss of the transgene in F1 progeny where maternal contribution of pat-3 is likely present; however, we were unable to obtain stable F2 lines having mosaic loss of pat-3 in the vulval cells or AC, possibly reflecting an essential function for stable pat-3 inheritance for reproduction or viability.
Images were acquired using a Yokogawa spinning disk confocal mounted on a Zeiss AxioImager microscope with a 100X Plan-APOCHROMAT objective run by iVision software (Biovision Technologies, Exton, PA) or with a Zeiss AxioImager microscope with a 100X Plan-APOCHROMAT objective equipped with a Zeiss AxioCam MRm CCD camera and run by Axiovision software (Zeiss Microimaging Inc., Thornwood, NJ). Images were processed and overlaid using Photoshop 8.0 (Adobe Systems Inc, San Jose, CA), and 3-Dimensional projections were constructed using IMARIS 6.0 (Bitplane Inc., Saint Paul, MN).
AC invasion was scored as previously described (Sherwood et al., 2005). Polarity in wild-type animals, ina-1 mutants, RNAi treatments and HA-βtail animals was determined using the ratio of the average fluorescence intensity from a five-pixel-wide linescan drawn along the invasive and non-invasive membranes of ACs expressing MIG-2::GFP, UNC-40::GFP, mCherry::moeABD, mCherry::PLCδPH and PAR-3::GFP strains (n ≥ 15 animals for each) using Image J 1.40g software. Scoring of apical AJM-1::GFP in the AC was performed as described (Ziel et al., 2009) (n = 20 animals for each). Fluorescence intensities of AC expression of transcriptional reporters for zmp-1 (syIs77), hemicentin (syIs129), cdh-3 (syIs157) and translational reporters for fos-1a (syIs123) and hemicentin (rhIs23) were determined using Image J 1.40g software (n = 15 animals for each). In all cases a two-tailed, unpaired Student’s t-test was used to determine statistical significance of changes in expression or polarity.
3-Dimensional reconstructions were generated from confocal z-stacks of ACs expressing the F-actin probe, mCherry::moeABD, using Imaris 6.0 (Bitplane Inc., Saint Paul, MN). Isosurface renderings of mCherry::moeABD were created by setting a threshold fluorescence intensity value such that the renderings were constructed only in place of the dense network of F-actin at the invasive membrane of wild-type ACs. Identical settings were used in unc-6(ev400) and ina-1(gm39) mutants. Quantitative measurements of the volume and integrated fluorescence intensity inside isosurface renderings were made using the Imaris 6.0 software package (n = 15 animals for each background). Quantification of the total levels of mCherry::moeABD present in the AC in wild-type, unc-6 and ina-1 mutants confirmed similar levels of expression in all three backgrounds.
We are grateful to J. Schwarzbauer for the pat-3 HA-βtail vector and PAT-2::GFP strain, J. Plenefisch for the βPAT-3::GFP vector, S. Johnson for imaging advice, the Caenorhabditis Genetics Center for providing strains, and N. Sherwood, J. Croce and D. Matus for comments on the manuscript. This work was supported by a Basil O’Connor Award, Pew Scholars Award and NIH Grant GM079320 to D.R.S.
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