If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Basement membrane linkages (B-LINKs) attach adjacent tissues in C. elegans
Hemicentin is required to link tissue through basement membranes
Intracellular plakin tethers hemicentin to the region of B-LINK
Integrin promotes hemicentin punctae formation and B-LINK
Basement membrane (BM), a sheet-like form of extracellular matrix, surrounds most tissues. During organogenesis, specific adhesions between adjoining tissues frequently occur; however, their molecular basis is unclear. Using live-cell imaging and electron microscopy, we identify an adhesion system that connects the uterine and gonadal tissues through their juxtaposed BMs at the site of anchor cell (AC) invasion in C. elegans. We find that the extracellular matrix component hemicentin (HIM-4), found between BMs, forms punctate accumulations under the AC and controls BM linkage to promote rapid invasion. Through targeted screening, we identify the integrin-binding cytolinker plakin (VAB-10A) and integrin (INA-1/PAT-3) as key BM-BM linkage regulators: VAB-10A localizes to the AC-BM interface and tethers hemicentin to the AC while integrin promotes hemicentin punctae formation. Together, plakin, integrin, and hemicentin are founding components of a cell-directed adhesion system, which we name a BM-LINKage (B-LINK), that connects adjacent tissues through adjoining BMs.
Basement membrane (BM) is a conserved, sheet-like extracellular matrix that surrounds most tissues, providing structural support, barrier functions, and a surface for signaling activities (
), little is known about mechanisms regulating adhesion between BMs of neighboring tissues. Though poorly characterized, connections between juxtaposed BM-encased tissues frequently occur. During organogenesis, for example, the BMs lining the sides of the developing optic cup meet, remodel, and fuse to close the optic fissure (
). Whether the AC utilizes a mechanism to stably link the adjacent BMs and fix the position of the uterine and vulval tissues during invasion, is unknown.
A potential candidate for regulating BM-BM interactions is hemicentin (known as HIM-4 in C. elegans), a large (5,198 residue) extracellular matrix protein. Hemicentin accumulates between BMs of neighboring organs in C. elegans, and its loss leads to defects in the alignment and association between tissues (
). The specific function(s) of hemicentin between tissues, however, is unclear, as it has not yet been possible to dynamically characterize the interactions of BM-encased tissues where hemicentin is found. During C. elegans uterine-vulval attachment, hemicentin is secreted by the AC and localizes to punctae in the BM under the AC prior to invasion. In worms lacking hemicentin function, AC invasion is delayed, indicating that hemicentin facilitates invasion (
Using genetic analysis, transmission electron microscopy, tissue shifting experiments, and live-cell imaging of BM dynamics during AC invasion, we find that the matrix component hemicentin mediates a specific, spatially restricted attachment between the gonadal and ventral BMs under the AC prior to and during invasion. With live-cell imaging we show that this BM-BM linkage is critical for rapid, coordinated invasion through the juxtaposed gonadal and ventral BMs. Through a focused RNAi screen we also identify the plakin isoform VAB-10A (
), which links receptors such as integrin to the cytoskeleton, as a cellular regulator of BM-BM linkage. We show that VAB-10A within the AC is polarized toward the BM, required for BM-BM adhesion, and anchors hemicentin punctae to the region of BM beneath the AC. We also demonstrate that the extracellular matrix receptor integrin (INA-1/PAT-3) is required for the assembly of hemicentin punctae and BM-BM linkage. Finally, we reveal a similar BM-BM connection that appears to link the uterine utse and epidermal seam cells together, fastening the uterine tissue to the epidermis. Together, these data reveal founding members of an adhesion system we name a BM LINKage (B-LINK), which forms a cell-directed connection between the BMs of neighboring tissues.
A BM-BM Linkage Forms Specifically under the Anchor Cell prior to Invasion
AC invasion is precisely timed and coordinated with divisions of the underlying P6.p vulval precursor cell and its descendants (
). AC invasion initiates at the late P6.p two-cell stage, and is completed approximately 90 min later during the P6.p four-cell stage. To determine how the AC invades across the neighboring gonadal and ventral BMs (Figure 1A), we first examined the association of these BMs under the AC using a functional GFP translational fusion of the lone laminin β subunit (laminin::GFP), whose localization is indistinguishable from immunolocalized laminin (
). The gonadal and ventral BMs are usually tightly juxtaposed and cannot be resolved by light microscopy. In rare cases, however, we observed worms in which the uterine and vulval tissues were slightly separated allowing resolution of each BM. Approximately 2 hr prior to BM breach (P6.p one- and early two-cell stage), we were able to identify the ventral and gonadal BM as individual, unattached structures (n = 7/9 animals; Figure 1B). This is consistent with observations that the uterine and vulval tissues slide independently at this time (
). During the P6.p two-cell stage, however, more animals showed BM-BM linkage, (13/24 animals; Figure 1B). By the late P6.p two-cell stage (when the AC initiates invasion) most animals had BM-BM linkage under the AC (4/6 animals). Following invasion (P6.p four-cell to P6.p eight-cell stage), the gonadal and ventral BMs fused at the edge of the BM breach created by the AC (n = 19/19; Figure 1B) (
). These observations suggest that the two BMs are connected prior to invasion.
To further examine if the BMs might be connected prior to invasion, we photobleached 1–2 μm wide landmarks in the region of the juxtaposed BMs under the AC. Following photobleaching, we laterally slid the worms to induce transient misalignment between the uterine and vulval tissues. This optical marking strategy allowed us to determine whether the bleached regions of BM remained juxtaposed (implicating linkage) or separated (indicating lack of connection). During the P6.p one-cell stage, the bleached regions of BM could shift relative to one another (n = 4/8 animals; Figure 1C). In contrast, at the mid P6.p two-cell stage, the bleached regions of BM remained aligned (n = 9/9, p < 0.05 compared to P6.p one-cell stage, Fisher exact test; Figure 1C). These optical highlighting experiments further support the idea that the uterine and vulval BMs become linked under the AC prior to invasion.
We hypothesized that the BMs could fuse into a single sheet or remain two distinct sheets held together by an adhesive structure. To distinguish between these possibilities, we examined the BM beneath the AC using transmission electron microscopy. We cut longitudinal serial sections across the entire width of the gonad, allowing us to use the surrounding tissue to identify the AC and establish the precise developmental stage of the worm. Electron micrographs revealed punctate electron dense structures between the gonadal and ventral BMs prior to invasion (n = 4/5 animals, P6.p two-cell stage; Figure 1D and Figure S1 available online). These structures spanned the space between the gonadal and ventral BMs and had an average width of 130 ± 10 nm (±SEM, n = 5 punctae with entire structure visible). Thus, prior to invasion, the gonadal and ventral BMs become linked under the AC by an adhesive structure.
Hemicentin Is Present at the Site of BM-BM Adhesion
We were next interested in determining the molecular mechanism of BM-BM adhesion. Because hemicentin is secreted by the AC prior to invasion and is known to localize between BMs, we hypothesized that hemicentin might link the BMs under the AC (
). Hemicentin was deposited at low levels throughout the ventral and gonadal BMs, and accumulated in track-like lines at the edges of the ventral BM (Figure 2A). Notably, prior to invasion (from P6.p one-cell stage to mid P6.p two-cell stage), hemicentin accumulated in punctae precisely under the AC (Figures 2A, 2B, and S2). The approximate size of the hemicentin structures was similar to the size of the BM-BM adhesions observed with electron microscopy (187.2 ± 0.8 nm, n = 104 structures in 23 worms; Figure S2). Time-lapse imaging revealed that hemicentin structures were static and new punctae continued to form throughout the course of AC invasion. Once formed, the hemicentin structures remained within the BM under the AC and did not disassemble during invasion (n = 132 punctae in 22 animals; Figure 2B; Movie S1). Optical highlighting of the BM components laminin and type IV collagen has recently shown that the AC physically displaces BM during invasion (
). Consistent with a strong association with the BM, the hemicentin punctae were also physically displaced by the invading AC (Figure 2A; Movie S2). Thus, hemicentin punctae are tightly associated with the BM and are present at the time and location of BM-BM adherence.
BM-BM Adhesion Is Hemicentin Dependent
To determine if hemicentin plays a role in BM-BM adhesion, we analyzed the gonadal and ventral BMs in animals harboring a null mutation in hemicentin (him-4(rh319)) (
). In contrast to wild-type animals, the BMs beneath the AC could be independently resolved prior to and during invasion (n = 10/10 animals; Figures 2C and 2D), suggesting that the specific linkage between the BMs is lost in hemicentin mutants. Interestingly, the BMs still fused into a continuous sheet following invasion, indicating that BM fusion is not dependent on hemicentin (n = 10/10 animals; Figure 2C).
To further examine the role of hemicentin in BM-BM linkage, we performed tissue-shifting experiments. In contrast to wild-type animals, loss of hemicentin allowed the gonadal and ventral BMs to shift beneath the AC at the P6.p two-cell stage (n = 3/4, p < 0.05 compared to wild-type, Fisher’s exact test; compare Figures 1C and 2E). Furthermore, electron microscopy revealed that the structures spanning the BMs underneath the AC were no longer present in him-4(rh319) mutants (n = 4/4 ACs at the P6.p two-cell stage; Figure 2F). We conclude that hemicentin is required for BM-BM adhesion.
Anchor Cell Invasion Is Delayed when BM-BM Adhesion Is Disrupted
To explore the role of hemicentin and BM-BM adhesion in invasion, we visualized BM removal during AC invasion, utilizing time-lapse analysis from the ventral perspective for increased resolution (
). We found that wild-type animals usually breached the gonadal and ventral BMs before widening a gap in both BMs (n = 6/10 time-lapsed animals; Figure 3A). While hemicentin mutants (him-4(rh319)) initiated invasion at the same time as wild-type animals, they initially breached only the gonadal BM and widened the gap in the gonadal BM alone (n = 10/10 time-lapsed animals; Figures 3A and S3). him-4(rh319) mutants eventually breached the ventral BM, but in a delayed manner (Figure 3B; Movie S3). To further quantify the extent of this defect, we assessed ventral BM breach in relation to the size of gonadal BM breaches. We found that in worms with gonadal BM breaches between 1–5 μm2 in area (the smallest detectable size), the majority of wild-type animals had already breached both BMs (Figure 3C). In contrast, only a few him-4(rh319) mutants with similarly sized gonadal BM breaches had breached both BMs (Figure 3C). Further, in worms with larger gonadal BM gaps (10–25 μm2), only 67% of hemicentin mutant worms breached the ventral BM, while all wild-type worms had breached both BMs (Figure 3C). Notably, ACs lacking hemicentin opened the gonadal BM at approximately half the rate that wild-type worms opened both BMs (Figures 3D and 3E). Once the AC of hemicentin mutants initiated ventral BM breach, the ventral BM gap opened quickly (Figures 3B, 3D, and 3E; Movie S3). Our data suggest that removing hemicentin eliminates BM-BM adhesion, and that this adhesion is required for efficient invasion.
Our tissue shifting, transmission electron microscopy (TEM), and visual analysis suggested that removing hemicentin completely eliminates BM-BM adhesion, and that this adhesion is required for efficient invasion. To test the idea that the hemicentin dependent BM-BM linkage promotes efficient invasion, we examined genetic interactions between hemicentin and the netrin receptor UNC-40/DCC. Loss of unc-40 results in a delay in invasion from a distinct invasion defect, an inability to generate a protrusion that crosses the BM (
). We hypothesized that if the hemicentin dependent BM-BM adhesion promotes efficient invasion, the defect in invasion would be dramatically enhanced in animals harboring mutations in both him-4 (hemicentin) and unc-40. Supporting this idea, AC invasion was nearly abolished in unc-40; him-4 double mutant animals (93% failed to invade in unc-40(e271); him-4(rh319) versus 43% in unc-40(e271); n = 28 and 58, respectively; p < 0.0005). Thus hemicentin and BM-BM adhesion promote rapid, coordinated breaching and clearance of the gonadal and ventral BMs.
VAB-10A Plakin Promotes BM-BM Adhesion
Previous work in C. elegans has shown that hemicentin is required to organize hemidesmosomes in the epidermis around the mechanosensory neurons and the utse syncytium (
). To identify possible additional components of the BM-BM adhesion system, we performed a targeted screen examining mutant alleles or RNAi mediated knockdown of genes associated with hemidesmosomes for defects in AC invasion or hemicentin punctae formation (Tables 1, S1, and S2) (
) had a 35% invasion defect (n = 56; Table 1), which is similar to the invasion defect caused by loss of hemicentin (him-4(rh319)). In contrast, RNAi-mediated knockdown of mua-3 caused a less penetrant defect (11% invasion defect, p < 0.05, n = 65; Table 1) and the only viable mua-3 allele, which disrupts hypodermal hemidesmosomes (
), did not affect AC invasion. Additionally, RNAi targeting mua-3 did not appear to cause a defect in BM-BM adhesion, as 86% of worms penetrated both BMs before widening the gap (n = 14 worms with gonadal BM breaches 1–5 μm2 in area). We thus focused on the role of vab-10.
). RNAi targeting vab-10a produced a similar reduction in invasion efficiency, providing further support that vab-10a plays a role in AC invasion (Table 1). Genetic loss of vab-10b is embryonic or early larval lethal (
). We thus utilized RNAi to target vab-10b during midlarval stages and found that reduction of vab-10b did not significantly affect AC invasion (Table 1). Together, these data suggest that the vab-10a isoform promotes AC invasion.
To determine if VAB-10A (plakin) plays a role in BM-BM adhesion, we examined worms in which the gonadal and vulval tissues were slightly separated, allowing independent resolution of the gonadal and ventral BMs. Similar to hemicentin mutants, the BMs were resolvable after treating with RNAi targeting vab-10a just prior to invasion, indicating an absence of adhesion between the two BMs (Figure 4A). To confirm that VAB-10A plays a role in linking BMs during AC invasion, we assayed worms for a delay in breaching the ventral BM following vab-10a knockdown. As in hemicentin mutants, we found that RNAi knockdown of vab-10a resulted in a specific delay in breaching the ventral BM (Figure 4B). Importantly, invadopodia, which are F-actin rich structures within the AC that mediate BM breaching (
). Additionally, we observed that vab-10a was enriched at the invasive cell membrane of the AC (Figures 4C and S5), suggesting that VAB-10A functions within the AC at the invasive membrane to promote BM-BM adhesion. To directly test whether VAB-10A functions within the AC, we used a C. elegans strain in which only uterine tissue, including the AC, is sensitive to RNAi (
). We found that knocking down vab-10a only in the uterine cells led to a 34% invasion defect, similar to the invasion defect caused by whole-body vab-10a knockdown (Table 1). In contrast, knocking down vab-10a in a strain where only the vulval cells are sensitive to RNAi did not significantly affect invasion (Table 1). Furthermore, expression of vab-10a::GFP specifically within the AC rescued the invasion defect of the vab-10 mutant (Table 1; Figure 4D). We conclude that VAB-10A functions within the AC to regulate BM-BM adhesion.
VAB-10A connects hemidesmosome-like fibrous organelles to the intermediate filament cytoskeleton in the C. elegans epidermis (
). To determine if VAB-10A might similarly connect the hemicentin based BM-BM adhesions to intermediate filaments, we conducted a preliminary RNAi screen examining each of the 11 intermediate filament genes for AC invasion defects, followed by a more extensive targeted screen in the RNAi sensitive rrf-3(pk1426) strain (Tables S3 and 1). RNAi mediated knockdown of IFC-2, IFD-1, and IFD-2 individually produced few AC invasion defects, however, when IFC-2, IFD-1, and IFD-2 were reduced in combination, the number of AC invasion defects increased, suggesting these proteins may function redundantly to promote invasion (Table 1). To more directly test if VAB-10A requires intermediate filaments to function in the AC, we created transgenic animals with an AC-expressed form of VAB-10A lacking all plectin repeat domains, which are responsible for intermediate filament binding (cdh-3>vab-10aΔPRD::GFP). Similar to full-length VAB-10A, VAB-10AΔPRD was enriched at the invasive membrane (Figure S4), however, VAB-10AΔPRD was not able to rescue AC invasion in the vab-10(e698) background (Table 1). Taken together, these data suggest that VAB-10A might mediate a connection between the hemicentin dependent BM-BM adhesions and intermediate filament proteins in the AC.
VAB-10A Anchors Hemicentin Punctae under the Anchor Cell
Reduction of vab-10a (plakin) and loss of him-4 (hemicentin) resulted in similar defects in BM-BM linkage, suggesting that they function together to link the gonadal and epidermal BMs under the AC. To further test this notion, we examined genetic interactions between both genes. If him-4 and vab-10a operated in the same genetic pathway to promote BM-BM adhesion, loss of vab-10a would not be expected to increase the invasion defect of him-4(rh319) animals. Consistent with this notion, RNAi depletion of vab-10a in him-4(rh319) worms did not enhance the AC invasion defect (Table 1).
To understand how hemicentin and VAB-10A might cooperate to regulate BM adherence, we next analyzed the effect of vab-10a RNAi on hemicentin localization. After vab-10a knockdown, hemicentin was still secreted and formed punctae in the BM under the AC. Unlike wild-type animals, however, where hemicentin punctae remained in the BM under the AC, we noted a change in hemicentin localization. In worms treated with vab-10a RNAi, hemicentin aggregates were no longer restricted to the footprint of the AC (Figures 4E and 4F). In contrast, treating worms with him-4 RNAi did not effect VAB-10A::GFP localization (n = 11/11 animals; Figure S4). Together, these data suggest that VAB-10A regulates BM-BM adhesions by indirectly anchoring hemicentin punctae to the region of BM beneath the AC.
Integrin Is Required to Assemble Hemicentin Punctae
The presence of an intracellular component (VAB-10A) and an extracellular component (hemicentin) required for BM-BM linkage under the AC suggested that a transmembrane receptor was also likely a requirement. We thus screened all known BM receptors in C. elegans for AC invasion defects using a combination of RNAi treatment, mutant analysis, and previously published data (
) to identify AC invasion defects (Table S3). While mutations or RNAi targeting most of the BM receptors did not affect AC invasion, RNAi targeting either subunit of the INA-1/PAT-3 integrin dimer disrupted AC invasion (Table S3). Like VAB-10A, integrin is enriched along the entire AC-BM interface, suggesting that integrin and VAB-10A both localize to the AC-BM interface adjacent to the extracellular hemicentin punctae (Figure S5). Reducing the function of integrin results in a failure to form invadopodia that breach the BM, causing a highly penetrant invasion defect (
). To determine how integrin regulates hemicentin deposition, we examined hemicentin accumulation after the specific disruption of integrin activity in the AC using a previously characterized AC-specific dominant-negative integrin construct (zmp-1>HA-βtail; (
). Notably, hemicentin did not accumulate intracellularly, indicating that integrin is not required for hemicentin secretion (n = 10/10 animals observed; Figure 5A). Furthermore, hemicentin was not deposited inappropriately along the lateral membrane of the AC, which occurs when AC polarity is disrupted (
). Increased levels of hemicentin, however, were found diffusely in the surrounding BM under the AC (Figures 5A, 5B, and S5). This suggests that integrin activity is required for punctae formation at the site of BM-BM adherence, but not for general secretion or deposition in the BM. Additionally, integrin has been shown to bind to the VAB-10A homolog plectin in vertebrates (
), thus we hypothesized that integrin might also be required for VAB-10A localization. Consistent with this, loss of integrin function in the AC (zmp-1>HAβtail) disrupted the polarized localization of VAB-10A along the AC-BM interface and resulted in diffuse VAB-10A throughout the cytoplasm (compare Figure 5C with 4C). Together, this data suggest that integrin is required to connect both hemicentin and VAB-10A to the site of BM-BM adhesion.
To determine if integrin regulates BM-BM adherence, we assayed worms treated with ina-1 RNAi for a delay in breaching the ventral BM relative to gonadal BM breach. Similar to loss of hemicentin and vab-10a, we found that following RNAi-mediated knockdown of ina-1 there was a delay in breaching the ventral BM (Figure 5D). Taken together, these data suggest that integrin regulates the formation and function of the BM-BM adhesion under the AC.
Hemicentin, VAB-10A, and Integrin Mediate Utse-Seam Cell Connection
We next wanted to determine if hemicentin, VAB-10A, and integrin link adjacent BMs in other contexts. We thus examined the BM-BM connection between the uterine utse and hypodermal seam cell, a linkage that helps maintain the uterus inside the animal during egg-laying (Figure 6A) (
). Notably, hemicentin localizes to the site of BM-BM attachment and eliminating hemicentin causes the utse and the seam cell to separate between their BMs, resulting in uterine prolapse or rupture through the vulva (
). Importantly, we found that in addition to hemicentin, VAB-10A and integrin were enriched at the site of utse and seam cell attachment (Figure 6B). Further, RNAi knockdown of vab-10a or ina-1 (integrin) in either the entire worm or specifically in the uterine tissue resulted in uterine prolapse (Figures 6C and 6D). Together, these results indicate that regulators of BM-BM adhesion under the AC also control the linkage between the BMs surrounding the utse and the seam cell.
Utilizing electron microscopy, live-cell imaging, and focused screening in C. elegans we identify here a cell-directed BM-BM adhesion system, which we name a B-LINK, that links tissues through their adjoining BMs (Figure 6E). Notably, the molecular regulators of this adhesion system—hemicentin, plakin, and integrin—are highly conserved. The widespread observation of connections between juxtaposed BMs and the conserved nature of the components suggest B-LINKs might be broadly utilized to attach adjacent tissues through BMs.
Previous research has indicated that the extracellular matrix protein hemicentin is required to mediate interactions between BM-lined tissues in C. elegans and zebrafish larvae (
). The exact function of hemicentin in these cases, however, has remained unclear because the timing of hemicentin activity and the exact nature of its role between BMs have been difficult to establish. We show here that the C. elegans gonadal AC secretes hemicentin into the BM just prior to its invasion into the vulva. Using tissue shifting experiments and live-cell imaging we find that hemicentin punctae link the juxtaposed gonadal and ventral BMs to facilitate the AC’s rapid and coordinated invasion through these BMs, a process critical for initiating direct uterine-vulval connection (
). Our data indicate that VAB-10A is required within the AC to form a functional B-LINK. In the absence of VAB-10A, hemicentin is still secreted and accumulates into punctae, but the structures cannot hold together the uterine and vulval tissues. VAB-10A might act in part to tether hemicentin to the AC, as reducing VAB-10A results in hemicentin punctae outside the AC footprint. In both vertebrates and C. elegans, plectin and VAB-10A have been implicated in linking transmembrane receptors to the intermediate filament cytoskeleton (
). Because the intermediate filament-binding domain of VAB-10A is required for VAB-10A function in the B-LINK, VAB-10A may function to connect the B-LINK to the intermediate filament network. Interestingly, the plectin (VAB-10A) knockout mouse exhibits defects in muscle attachment and destabilized hemidesmosomes in the epidermis, suggesting a conserved role for plectin in strengthening cell-matrix adhesion (
), is required to establish hemicentin punctae, recruit VAB-10A, and form a functional BM-BM linkage. A vertebrate homolog of VAB-10A, plectin, is known to directly interact with α6β4 integrin, a laminin-binding integrin present at hemidesmosomes (
). The conservation of integrin association with plectin, and role of INA-1/PAT-3 in hemicentin punctae formation, suggests that integrin may not only be required for B-LINK formation, but also to anchor B-LINKs to the AC’s cytoskeleton.
The B-LINK may represent a developmental switch in matrix function from a protective layer that facilitates tissue sliding to an adhesive link between tissues. Suggesting that the B-LINK may additionally reflect a common strategy for stably linking BMs, we found that the B-LINK components are required to connect the utse to the seam cells. In C. elegans, the muscle and epidermis are also connected through an intervening matrix (
). Molecularly, this attachment, referred to as a fibrous organelle (FO) has similarities to a B-LINK, including a requirement for VAB-10A and intermediate filaments, but also important distinctions from the B-LINK (
), and hemicentin, a critical component of the B-LINK, is not localized to FOs nor required for FO function. Intriguingly, the matrix-spanning attachment between the muscle and epidermis is required to transmit signals from the muscle to the epidermis, reinforcing cell-matrix adhesion in response to mechanical signals from the muscle (
). Thus it will be interesting to examine if the B-LINK provides more than a physical connection between tissues.
We expect that BM-BM adhesion is a common mechanism for coordinating interactions between neighboring tissues. BM-BM adhesion structures are likely important when BM lined tissues fuse to form a single structure, as occurs in optic cup development (
). The molecular identification of the B-LINK might thus have important implications in numerous developmental events, tissue structures, and human pathologies where BM-BM adhesions regulate organ construction and tissue functions.
Strains and Culture Conditions
Culturing and handling of C. elegans was performed as previously described (
). Wild-type animals were strain N2. In the text and figures, we designated linkage to a promoter with a (>) symbol and use a (::) symbol for linkages that fuse open reading frames. The following alleles and transgenes were used in this study: qyIs102(fos-1a > rde-1); qyIs127(laminin::mCherry); rol-6(su1006); urIs(rol-6(su1006), laminin::GFP); qyEx459(cdh-3 > vab-10aΔPRD::GFP, myo-2::GFP); qyEx460(cdh-3 > vab-10aΔPRD::GFP; unc-119(+))LGIvab-10(ju281); LGIIrrf-3(pk1426); qyIs23[cdh-3 > mCherry::PLCδPH]; vab-19(e1036); LGIIIunc-119(ed4), rhIs23[GFP::hemicentin]; syIs129(ΔSP-GFP-hemicentin); mua-3(rh195)LGIVmfIs70(lin-31 > rde-1); qyIs10[laminin::GFP]; eps-8(ok539); qyIs42[pat −3::GFP; genomic ina-1]; qyIs15[zmp-1 > HA–β -tail ]; LGVrde-1(ne219); qyIs50[cdh-3 > mCherry::moeABD]; LGXhim-4(rh319); pak-1(ok448); pix-1(gk416) and; git-1(tm1962).
Light Microscopy, Image Acquisition, Processing, and Analysis
Images were acquired using a Hamamatsu electron multiplied charge-coupled device (EM-CCD) camera and a Yokogawa CSU-10 spinning disk confocal mounted on a Zeiss AxioImager microscope with a 100 × Plan-Apochromat objective. The microscope was controlled by iVision software (Biovision Technologies) or microManager (
). Acquired images were processed to enhance brightness/contrast using ImageJ 1.40 g and Photoshop (CS6 Extended Adobe Systems), and smoothened using a 0.8 pixel radius Gaussian blur filter. 3D reconstructions were built from confocal Z-stacks, analyzed, and exported as (.mov) files using IMARIS 7.4 (Bitplane). Figures and graphs were constructed using Illustrator (CS3 Extended Adobe Systems) and JMP (Version 10, SAS Institute). Movies were annotated using Photoshop. Time-lapse imaging and invadopodia analysis was performed as described previously (
). Images were acquired on a Phillips CM12. The transverse section in Figure S1 is part of an archival series originally collected in the laboratory of Sydney Brenner (Medical Research Council Laboratory of Molecular Biology [MRC/LMB], Cambridge, England) and now curated by the Hall lab.
Landmark Photobleaching and Tissue Shifting
Worms were photobleached on an inverted Zeiss LSM 510 confocal equipped with a 100× Plan-Apochromat objective. Regions of interest were bleached using 500 iterations of a 488 Argon/2 laser at 100% power. Worms were then imaged on the Yokogawa CSU-10 spinning disk confocal, moved approximately 1 mm across the agar pad by sliding the coverslip along the longitudinal axis of the worm, and imaged again to detect a transient shift in tissue alignment.
Uterine-Vulval Tissue Separation
Worms were incubated on 5% agar with 0.01 M NaN3 for up to one hour. Under these conditions, the uterine and vulval tissues will occasionally separate, allowing visualization of the distinct BMs. To measure the average distance between the BMs, the BMs were traced in ImageJ and connected by perpendicular lines. The area of the resulting rectangle was divided by the Feret’s diameter, which was assumed to approximate the width of the rectangle.
Analysis of Anchor Cell Invasion and Hemicentin Punctae
The efficiency of AC invasion was analyzed at the P6.p four-cell stage as previously described (
). Worms were staged using a combination of divisions of P6.p and the ventral uterine cells, distal tip cell migration, and development of the surrounding tissues. Invasion was scored as “complete invasion” if the BM was removed beneath the AC, “partial invasion” if the BM was breached but not cleared beneath the AC, and “no invasion” if there was no BM breach. The area of BM breach was measured in ImageJ by creating a maximum projection from a ventral perspective confocal Z-stack and using an intensity threshold to select the area of gonadal BM breach as indicated by decreased laminin::GFP signal.
To measure the volume of GFP::hemicentin punctae, 3D reconstructions of the AC-BM interface were made from confocal Z-stacks. Isosurface renderings of GFP::hemicentin were created by setting a consistent threshold fluorescence intensity value such that the diffuse BM signal was excluded, but the bright hemicentin punctae were not. The line-shaped structures on either side of the ventral BM were excluded from our analysis, as they appear to represent a different population of hemicentin. Quantitative measurements of hemicentin volume were performed in Imaris 7.4.
All statistical analysis was performed in JMP version 9.0 (SAS Institute), using either a two-tailed unpaired Student’s t test, a Wilcoxon Rank-Sum test, or a two-tailed Fischer’s exact test. Confidence intervals reported are 95% confidence intervals with a continuity correction. Figure legends specify which test was used.
We thank N. Medvitz and S. Miller at Duke Electron Microscopy Services; K. Nguyen at the Center for C. elegans Anatomy; B. Vogel and M. Labouesse for strains and discussions, and L. Kelley, L. Lohmer, D. Matus, and J. Pelton for comments on the manuscript. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The TEM image shown in Figure S1 comes from the laboratory of Sydney Brenner at MRC/LMB in Cambridge England, provided courtesy of John White and Jonathan Hodgkin. Curation of the Brenner EM archive in David Hall’s laboratory in New York is supported by NIH grant OD010943 to D.H.H. This work was supported by The Pew Scholars Program in the Biomedical Sciences and NIH Grants GM079320 and GM100083 to D.R.S. and a National Science Foundation Graduate Research Fellowship to M.A.M.