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Vol. 18, Issue 4, 1153-1166, April 2007
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*Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel; Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and ||Center for C. elegans Anatomy, Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461
Submitted September 25, 2006;
Revised January 2, 2007;
Accepted January 8, 2007
Monitoring Editor: Peter Walter
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Heiman and Walter, 2000; Wassarman et al., 2001; Abmayr et al., 2003; Shemer and Podbilewicz, 2003; Chen and Olson, 2005). In C. elegans, diverse cell fusions essential for organ formation and cell fate determination have recently become paradigms for developmental cell fusion (Podbilewicz and White, 1994; Mohler et al., 1998; Nguyen et al., 1999; Sharma-Kishore et al., 1999; Podbilewicz, 2000; Alper and Kenyon, 2002; Mohler et al., 2002; Shemer and Podbilewicz, 2002, 2003; Witze and Rothman, 2002). Cell fusion functions to sculpt organs and to accomplish defined body shapes (Sharma-Kishore et al., 1999; Witze and Rothman, 2002; Shemer and Podbilewicz, 2003). eff-1 (epithelial fusion failure) was identified as a gene encoding type I membrane proteins required for diverse epithelial cell fusion reactions. Mutations in eff-1 result in failure of epithelial cell fusion and developmental defects in organs where cell fusion normally occurs (Mohler et al., 2002; Shemer, 2002; Shemer and Podbilewicz, 2002). The activity of eff-1 is strongly regulated by homeobox containing genes (Shemer and Podbilewicz, 2002, 2003; Cassata et al., 2005). Other transcription and signaling factors have been shown to control the cell fusion process in C. elegans (Nilsson et al., 1998; Ch'ng and Kenyon, 1999; Shemer et al., 2000; Alper and Kenyon, 2001; Chen and Han, 2001; Koh and Rothman, 2001; Alper and Kenyon, 2002; Koh et al., 2002; Zhao et al., 2002; Shemer and Podbilewicz, 2003). The specific roles of different proteins that mediate and control cell fusion and the pathway of this fusion reaction remain unexplored. eff-1 induces cell fusion at distinct developmental times in different organs. Expression of eff-1 using a heat-shock promoter results in cell fusion between different cell types (Shemer et al., 2004; del Campo et al., 2005).
Interestingly, genes homologous to eff-1 have not been identified in Drosophila or vertebrates (Shemer and Podbilewicz, 2003; Podbilewicz and Chernomordik, 2005; Podbilewicz, 2006; Podbilewicz et al., 2006). Genetic screens in Drosophila have identified several genes required for myoblast fusion. Using elegant ultrastructural and developmental studies, it has been determined that the steps affected by these mutants include myoblast differentiation, acquirement of fusion competence, and recognition and adhesion between myoblasts (Doberstein et al., 1997; Abmayr et al., 2003; Chen et al., 2003). However, genes necessary and sufficient for the actual merger of two plasma membranes into one have not been reported in other developmental cell fusion reactions outside syncytin-mediated fusion between human trophoblasts (Mi et al., 2000) and eff-1–mediated cell fusion (Shemer et al., 2004; del Campo et al., 2005; Podbilewicz, 2006; Podbilewicz et al., 2006).
Here we hypothesized that since embryonic cell divisions in C. elegans are tightly controlled and invariant (Sulston et al., 1983), we may also find an ordered pattern of cell–cell fusions within the same large syncytium that will be nearly constant between individuals. We apply experimental approaches used for model fusion reactions (Stegmann et al., 1990; Phalen and Kielian, 1991; Frey et al., 1995; Hoekstra et al., 2002; Blumenthal et al., 2003; Chernomordik and Kozlov, 2003; Gibbons et al., 2003; Hu et al., 2003; Jahn et al., 2003; Bonifacino and Glick, 2004; McInerney et al., 2004) to address this hypothesis in living C. elegans. We analyzed cell fusion kinetics in developing embryos and dissected cell membrane fusion into defined stages. Surprisingly, we demonstrate that in the embryonic epidermis of C. elegans a variable cell fuses first, and for each fusogenic cell the anterior and posterior membrane domains fuse independently and asymmetrically. In addition, we found that stable intermediates in late stages of epidermal syncytia formation can be found in larvae and adults of partial loss-of-function eff-1 mutants. Thus, we show that eff-1 is required to initiate, expand and complete syncytia formation in the epidermis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The resolution of our confocal microscope is 
250 nm. We used and show projections (flattened images) for each time point in all kinetic analyses. Four-dimensional stacks were also analyzed and archives of the complete original data sets are available for further analyses.
The cells responsible for the elongation of the embryo are the hypodermal cells (Sulston et al., 1983; Priess and Hirsh, 1986) and we measured the elongation of the lateral seam cells of the head (Rabin and Podbilewicz, 2000). The whole body elongation rate is 2.5-fold faster than the head elongation rate (Priess and Hirsh, 1986).
The largest syncytium (hyp7) is initiated in the embryo during elongation, and the events occurring from the comma stage to the 1.5–fold stage have been characterized here (Figures 1 and 2). During this time window the wild-type embryo does not move, allowing us to follow the kinetics as described below.
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Kinetics of Cell Fusion: The Normal Sweep Method
We developed a semiautomated method to monitor membrane fusion, leading to disappearance of GFP-marked cell junctions in a single C. elegans embryo (Figure 3 and Supplemental Materials and Methods). Using the semiautomated normal sweep method, we could quantify the membrane fusion for each junction as follows:
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Quantifying the Kinetics of Cell–Cell Fusion
As shown in Figure 3A, first we defined the junctions (blue lines) as circular arcs fitted through three points (red circles defined roughly along the dorsal midline and at the edges of the cells in the dorsal view) that were determined manually for each junction in each individual frame. This is based on the simple mathematical concept that a unique circle passes through every three noncollinear points. Thus, the arc defining each junction was a part of unique circle passing through manually defined points. Because it is impossible to assign fixed geometry to a live biological specimen, especially when monitoring it in real time, each frame had its own set of arcs characterizing the individual junctions of the embryo. Knowing the total number of pixels along each arc (junction), we measured the apical junction (AJ) discontinuity, representing expanding fusion pores, by looking at appearance of blank pixels (pixel value < threshold; see below), as shown in Figure 3, B and C, by yellow circles along the arcs (blue lines). Because of junctions in live embryos not following strict circular-arc geometries, for each pixel coordinate along each arc we applied a normal sweep (depending on how good the "blue" arc described a junction by eye): a pixel is scored as blank (i.e., shown in yellow, Figure 3) only if 2–4 pixels above it and below it along the normal from the center of the circle corresponding to that arc and if the pixel value itself is lower than the threshold. Figure 3B shows the normal along a single point on the arc, which is always perpendicular to the tangent at that point on the arc. The threshold was uniformly selected for each junction for all frames as being 1–2 times the average intensity of the whole image, depending on the brightness of the junction (because all the junctions do not have the same GFP intensity). Figure 3C shows the "initiated macrofusion" represented by the yellow circles in absence of the blue line (junction). Supplementary Figure S1 shows four frames from a movie of a developing embryo in which yellow circles are seen to appear as the junctions "dissolve."
Using our normal sweep method, we found that each fusion process for different junctions in different embryos grown at different temperatures follows sigmoidal kinetics (see examples in Figures 4 and Supplementary Figure S2). To extract the kinetic parameters of the curves we defined the fusion onset, i.e., the lag time (t1) and the time required to reach the sigmoidal saturation (t2). From these, the time required for the termination of the fusion event after the onset was determined as the macrofusion time (t2 – t1). This kinetic parameterization for understanding sigmoidal curves was introduced and has been explained in detail (Mittal et al., 2003).
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50 nm/min at 11°C to 1000 nm/min at 25°C. The Arrhenius plot obtained by this method gave a slope used to estimate an apparent activation energy of 29.8 kcal/mol (r2 = 0.9474).
Electron Microscopy
Transmission electron microscopy was performed as described on fourth larval stage and adult eff-1(hy21ts) mutant animals grown at the semipermissive temperature 20–23°C (Shemer et al., 2004). Two tests were performed to determine whether a candidate microfusion site is indeed a cytoplasmic bridge that may have resulted from an incomplete membrane fusion event. First, the specimen was tilted in the transmission electron microscope (TEM) by ±10° to sharpen up the view of the plasma membrane bordering an apparent cell bridge, further tilting by ±50° showed whether the cell bridge is real. That is to say, if even when the specimen was tilted to extreme angles, we did not find an intact plasma membrane blocking the bridge, this was recorded as a bona fide cytoplasmic bridge. The second test was based on reconstructions in serial sections as previously described (Nguyen et al., 1999). In many instances we found apparently good bridges that failed to meet these criteria.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The dynamic behavior of AJs in C. elegans is a reliable assay for cell fusion of epithelial cells (Kenyon, 1986; Priess and Hirsh, 1986; Baird et al., 1991; Podbilewicz and White, 1994; Mohler et al., 1998, 2002; Nguyen et al., 1999; Sharma-Kishore et al., 1999; Alper and Kenyon, 2002). To follow cell fusion by confocal microscopy, we recorded time lapse 4D movies and followed the disappearance of the AJM-1::GFP reporter from the AJ between fusing cells (Hird and White, 1993; Mohler et al., 1998; Rabin and Podbilewicz, 2000; Koppen et al., 2001; Figure 1, A and B, Supplementary Movies S1–S10). Loss of the cell contact zone upon fusion was also followed using immunofluorescence and immunoelectron microscopy with the mAb MH27 that recognizes both endogenous AJM-1 and transgenic AJM-1::GFP proteins (Francis and Waterston, 1991; Koppen et al., 2001). In addition, cytoplasmic content mixing and membrane loss precede AJ disappearance (Mohler et al., 1998, 2002; Shemer et al., 2004), validating AJM-1::GFP loss as an assay for cell–cell fusion in C. elegans.
To quantitatively analyze cell fusion during elongation of C. elegans embryos, we determined the number of cell pairs that initiated fusion over time. We found a gradual increase in the accumulated number of cells with detectable disruption of AJ continuity. Figure 1C shows the time course for initiation of cell fusion events in a single elongating embryo incubated and imaged at 23°C (see also Figure 2 and Supplementary Movies). Embryonic elongation in C. elegans is characterized by defined stages starting with a comma shape (50-µm length; time = 0 in Figure 2) stage through 1.5-, 2-, 3-, and 4-fold (200-µm length) stages of elongation (Sulston et al., 1983; Priess and Hirsh, 1986). Most embryonic epidermal cell fusion events occur from comma to twofold stages (Podbilewicz and White, 1994).
idf-1 Mutant and Low Temperatures Block Cell Fusion
idf-1(zu316), an embryonic lethal mutant, was isolated in a screen for elongation defective embryos (Costa and Priess, personal communication). We found immunostained idf-1(zu316)–arrested embryos to have fewer dorsal fusions than in wild-type using the MH27 mAb. We genetically mapped idf-1(zu316) to the left arm of chromosome X and found that idf-1 acts recessively. Mutant embryos arrest between the 1.5–3-fold stage of elongation with fewer cell fusions in the dorsal hypodermal cells and posterior defects (Figure 1E; n > 1000). To test whether idf-1(zu316) is a hypomorph or a knockout, we constructed an idf-1 over deficiency (deletion) strain and found that the arrested idf-1(zu316)/deficiency embryos arrested earlier than the homozygous idf-1(zu316) embryos (data not shown). Thus, idf-1(zu316) is a hypomorph. The molecular identity of idf-1 has not been determined. Irregular cell fusion events occurring in idf-1 mutants were retarded compared with the timing of expected fusion events in wild type. idf-1(–) cells fused after the embryos began twitching and not before activation of the body wall muscle contraction (n > 100; Supplementary Movie S9). When comparing the dorsal cell fusion defects between different idf-1(–) arrested embryos we found that cells in the anterior dorsal hypodermis have a higher probability of remaining unfused than cells in the posterior hypodermis (Figures 1E, 2F, and 7A). However, all 21 dorsal epithelial cells that normally form the two major syncytia, hyp6 and hyp7 (Figure 1), are able to express the Idf phenotype (Figure 7A), suggesting that idf-1 activity is involved as part of the dorsal fusion machinery or in its regulation and not as a regional regulator of cell fusion affecting specific cells along the anterior-posterior axis.
Low temperatures have been used to stabilize and identify important steps in viral fusion (Stegmann et al., 1990; Schoch et al., 1992; Chernomordik et al., 1998; Melikyan et al., 2000). To determine the effects of temperature on embryonic epidermal cell fusion in AJM-1::GFP embryos, we incubated comma stage embryos (time = 0; Figure 2) at different temperatures and recorded the changes in dorsal epidermal AJs upon fusion. Neither cell fusion nor embryonic elongation was observed at temperatures below 8.5°C, even after incubation for 24 h (n = 11; Figure 2A). Wild-type embryos incubated between 10 and 25°C reached the twofold stage (halfway through elongation) with most of the dorsal cells fused (n > 100; Figure 2, C and D). We found that cells failed to fuse in wild-type embryos grown at 8.5–10°C; these animals reached the twofold stage of elongation and the muscles twitched demonstrating that the embryos with cells that fail to fuse have some physiological activities (n = 19; Figure 2B).
To compare the "frozen" fusion phenotype obtained at 9°C to the cell fusion defects obtained in eff-1 and idf-1 mutants, we imaged embryonic elongation in eff-1(hy21) mutant embryos expressing AJM-1::GFP. In eff-1(hy21) embryos, dorsal epidermal cells completely failed to fuse at 15 and 25°C (Figures 1D and 2E). Although eff-1(–) embryos elongate and remain dumpy (short and fat) with bulged tail, lumpy body, and other morphological defects maintained during postembryonic development (Mohler et al., 2002; Shemer, 2002), wild-type embryos elongating at 9°C irreversibly arrest at the twofold stage. It seems that elongation is not dependent upon cell fusion, because eff-1 blocks cell fusion but not elongation. It appears that other defects unrelated to epithelial fusion failure may be responsible for the embryonic arrest observed in "frozen" and idf-1(–) embryos (e.g., microtubule depolymerization in the cold).
In summary, we can block cell fusion in wild-type cells at 9°C partially phenocopying eff-1 and idf-1 mutant embryonic cells.
Kinetics and Temperature Dependence of Cell Fusion In Vivo
To study intermediates of cell fusion we initiated a kinetic approach in the epidermis of the embryo. For a detailed characterization of cell fusion kinetics, we developed a computer-based method where cell fusion was measured by following the loss of AJ as the appearance of blank pixels in each junction and for each individual frame (see Materials and Methods; Figure 3 and Supplementary Figure S1). Using this semiautomated method, we found that each fusion process for six to nine distinct junctions, in embryos grown at different temperatures, follows sigmoidal kinetics (n = 74 cell pairs; Figures 4 and 5A and Supplementary Figure S2). To extract the kinetic parameters of the curves we defined the delay time or lag (t1) and the time required to reach the sigmoidal saturation (t2). From these, the time required for the termination of the fusion event after the onset was determined and defined as the macrofusion time (t2 – t1). This novel semiautomated method was validated by a different manual method to measure macrofusion (see Materials and Methods). Using the semiautomated method, we found that at the fusion-permissive temperatures the macrofusion rate [kMacrofusion = 1/(t2 – t1)] for each cell pair increased with temperature (Figure 5B). We have analyzed 5–6 pairs of fusing cells in 10 different embryos grown from 13 to 25°C (Figure 4).
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0.91; a similar linear slope was independently obtained using a different method to estimate the macrofusion rates over the range of 11–25°C (Figure 5C; see Materials and Methods).
To determine whether our kinetic analyses of cell fusion in vivo have a distinct behavior from other temperature-sensitive processes, we measured the rate of embryonic elongation at different temperatures using the same embryos where we measured the cell fusion rate. The initial rates of embryonic elongation changed from 1.3 nm/min at 8.2°C to 193.5 nm/min at 24°C (Rabin and Podbilewicz, 2000). The temperature dependencies of embryonic elongation have different slopes in different temperature ranges (Figure 5D). This is in contrast to a single apparent rate-limiting step for cell fusion in C. elegans embryonic hypodermis (Figure 5C), implying, along with the cold block of cell fusion (Figure 2B), that cell fusion and embryonic elongation are two distinct processes (see Discussion).
Kinetic Studies Reveal at Least Three Steps of Cell Fusion
Is fusion pore expansion (macrofusion) simply a continuation of the same processes that operate during the lag time? If this were the case, then one would expect the lag time to directly correlate with the macrofusion time. To test this, we plotted macrofusion versus lag times for each junction. For all temperatures studied we found no correlation between lag and macrofusion time, indicating that these are independent kinetic steps (Figure 5F). Moreover, the Arrhenius plot of the lag rates gave a very weak trend (Figure 5E) compared with macrofusion rates (Figure 5C), supporting the interpretation that the lag and macrofusion stages are different mechanistic steps in the process. Because lag and macrofusion are distinct stages, we infer a new stage that occurs during lag time. This microfusion stage cannot be resolved using live confocal microscopy of AJ disappearance. Thus, based on kinetics we have been able to dissect cell fusion into two distinct steps: an early step of microfusion followed by a stage of expanding gap or macrofusion, which we actually measure in our assay.
In various membrane fusion studies, the lag time has proved to be a very significant measurement to investigate when the system is "ready" to fuse (Stegmann et al., 1990; Bron et al., 1993; Danieli et al., 1996; Munoz-Barroso et al., 1998; Parlati et al., 1999). To analyze the lag phase in our system, we pooled the lag time parameters into a cumulative distribution showing a fraction of events that had already occurred by a given time (Supplementary Figure S3). To explain this lag distribution, we investigated different linear kinetic models (see Supplementary Material). We found that the simplest model that fits the data includes two distinct steps. Taken together, this kinetic model and the finding that the Arrhenius plot for lag does not show a linear relationship as would be expected for a single rate-limiting step process (Figure 5E), implying that initiation of cell fusion in C. elegans is at least a two-step process.
In summary, kinetic dissection of cell fusion in C. elegans embryos shows that this is at least a three-step process: Two steps during the lag stage leading into microfusion and a third step for the actual gap expansion between cells (macrofusion) that results in syncytia formation.
idf-1 and eff-1 Genetic Interactions during the Epidermal Cell Fusion Process
To investigate whether idf-1 and eff-1 interact genetically, we constructed a strain to study the double mutant idf-1; eff-1 and compared the embryonic phenotypes of the single mutants with the double mutants at 15 and 20°C (see Materials and Methods). In wild-type embryos most dorsal and ventral fusions take place by the twofold stage of elongation. idf-1(–) embryos arrest with the characteristic Idf phenotype, namely, irregular dorsal fusions. eff-1(–) embryos elongate with neither dorsal nor ventral epithelial fusions, and double mutants idf-1(–); eff-1(–) arrest at the 1.5–3-fold stage of elongation without any cell fusion (Figure 6). At 15 and 20°C the phenotype of the double mutant is a combination of the individual Idf and Eff phenotypes (see Materials and Methods). The lethality associated with idf-1(–) may not be directly related to cell fusion defects, but rather, might represent an additional function for idf-1.
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). To test whether idf-1 and eff-1 genes function redundantly to promote fusion, we constructed and tested hsp::eff-1; idf-1/+; ajm-1::gfp animals. We obtained the ectopic fusion phenotype in homozygous idf-1(–) embryos after heat-shock (n = 14/84). Taken together, these results are consistent with idf-1 and eff-1 genes acting independently to positively control cell fusion. An alternative explanation is that idf-1 positively controls eff-1.
Comparative Kinetics of Cell Fusion between idf-1(–) and idf(+) Embryos
To understand the kinetics of cell fusion, it would be useful to have mutations that affect the kinetic parameters. Even the weakest allele of eff-1 has a complete failure in the initiation of embryonic cell fusion, so we could not use eff-1 in kinetic analyses. However, in idf-1(–) embryos some dorsal epidermal cells are able to fuse several hours after the normal time of fusion during early embryonic elongation (Figure 7A). We imaged nearly 100 embryos and analyzed the kinetics of 10 fusion events from 2 independent embryos that were optimal for quantification using the semiautomated method. We found that these embryos arrested at the twofold stage with characteristic Idf phenotypes (Figure 1E) showing 10 pairs of dorsal cells fusing (Supplementary Movie S9 and Figure 2F). These cell fusions followed characteristic sigmoidal behaviors (see example in Supplementary Figure S2), showing longer lag and macrofusion times than in wild-type embryos imaged at the same temperatures (Figure 7, B and C, and green and blue triangles in Figure 5, C and E). Lag and macrofusion times in idf(–) embryos were significantly longer for the fusing pairs of cells than for wild-type (see Supplementary Material).
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; Mohler et al., 1998; Mohler et al., 2002). In summary, most junctions in the dorsal hypodermis can initiate a syncytium with a pioneer fusion event. Additional nonpioneer cells fuse to the initial intermediate binucleate syncytium that expands into a giant cell. Our comparison between kinetic parameters of pioneers and nonpioneers indicates that fusion initiation at one side of the cell affects neither the lag time of microfusion nor the macrofusion rate at the other side of the same cell. Autonomous fusion for adjacent junctions argues against the hypothesis that the cell fusion pathway is driven by the lateral tension in the membrane bilayer that might be generated by osmotic effects or by cytoskeleton activity.
Microfusion: Ultrastructural Intermediate of EFF-1–mediated Epidermal Cell Fusion
TEM has been previously used to identify structural cell fusion intermediates in yeast, worms, flies, and mammals (Kalderon and Gilula, 1979; Baron et al., 1986; Doberstein et al., 1997; Gammie et al., 1998; Mohler et al., 1998; Heiman and Walter, 2000). In C. elegans, the existence of a distinct stage of microfusion in the fusion pathway is independently supported by kinetic analyses (see above) and the phenotypes in myoepithelial cells observed by TEM in eff-1 conditional mutants that were grown at the semipermissive temperature where microfusion intermediates failed to expand (Shemer et al., 2004). We looked for similar intermediates in the hypodermis of the same fixed and sectioned specimens and found some initiated fusion events that failed to expand (Figures 8 and 9). We conclude that EFF-1 in the hypodermis is required both to initiate cell fusion but also to expand membrane gaps of 20–50 nm to complete macrofusion of around 20,000 nm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), strengthening the notion that during the lag phase there are two kinetic steps followed by expansion or macrofusion. It is conceivable that additional gene(s) may function in some cell fusion events in epidermal and myoepithelial cells of the pharynx, explaining the stable microfusions in eff-1 mutants (Shemer et al., 2004).
Genes and Kinetic Behavior Characteristic of Developmental Cell Fusion
In Figure 10 we propose a model for the cell fusion process in epithelial cells and hypothesize the roles of eff-1 and idf-1 in specific stages based on our findings. Epidermal cell fusion in the embryo is dependent on the activity of EFF-1 (Mohler et al., 2002). Expression of EFF-1 is enough to fuse cells in C. elegans and activated EFF-1 primes the system for cell fusion (Shemer et al., 2004; del Campo et al., 2005). We identified three steps in the cell fusion pathway. Two steps in the lag may involve activation of EFF-1 and the initiation of cell fusion or microfusion. Recently we found that expression of EFF-1 on the surface of insect Sf9 cells is enough to fuse cells via hemifusion (Podbilewicz et al., 2006). The 9°C cell fusion block we observed in C. elegans embryos may be analogous to the 4°C block in influenza virus fusion that freezes the membranes in a hemifusion state (Chernomordik et al., 1998). The discontinuity in the plasma membranes has to expand and this expansion of the microfusion is what we measure in the cell macrofusion assay as the disappearance of the AJ. Although the early stages of membrane fusion are rapid (from fractions of a second to 1 or 2 min; Kaplan et al., 1991; Plonsky and Zimmerberg, 1996; Mohler et al., 1998; del Campo et al., 2005), the complete disappearance of the membranes and the apical junctions associated to them is a temperature-dependent process that takes several minutes (Figure 4).
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; Clague et al., 1990; Oberhauser et al., 1992; Lee and Lentz, 1998; Earles et al., 2001).
idf-1 may affect the lag and the macrofusion stages (Figure 5, C and E). Macrofusion may involve vesiculation of the plasma membrane surrounding the microfusion gap. This is supported by TEM studies of embryonic (Mohler et al., 1998) and postembryonic (Nguyen et al., 1999) epidermal cells that have revealed the appearance of vesicles in the gap between fusing cells, suggesting that vesiculation after initial microfusion may be a critical step in syncytia formation and morphogenesis (Podbilewicz, 2000, 2006; Podbilewicz and Chernomordik, 2005).
Actin contraction may be the rate-limiting step for the actin-mediated elongation of C. elegans embryos (Priess and Hirsh, 1986; Costa et al., 1998; McKeown et al., 1998). Morphogenesis is strongly temperature dependent only between 8 and 15°C with an apparent activation energy of 110 kcal/mol (Figure 5D), similar to the apparent activation energy of F-actin movement on rabbit skeletal myosin (Anson, 1992). At temperatures in the range of 15–25°C, the rate of elongation is weakly temperature-dependent with an estimated apparent activation energy of 1.5 kcal/mol and an average elongation rate of 160 ± 23 nm/min. In contrast to elongation, the Arrhenius plots for the macrofusion step were linear at 13–25°C using the semiautomated method and at 11–25°C using the manual method, indicating that within this temperature range cell fusion in C. elegans has the same rate-limiting step.
Using stringent criteria including careful tiltings and reconstructions in serial sections (Materials and Methods), we found that microfusion events in eff-1 mutants do occur between epidermal cells that fail to complete syncytia formation. In multiple cases we were able to distinguish places where the membrane just twists into a new plane of section (detectable when tilting the TEM grid) from true microfusion sites where fields of cytoplasmic ribosomes span the gap across the cell border. Thus, although the unfused cells bear irregular and convoluted common borders not found in wild-type animals, we can confirm multiple microfusions that reveal another function for EFF-1 in the expansion from microfusion to complete fusion. There appear to be multiple chances for such microfusions to occur between the same hypodermal cells as their common borders are very extensive. Structurally similar microfusions have been identified in cells infected with human cytomegalovirus and measles virus (Firsching et al., 1999; Gerna et al., 2000; Ehrengruber et al., 2002). It is believed that viral induced microfusions may be involved in viral spreading. In addition, stable microfusion-like structures have been described between fibroblasts and cardiomyocytes (Driesen et al., 2005). Although in virus-cell fusion and intracellular membrane fusion a lot is known about the initial steps leading to fusion pore formation, relatively little is known about fusion pore expansion (Scepek et al., 1998; Dutch and Lamb, 2001; Haller et al., 2001; Gibbons et al., 2004; Jaiswal et al., 2004; Leikina et al., 2004; Nolan et al., 2006).
In summary, independent kinetic, genetic, and ultrastructural studies on cell fusion are consistent with at least three steps in the membrane fusion pathway in C. elegans, with eff-1 acting in early local fusion (microfusion) and late expansion (macrofusion) steps of the pathway.
Fusion Events Do Not Typically Occur Symmetrically
We have developed a sensitive and unique paradigm for studying kinetics of cell fusion in living embryos of C. elegans and show that it can be used as a model to analyze how molecules identified in genetic screens for cell fusion defective mutants affect specific steps in the dynamic process of cell fusion. Indeed, in a screen for mutants defective in embryonic morphogenesis (Costa and Priess, personal communication; Costa et al., 1998) idf-1(zu316) was identified, and here we show how this mutant gene slows down two distinct kinetic steps of cell fusion in vivo. Unexpectedly, idf-1 mutants affect some cell–cell fusions in a different manner, arbitrarily. Some cell pairs have a complete block, whereas others have a retarded initiation followed by a slow execution. This differential kinetic behavior of cell fusion in the same embryo may reveal intrinsic differences between cells and variability in the trigger of cell fusion in a developing tissue. One explanation for these results is that low density of active EFF-1 on the fusion sites may be sufficient to initiate pore formation but not for their expansion as shown for influenza virus fusion (Kozlov and Chernomordik, 2002; Chernomordik and Kozlov, 2003; Leikina et al., 2004). Alternatively, other genes may also be required to act along with EFF-1 to complete fusion. It is surprising that the arrest phenotype of the idf-1 mutant phenocopies the cold fusion block (9°C). idf-1 has a role in dorsal epidermal fusion and additional essential roles probably unrelated to cell fusion. Future TEM of idf-1–arrested embryos compared with the 9°C block, together with physiological tests to follow lipid and content mixing between fusing cells, should give us a better understanding of the intermediates in cell fusion and the specific roles of EFF-1 and IDF-1 in the process.
Membrane Domains Fuse Autonomously and Asymmetrically.
Early studies on epidermal cell fusion in C. elegans embryos suggested that there is a variable program in the sequence in which 23 syncytial precursor cells fuse to form the hyp7 syncytium. These studies were based on immunofluorescence of hundreds of fixed specimens at different stages in morphogenesis and reconstruction of the pathways of cell fusion during syncytiogenesis (Podbilewicz and White, 1994). More recently, using GFP reporter genes and membrane markers it was possible to follow syncytiogenesis of individual embryos in real time (Mohler et al., 1998, 2002; Shemer et al., 2004; del Campo et al., 2005). Taken together these studies showed that the final position, number, and identity of the cells that fuse is invariant during development though the fusion sequence is variable between individuals. In addition, cytoplasmic content mixing followed using GFP reporters is completed in 2–3 min, whereas the complete rearrangement of the plasma membranes and apical junctions takes about 40 min at 23°C (Mohler et al., 1998; Shemer et al., 2004; del Campo et al., 2005).
Here, for all fusion events in wild-type and the idf-1 mutant embryos, we observed sigmoidal kinetics of fusion and measured the characteristic parameters: Lag (microfusion) and macrofusion times. Each cell pair fuses with characteristic macrofusion rate in embryos monitored at different temperatures. It appears that the anterior membrane domain of a fusion competent cell fuses or fails to fuse independently of the posterior plasma membrane domain of the same cell. Thus, the anterior or posterior end can fuse faster that the opposite end of the cell, but which end fuses faster is random. Although the lateral membranes are fusion incompetent during embryogenesis in the wild-type, the anterior and posterior membranes develop their fusion competence autonomously and without any apparent symmetry. This control of the fusion competence of specific cells and membrane domains can be overruled by ectopic activity of EFF-1 in fusion-incompetent cells (Shemer et al., 2004). Ectopic expression of eff-1 followed by abnormal tissue-specific cell fusion can also be the result of inactivation of Engrailed/ceh-16–dependent transcriptional repression of eff-1 in lateral seam cells (Cassata et al., 2005), inactivation of vacuolar ATPase in the lateral hypodermis (Kontani et al., 2005), or inactivation of lin-39/Deformed repression of eff-1 in the ventral vulval precursor cells (Shemer and Podbilewicz, 2002).
Founder Cell Fusion Event Is Variable.
Most dorsal precursor cells of the hyp7 syncytium, except one structural junction, are competent to be pioneers or founder cells. Macrofusion and microfusion rates are not correlated with pioneer and nonpioneer cells having similar probabilities to fuse with the fastest or slowest macrofusion rates. These findings show that there is randomness in the initiation and completion of a genetically programmed sequence of cell fusion events. Localization of EFF-1 in the cell–cell contact zone above a certain threshold may explain these apparently stochastic events (del Campo et al., 2005). The concept of a stochastic epidermal founder cell in C. elegans described here is analogous to the founder myoblasts and fusion-competent myoblasts hypothesis in Drosophila (Rushton et al., 1995; Abmayr et al., 2003; Chen et al., 2003; Englund et al., 2003). However, our definition of the founder cell is strictly kinetic with respect to the first detectable cell fusion event that occurs after cell fate determination, migration, recognition, adhesion, differentiation, and patterning of the epidermis. In contrast, in the muscles of Drosophila, founder cells are pioneers for myogenesis that differ from fusion-competent myoblasts primarily by distinct phenotypes of mutations and differential expression of molecular markers required for recognition, signaling, adhesion, patterning, differentiation, and fusion competence. In contrast to myoblast fusion in Drosophila, in the epidermis of C. elegans, tightly regulated homotypic expression of EFF-1 initiates and expands cell fusion (Podbilewicz et al., 2006).
In summary, here we have used a new system to study the molecular and cellular mechanisms of cell membrane fusion in developing animals and showed how mutations, temperature blocks, ultrastructural, and kinetic analyses reveal that the first cell fusion event is variable and that membrane fusion events have independent and asymmetric anteroposterior kinetics. Moreover, eff-1 activity is required at early and late stages of the process of epidermal syncytiogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Present address: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi 110016, India.
Address correspondence to: Benjamin Podbilewicz (podbilew{at}tx.technion.ac.il)
Abbreviations used: AJ, apical junction; eff-1, epithelial fusion failure-1; idf-1,, irregular dorsal fusion-1; TEM, transmission electron microscopy.
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REFERENCES |
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) and the increase in body length (
) for the same wild-type embryo are shown. Developing embryo expressing AJM-1::GFP was recorded at 23.1°C. A fusion event is defined as appearance of detectable AJ discontinuity. (D) Wild-type embryo (
) compared with wild type embryos (n = 15; 
) at similar elongation stages at 15°C: 1.88 ± 0.14- and 1.89 ± 0.22-fold elongation for wild type and idf-1(zu316), respectively. Cells a–d are part of hyp6; cells 1–17 are part of hyp7 (
