Actin is required for specific processes during early embryogenesis
To ask if different processes in the early embryo that depend on an intact actin cytoskeleton are differentially sensitive to the amount of available actin, we performed a time-course analysis of early embryogenesis after injection of dsRNA targeting all actin genes using act-1(RNAi) (see Materials and Methods, Figure 1, and Additional File 1: Supplementary Figure 1). To verify and quantify the depletion of actin, we used an anti-actin antibody to detect the amount of protein in the gonad over time. We observed that protein levels were reduced by about half at approximately 6 hours post-injection, declined sharply between 7–8 h, and finally tapered off (Figure 1A and 1B). We will refer to the embryos produced by treated animals as actin(RNAi) embryos. Analysis of actin(RNAi) embryos at distinct time points revealed a phenotypic series analogous to what might be seen if we had an allelic series for a family of redundantly functioning genes (Figure 1B and 1C).
The wild-type (WT) embryo undergoes a series of canonical events that are easily observable using time-lapse DIC microscopy (Figure 1C, Additional File 4: Movie 1). We review them here and compare them with events that occur in actin(RNAi) embryos (Figure 1C, Additional Files 5, 6, 7, 8 Movies 2–5). After the completion of meiosis and the extrusion of two polar bodies, approximately 25 minutes after fertilization, the maternal and paternal pronuclei form at anterior and posterior ends, respectively. Concurrently, the cortex of the embryo undergoes intense contractions that cease at the posterior end of the embryo when the paternal pronucleus closely associates with it. These contractions culminate in a deep invagination near the middle of the embryo referred to as the pseudocleavage furrow. At this time, the maternal pronucleus migrates toward the paternal pronucleus. The pseudocleavage furrow relaxes as the pronuclei meet in the posterior half of the embryo. The pronuclei do not immediately fuse upon meeting, but move together toward the middle and rotate, such that the division axis is established perpendicular to the long axis of the embryo before nuclear envelope breakdown. The spindle is displaced at anaphase to the posterior of the embryo, giving rise to two daughter cells of different size. These cells have distinct fates giving rise to different parts of the adult worm. The distinct identities of these cells are characterized by the way they divide in the second round of mitosis: they undergo mitosis at different times, with the larger anterior AB cell dividing before the smaller posterior P1 cell, and their division axes are perpendicular to each other (Figure 1C, Figure 2A, Additional File 4: Movie 1).
The effect of actin(RNAi) on these canonical early embryonic events depends on the time elapsed between injection of dsRNA and initiation of embryogenesis, due to a progressive depletion of actin over time in the treated animal (Figure 1). Eventually, actin(RNAi) leads to a drastic reduction of fertilized embryos, approximately 12–15 hours after injection; in contrast, control animals produce fertilized embryos for at least twice as long (see Additional File 2: Supplementary Figure 2; [18]). These results are consistent with a recent analysis showing that the lateral plasma membrane collapses in gonads cultured with high concentrations of actomyosin inhibitors [36], which could lead to disruption of oocyte integrity and fertilization. Thus, the existence of any embryos indicates that oocyte integrity is maintained by residual actin activity, and therefore represents a hypomorphic condition. Prior to this study, the only hypomorphic phenotypes reported in actin(RNAi) embryos, as part of a large scale study, were failure to extrude polar bodies, loss of spindle displacement, and loss of cytokinesis [18]. By selecting embryos for analysis from RNAi-treated animals in a carefully timed way, we were able to observe a reproducible series of defects that we could calibrate for each batch of dsRNA (see Materials and Methods). We grouped embryos showing similar ranges of progressively more severe defects into four phenotypic classes (Classes I-IV; Figures 1C and 2). Specific phenotypes appearing together within a class thus represent processes that require similar levels of actin in the cell. The range of phenotypes observed in the four phenotypic classes is summarized in Additional File 3: Supplementary Table 1.
Class I defects (Figures 1C (n = 11) and 2B (n = 5), Additional File 5: Movie 2) highlight the processes that are affected by the mildest reduction in available actin, and thus require the highest levels of actin. In the first cell cycle, cortical ruffling and pseudocleavage were absent. Spindle movements in P0 appeared normal and the first cell division was asymmetric, whereas the AB and P1 cells divided in the correct orientation but with abnormally synchronous timing. Thus, Class I defects appear to separate two processes, spindle orientation and timing of the second division, which are thought to be coupled and to depend on proper polarity establishment in the one-cell stage. In Class II embryos (Figures 1C (n = 12) and 2C (n = 5), Additional File 6: Movie 3), the paternal pronucleus failed to maintain its close association with the posterior cortex, causing the pronuclei to meet more centrally. After nuclear fusion, the spindle failed to move toward the posterior prior to cytokinesis, giving rise to more symmetric daughter cells than in WT. Finally, the second mitotic cell divisions were both synchronous and oriented parallel to each other, transverse to the AP axis (Figure 2C). Class III embryos (Figures 1C (n = 12) and 2D (n = 4), Additional File 7: Movie 4) began to show cytokinesis defects, as well as disorganized furrows during subsequent cellular divisions that gave rise to polynucleated cells. Nuclear reformation occurred close to the contractile ring, an indication of reduced elongation of the central spindle during anaphase B. Severely depleted Class IV embryos (Figures 1C (n = 14) and 2E (n = 4), Additional File 8: Movie 5) failed both to extrude both polar bodies and to execute cytokinesis, and contained small karyomeres. Interestingly, even among the most strongly affected embryos, the rotation of the spindle in P0 occurred normally.
The reformation of the nuclei near the cell division remnant and the presence of karyomeres in actin(RNAi) embryos suggested that they might also experience chromosome segregation defects. To further explore the effect of actin depletion on chromosome segregation, we analyzed the same time points in a line expressing a GFP-histone fusion (GFP::HIS) [37], which reveals chromosomal movements and dynamics. In addition to confirming the orientation of mitotic chromosomes and cell cycle timings, we also observed that anaphase figures showed partially decondensed chromosomes, lagging chromosomes, and chromosome bridges in Class III and IV embryos (Figure 2D and 2E). These data indicate that severe actin depletion interferes with the proper resolution of chromosomal pairing and/or movement of chromosomes to the spindle poles.
Cortical F-actin undergoes three phases of morphological transitions in the one-cell embryo
To visualize actin dynamics in vivo in the early embryo we fused GFP with the D. melanogaster Moesin F-actin binding domain [38]. This GFP::MOE marker has been shown to faithfully report the distribution of microfilaments in both D. melanogaster [39] and C. elegans [38]. All the structures that we observe with our GFP::MOE line are therefore F-actin-rich structures decorated with GFP molecules. Previous analyses using phalloidin-stained fixed specimens [19, 20] or in vivo visualization of NMY-2::GFP dynamics [23] during the first cell cycle of embryogenesis have shown that actomyosin forms a cortical lattice that appears to contract asymmetrically to the anterior half of the embryo [23]. GFP::MOE embryos similarly show a dynamic meshwork of cortical actin fibers and dense focal accumulations, as well as numerous small punctate structures. These structures all become enriched to the anterior with similar kinetics as NMY-2::GFP. However a persistent collection of small actin-rich puncta remains at the extreme posterior throughout the first cell cycle. The asymmetric redistribution of microfilaments does not appear to result from collapse of the meshwork under tension; rather, the dynamic formation and dissipation of F-actin structures that we observe in the anterior indicates that microfilaments are in rapid flux. This suggests that the capacity for filament assembly becomes segregated to the anterior domain while assembly is reduced in the posterior. At pronuclear meeting, the fibrous actin meshwork revealed by GFP::MOE transforms into a collection of discrete puncta in the anterior half of the embryo that persists until just prior to contractile ring formation, at which point cortical F-actin rapidly dissipates (Figure 3, Additional File 9: Movie 6).
Thus, there appear to be three phases involving a major reorganization of the cortical actin cytoskeleton that are coordinated with the cell cycle in the one-cell embryo: first, the asymmetric redistribution of F-actin structures to the anterior (we refer to this as "Phase I"); second, concomitant with pronuclear meeting and centration, a shift in the overall morphology of predominant cortical F-actin structures from a dynamic meshwork to discrete puncta ("Phase II"); and finally, a rapid clearance of cortical F-actin in anticipation of cytokinesis ("Phase III").
The proper anterior redistribution of microfilaments depends on PAR proteins
Previous work has shown that the distribution of microfilaments to the anterior half of the embryo, as visualized in fixed preparations, is affected by mutations in par genes [20]. To investigate this relationship in vivo, we performed time-lapse microscopy of GFP::MOE embryos depleted of different PAR proteins using RNAi. For all par genes we analyzed (par-6, par-2, and par-1), we compared RNAi phenotypes with published phenotypes for the strongest reported genetic alleles to confirm that the phenotypes we obtained are as strong as the genetic alleles. Upon RNAi of PAR-6, a member of the anterior PAR complex, F-actin initially assembles on the cortex as a filamentous meshwork, similar to WT (n = 8; Figure 3, Additional File 10: Movie 7). par-6(RNAi) embryos show a weak clearing of the posterior domain prior to pronuclear meeting. The actin meshwork in par-6(RNAi) embryos resolves into discrete puncta during Phase II at a similar time as in WT; however, these puncta do not concentrate in the anterior as in WT, but are distributed throughout the cortex (n = 8; Figure 3). Thus any weak early asymmetries are lost by pronuclear centration and rotation. Similarly, par-2(RNAi) embryos show an initial weak depletion of cortical F-actin from the posterior (Phase I), but F-actin becomes uniformly distributed throughout the cortex and resolves into discrete punctate structures by the time pronuclei rotate and centrate (n = 5; Figure 3, Additional File 11: Movie 8). In comparison with par-2(RNAi), actin structures appear less prominent at the cortex in par-6(RNAi) embryos. This is most notable during Phase II in par-6(RNAi) embryos, when PAR-2 is uniformly distributed throughout the cortex) [10]. Cortical NMY-2::GFP is similarly reduced in par-3(RNAi) but not par-3; par-2(RNAi) embryos [23]. In par-1(RNAi) embryos, abnormal accumulations of actin appear in the meshwork during Phase I, and by pronuclear meeting F-actin becomes hyper-asymmetrically enriched in the anterior (n = 11; Figure 3, Additional File 12: Movie 9). As pronuclei centrate and rotate, a morphological transition to discrete puncta is clearly discernable (Phase II) and the posterior F-actin boundary expands slightly toward the midline of the A-P axis (Figure 3). PAR-1 might thus be required to set up the border of the anterior F-actin domain, similar to its role in establishing the PAR boundary [6, 10, 20]. In all three RNAi treatments, actin-rich puncta are evident at the extreme posterior of the embryo during Phase I, similar to WT. We conclude from these results that PAR-2 and PAR-6 proteins are primarily required to constrain actin-rich structures to the anterior half of the embryo, whereas PAR-1 contributes to the proper coordination of actin dynamics and establishment of the correct boundary of the anterior F-actin domain.
Additional File 12: Movie 9 – par-1(RNAi) of GFP::MOE strain (MOV 5 MB)
The diversity of actin-rich cortical structures and regulated transitions in their morphology led us to investigate the role of F-actin regulators on their dynamics. Regulators of the actin cytoskeleton have been shown to influence the localization of PAR proteins in C. elegans and other organisms [6, 24]. Severe depletion of CDC-42, which binds PAR-6 [40], leads to sterility and osmotic sensitivity, and the actin cytoskeleton fails to assemble on the cortical surface (data not shown) [24, 41, 42]. Less severe depletion of CDC-42 does not affect the initial polarization of F-actin to the anterior during Phase I, but by pronuclear meeting actin becomes hyper-asymmetrically concentrated in the anterior (n = 6; Figure 3, Additional File 13: Movie 10), similar to par-1(RNAi) embryos. Unlike par-1(RNAi), however, this hyper-asymmetry persists throughout Phase II, and the domain of F-actin-rich puncta at the extreme posterior appears to be expanded relative to WT during Phase II. Overall, the association of F-actin structures with the cortex appears weaker than in WT, and the contractile ring is very faint. Therefore, CDC-42 is needed to maintain the initial asymmetric distribution of F-actin and for a strong association of F-actin with the cortical surface.
Actin-binding proteins and nucleators of actin affect the morphology of cortical microfilaments
Profilins are actin-binding proteins that also interact with other regulators of the actin cytoskeleton and can affect actin dynamics in a variety of ways depending on the molecular context (reviewed in [43]). C. elegans contains three profilins with similar biochemical properties that all behave as classical nonvertebrate profilins [44]. Only one of these, PFN-1, is essential for embryogenesis, where it is required for cytokinesis and polarity in the one-cell embryo and is also required for the proper localization of F-actin and NMY-2 to the cortex [16]. To investigate profilin's role in polarity and its effect on cortical microfilaments in more detail, we analyzed embryos depleted of PFN-1 by time-lapse microscopy. We found that pfn-1(RNAi) prevented the appearance of an actin-rich meshwork at the cortex, while actin-rich puncta appeared much earlier and moved more freely around the cortex than in WT (n = 6; Figure 3, Additional File 14: Movie 11). These puncta began to coalesce into large actin-rich clumps between pronuclear meeting and centration (when the Phase II transition normally occurs in WT). Cytokinesis never occurred, although some residual contractile activity could be observed on the cortex during mitosis (Additional File 14: Movie 11 shows a striking ring-like formation of cortical actin-rich structures that appears to contract into a single aggregate). These observations indicate that the early embryo contains two genetically separable populations of cortical actin-based structures, a fibrous meshwork and distinct punctate accumulations, and that profilin appears to be required exclusively for the assembly of filaments that form the meshwork. Profilin is also required to correctly position and coordinate contractile activity required for cytokinesis with the mitotic spindle, as has been observed previously in C. elegans and other systems [45].
NMY-2 encodes the heavy chain of a non-muscle myosin motor protein that is required for cytokinesis and has been shown to affect polarity in the C. elegans one-cell embryo [13]. nmy-2(RNAi) embryos can be classified by the severity of phenotypes resulting from variable depletion of NMY-2 activity, from weak to strong) [10, 13]. Weak "Class II" NMY-2 depletion gives a PAR-like phenotype, and can thus be used to study the connection between polarity and actin. GFP::MOE dynamics revealed that the overall organization of cortical F-actin structures was disturbed in nmy-2(RNAi) Class II embryos and that association of microfilaments with the cortex appeared to be reduced. The contractile meshwork was absent and no asymmetric contraction occurred (Phase I); punctate accumulations were evenly distributed throughout the cortex at pronuclear meeting, and the morphological transition to Phase II did not occur (n = 4; Figure 3, Additional File 15: Movie 12). The cortex cleared prior to contractile ring formation as in WT, but the first division was symmetric and the spindle failed to rotate in P1 (a clear par-like phenotype). Thus, like PAR proteins, the asymmetric localization of actin during the first cell cycle depends on NMY-2 function. These data demonstrate that the proper assembly and organization of a variety of actin-based cortical cytoskeletal structures depends on myosin function.
Additional File 15: Movie 12 – nmy-2(RNAi) of GFP::MOE strain (MOV 9 MB)
The Arp (actin-related protein) 2/3 complex promotes rapid actin polymerization in response to various signals and results in the formation of branched filaments, which allow for cytoskeletal reorganization in the cell [5, 46–49]. In C. elegans, depletion of either ARX-1 (Arp2) or ARX-2 (Arp3) leads to embryonic lethality; while no early embryonic phenotypes have been reported, ventral closure and postembryonic defects have been observed [50, 51]. Disruption of the Arp2/3 complex by depletion of ARX-1 prevented the formation of an F-actin meshwork at the cortex; a few transient F-actin structures were observed, but no foci formed upon pronuclear meeting (n = 4; Figure 3, Additional File 16: Movie 13). Despite the lack of F-actin enrichment in the anterior, the cell was able to undergo an asymmetric cell division, indicating that other factors required for the establishment of polarity and the boundary between anterior and posterior cortical domains were unaffected by depletion of ARX-1. F-actin was properly recruited to the contractile ring and cytokinesis occurred as in wild type.
The C. elegansembryo contains highly dynamic actin-rich "comets"
In the course of characterizing actin dynamics in the GFP::MOE strain, we were struck by the presence of "comet tails" that moved rapidly through the cytoplasm and near the cortex in the one-cell embryo. Actin-rich comets have been described in association with both endocytic vesicles [5, 26–28, 30] and intracellular pathogens such as Listeria, Shigella, and Rickettsia (reviewed in [5, 31–33]). In C. elegans, we noticed that the comets have no consistent direction – often appearing as curvy or curly structures – and that they vary in length, speed and number throughout the first cell cycle (Figure 4, Additional File 17: Movie 14). We also observed comets in developing and maturing oocytes and in later stages of embryogenesis (data not shown). The observation of similar unknown structures has been reported in fixed samples [19], but their dynamic nature has not previously been described. To confirm that actin comet tails were not artifacts of the GFP::MOE transgenic line, we visualized F-actin in fixed WT and GFP::MOE embryos with rhodamine-conjugated phalloidin (Rh-phalloidin). Rh-phalloidin clearly labeled comet tails in both lines, and co-localized with GFP antibodies in the GFP::MOE line (Figure 4B). DAPI did not stain the heads of the comet tails, dispelling the notion that they are associated with an intracellular bacterial pathogen (Figure 4B).
To begin to elucidate the potential role of actin comets, we characterized their behavior. Since these comets are more numerous during the first cell cycle, we quantified the number of comets present during this time and observed that the number of comets peaks around prometaphase (Figure 4C). Additionally, during the period from pronuclear meeting to prometaphase, the comets appear to coalesce around the pronuclei and sometimes to associate with the centrosomes (Additional File 11: Movie 8). This suggests that an accumulation of any cargo propelled by the comets may ultimately be required at some location near or defined by the nuclei and/or mitotic spindle. We measured speeds of actin comets in C. elegans and found that they travel at an average of 0.22 ± 0.10 μm/s (Figure 4D), similar to the mean speed and range of endocytic vesicles in transfected rat basophilic leukemia (RBL) cells (0.24 ± 0.10 μm/s) [27]. Although we observed that some comets were thicker than others, we found no correlation between their widths and speed (Figure 4E). We note that the trajectories and speeds of actin comets are quite distinct from those observed for an early endosome marker in the early embryo [34]; thus we believe they represent a different population of cellular structures.
We next asked if the actin comets were dependent on known regulators of F-actin assembly such as CDC-42, ARX-1 (Arp2), and PFN-1, which are required for the proper formation of actin comets in other systems. The Arp2/3 complex nucleates microfilaments, and CDC-42 promotes Arp2/3 activity by stimulating nucleation promoting factors (NPFs) that interact with Arp2/3 (reviewed in [5, 31–33]). Depletion by RNAi showed that all three actin regulators are required to achieve the maximal number of comets observed at prometaphase in WT (Figure 4C). Depletion of PFN-1, which promotes polarized filament growth and increases the rate of movement of comets in other systems [31, 32], caused the most dramatic reduction in the basal number of comets, which rose only slightly at prometaphase (Figure 4C). We cannot distinguish between the possibilities that further reduction of these proteins would lead to complete loss of comets, or that other cellular factors may be able to promote a basal level of filament nucleation in these comets. In contrast, depletion of PAR-6 (data not shown) or NMY-2 (Figure 4C) had no significant effect on either basal or maximal comet numbers, although in nmy-2(RNAi) embryos comets persisted well beyond telophase, particularly when the contractile ring was weak or failed to form (Figure 4C and data not shown).
We noticed no qualitative change in the speed or length of the comet tails in arx-1(RNAi), cdc-42(RNAi), pfn-1(RNAi), or nmy-2(RNAi) embryos (data not shown). We conclude that actin-rich comets in the C. elegans one-cell embryo are influenced by a cell cycle-dependent process that is coordinated with the activity of actin cytoskeletal regulators and that their speed is consistent with a potential role in endocytosis, but further characterization is needed to determine their precise role during development.