Qian Shen, Bin He, Nan Lu, Barbara Conradt, Barth D. Grant and Zheng Zhou.
C. elegans is one of the most widely studied model organisms – in fact, the developmental fate of every single somatic cell in this organism has been mapped. Below is a time-lapse video of the entire embryonic development of C. elegans:
Watching this video, you can see many cell divisions and the progression from one cell to about 1000. But in addition to creating new cells, these embryos are also destroying cells through a process known as apoptosis – “programmed cell death”. After apoptosis, however, the remains of the cell must be efficiently eliminated. This is where phagocytes come in – they engulf and then degrade apoptotic cells. Without this process known as phagocytosis, correct body symmetry in the embryo cannot be established and organs cannot can properly form.
Phagocytic cells use distinct cell-surface receptors to recognize surface features of apoptotic cells and initiate extension of pseudopods around their target for engulfment (Elliott and Ravichandran, 2010). The driving force of this pseudopod extension is the assembly and growth of actin filaments underneath the plasma membrane (Caron, 2001). But how do phagocytic receptors orchestrate the actin assembly needed to extend pseudopods and engulf target cells? This is the question the paper “Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans” by Shen, Q. et al. seeks to answer.
What is already known about the mechanisms of phagocytosis?
It is already known that the Rho family small GTPases are important regulators for the rearrangement of actin during phagocytosis (Ravichandra and Lorenz, 2007). However, additional regulatory pathways for actin polymeriation for the engulfment of apoptotic cells are not well-known. In particular, a key unanswered question is: how does a phagocytic receptor establish a spatial cue that attracts actin molecules to the engulfment region where the pseudopod extends?
Of the 113 somatic cells that undergo apoptosis during C. elegans embryogenesis, most of these events occur during the mid-embryogenesis stage (approximately at minutes 1:07-2:14 in the video above). Through genetic screening, many genes have been implicated as regulators of apoptotic-cell engulfment/degradation. Analysis has placed genes involved in the engulfment process in two partially redundant and parallel pathways (Reddien and Horvitz, 2004; Mangahas and Zhou, 2005; Yu et al., 2006).
Pathway 1: CED-1. One of the pathways is led by CED-1, a transmembrane protein that is the prototype of a phagocytic receptor family for apoptotic cells. CED-1 (along with CED-6) recruits a downstream mediator DYN-1 (dynamin) to budding pseudopods. During phagocytosis, DYN-1 supports pseudopod extension by promoting recruitment and fusion of intracellular vesicles to the plasma membrane.
Pathway 2: CED-10. In the second pathway, CED-5/Dock180 and CED-12/ELMO1 together activate CED10/Rac1 GTPase to promote reorgnization of the cytoskeleton (Reddien and Horvitz, 2004). CED-2/CrkII has been proposed to connect a phagocytic receptor with this CED-5/CED-12 complex.
It has been hypothesized that both of these pathways converge at CED-10, which mediates the actin-reorganization activity of CED-1. However, the results of this paper point to an alternative conclusion.
EPN-1 is important for cell corpse removal
After screeening for mutants containing excessive cell corpses (the “Ced phenotype”), en47 and en48 recessive alleles were isolated. The gene defined by the en47 and en48 mutations was found to be epn-1, which encodes a C. elegans homolog of epsin. In humans, epsins are a family of membrane proteins that function in establishing membrane curvature. To learn more about epsin in humans, click here.
A strain was constructed that generated en47(m-z-) progeny, which are progeny that have lost both maternal and zygotic epn-1 (m = maternal gene product and z = zygotic gene product). Approximately 1/3 of these progeny underwent embryonic developmental arrest and those remaining underwent L1 larval arrest. The number of persistent cell corpses retained in the m-/z- mutant was over threefold of that of those that were missing only the zygotic product (Fig 1B). These results indicate that depleting both maternal and zygotic epn-1 products resulted in strong loss-of-function phenotypes. Additionally, expression of epn-1 cDNA in cell types that can be phagocytic resulted in efficient rescue of the Ced phenotype in epn-1 mutants.
These results demonstrate that EPN-1 plays an essential role in efficient removal of cell corpses.
Inactivation of clathrin heavy chain causes cell-corpse accumulation
Because epsin is an adaptor for clathrin, the role of clathrin in phagocytosis was investigated. The only C. elegans coding gene for clathrin heavy-chain, chc-1, was examined for involvement in apoptotic cell removal. chc-1 gene expression was inhibited using a technique called RNA interference (RNAi); this technique essentially is using RNA molecules to inhibit gene expression, typically by destroying specific mRNA molecules. chc-1 (RNAi) caused cell-corpse accumulation in embryos. Additionally, Ced and lethal phenotypes were observed in deletion mutant embryos, chc-1(m-z-) (Fig 1E).
Thus, like EPN-1, CHC-1 also plays an essential role in cell-corpse removal.
Inactivating chc-1 or epn-1 specifically impairs cell-corpse engulfment
An engulfing cell-specific CED-1ΔC::GFP reporter (this means that the intracellular domain of CED-1 is replaced with GFP) allows the tracking of both the engulfment and degradation of cell corpses. The reporter is capable of recognizing neighboring apoptotic cells and is enriched on extending pseudopods. In chc-1 mutants and epn-1(m-z-) embryos, pseudopod extension took significantly longer to complete (Fig 3F, h-x and Fig 3C). However, initiation of pseudopod budding and degradation of engulfed cell contents occurred at normal time points (Fig 3F). These results indicate that pseudopod extension specifically is affected by chc-1 or epn-1.
These experiments show that the phenotypes found from Figure 1B and Figure 1E are specifically a result of the impairment of extension of the pseudopod in phagocytosis and not the result of some other factor.
epn-1 and chc-1 both act in the ced-1 pathway
To investigate if epn-1 and chc-1 act in one of the two known cell-corpse engulfment pathways, ced-5 and ced-12 mutants were examined (these are strong loss-of-function mutants that belong to the ced-10 pathway, see the introduction for reference). chc-1(RNAi) in these mutants increased the number of cell corpses by 32-49%. However, using chc-1(RNAi) in null mutants or ced-6 mutants (mutants in the ced-1 pathway) did not enhance the Ced phenotype (Figure 4Aa).
Additionally, double mutants were constructed containing the mutant epn-1(en47)(m-z-) coupled to six ced mutants, three from each of the two pathways. epn-1(e47) enhanced the Ced phenotype of mutants in the CED-10 pathway significantly but did not significantly enhance the Ced phenotype of mutants in the CED-1 pathway (Figure 4Ab).
Together, these results imply that both epn-1 and chc-1 act in the CED-1 pathway; this is independent and parallel to the CED-10 pathway.
EPN-1 is enriched on extending pseudopods in CED-1 pathway-dependent manner
To visualize EPN-1 expression, the researchers used EPN-1::GFP, which is fluorescently tagged EPN-1 using GFP. In embryos, EPN-1::GFP is enriched on extending pseudopods and remains on the surfaces of nascent phagosomes for 4-6 minutes before disappearing (Fig 4C). In ced-1 pathway mutants, EPN-1::GFP failed to enrich on pseudopods; however, this enrichment was noted in ced-10 pathway mutants (Fig4C). This indicates that EPN-1 acts in the ced-10 pathway. To determine where in the ced-10 pathway EPN-1 acts, CED-1::GFP was observed in epn-1(m-z-) mutants. The clustering of CED-1 on pseudopods is normal in these mutants, which indicates EPN-1 acts downstream of the CED-1 signalling pathway.
In summary, it has thus far been determined that EPN-1 acts in the ced-1 pathway, downstream of ced-1.
EPN-1 recruits CHC-1 to extending pseudopods in response to CED-1 signalling
Next, the researchers investigated possible mechanisms of interaction between EPN-1 and CHC-1. Similar to the GFP-tagging used previously, the researchers employed a CHC-1::YFP tag for visualization. In epn-1(RNAi) embryos, recruitment of CHC-1::YFP to pseudopods was defective (Fig 5D,E). However, the enrichment of EPN-1::GFP on pseudopods remained relatively normal. Thus, enrichment of CHC-1 to pseudopods relies on EPN-1, but not vice versa. In ced-10 pathway mutants, CHC-1::YFP was persistently enriched on pseudopods; this indicates that the ced-10 pathway is not involved in recruiting CHC-1 to pseudopods. In contrast, the enrichment of CHC-1 to pseudopods was severely reduced or blocked in ced-1 pathway mutants (Fig 5Cbc, Dd, E); this means that the CED-1 pathway recruits CHC-1 to the engulfment site.
These results show that CHC-1 relies on recruitment by EPN-1, and the signalling pathway involved is the CED-1 pathway.
EPN-1 and CHC-1 promote actin polymerization during pseudopod extension
A GFP-tagged actin-binding domain of Drosophila moesin was used as a reporter for polymerized actin filaments (F-actin) expressed in engulfing cells (Lu et al., 2011). In wild-type embryos, F-actin first appeared at the site where pseudopods budded and then extended around the apoptotic cell until the phagocytic cup was closed. After engulfment, F-action disassembled asymmetrically. However, when epn-1 or chc-1 was RNAi inactivated, the extension of F-actin was slowed down (Fig 6B and C). As a result, engulfment took 2-4 times as long as in wild-type embryos. Thus, EPN-1 and CHC-1 are essential for the extension and stability of F-actin underneath pseudopods.
Impairing actin depolymerization partially suppresses epn-1 and chc-1 phenotypes
C. elegans unc-60 encodes members of the ADF/cofilin family of actin-depolymerization factors (McKim, et al. 1994). UNC-60A strongly inhibits actin polymerization in vitro and inactivation of unc-60 causes actin organization defects. Because of this, unc-60 became a target for determining the potential link between epn-1/chc-1 and actin polymerization. Inactivation of unc-60 (null mutation OR RNAi) reduced the number of cell corpses generated by epn-1(RNAi) or chc-1(RNAi). Additionally, inactivation of unc-60 partially reverted the delayed actin filament extension phenotype observed in epn-1 or chc-1 mutants (Fig 7B-D). This suggests that inactivation of epn-1 and chc-1 directly influences the extension and stability of actin filaments.
EPN-1 and CHC-1 are not only promoters of actin polymerization in pseudopod extension – they also are directly involved with with stability and extension of actin filaments.
ced-1, ced-6 and dyn-1 mutants (in CED-1 pathway) are defective in promoting actin polymerization for cell-corpse engulfment
If the CED-1 pathway regulates CHC-1 localization during engulfment through EPN-1, then inactivating components of the CED-1 signalling pathway (CED-1, CED-6 or DYN-1) should impair the rearrangement of actin around apoptotic cells. In ced-1 mutant embryos, the extension of F-actin was much slower than in wild-type embryos (Fig 8) – this was also observed in ced-6 and dyn-1 mutants. The actin-related defects observed from these mutants are similar to those observed in epn-1(RNAi) and chc-1(RNAi) embryos (compare Fig 6 and 8), which indicates the CED-1 pathway regulates actin assembly underneath the growing pseudopods through EPN-1 and CHC-1.
Clathrin and EPN-1 form an actin-organizing center to facilitate pseudopod extension
This study reveals the essential function of clathrin heavy chain and its adaptor epsin in apoptotic cell engulfment in C. elegans. Additionally, observations from this study suggest that clathrin-actin crosstalk directs actin polymerization and drives pseudopod extension around apoptotic cells.
This work also identifies the novel physiological role of EPN-1 in apoptotic cell engulfment. A proposal from the results of this study is that EPN-1 induces membrane curvature (curving of the pseudopod to match the shape of its prey) during engulfment and the associating clathrin and attached F-actin further stabilize the curvature. In essence, the cooperative actions of EPN-1, CHC-1, and F-actin result in the extension of pseudopods along apoptotic cell surfaces.
The CED-1 signaling pathway orchestrates the remodeling of both the plasma membrance and cytoskeleton during apoptotic cell engulfment
This study reveals F-actin assembly to be an important event in pseudopod extension that is regulated by CED-1. Together, the results that actin defects of ced-1 pathway component mutants and epn-1/chc-1 mutants are similar along with the essential role of CED-1 pathway components in recruiting EPN-1 and CHC-1, indicate that the CED-1 pathway drives EPN-1 and CHC-1 to regulate actin remodeling underneath the pseudopod membrane.
Two parallel engulfment pathways in C. elegans were proposed in previous work to converge at the point of CED-10 (Kinchen, et al., 2005). However, the results of this experiment indicate that not only do CED-1 pathway components act in parallel to CED-10 pathway components, but CHC-1 and EPN-1 also belong to the CED-1 pathway. Furthermore, the enrichment of EPN-1 and CHC-1 in pseudopods is independent of the CED-10 pathway. A summary of the proposed pathway from this study can be seen in Figure 9.
In summary, the work presented in this paper reveals the roles of clathrin and epsin in apoptotic cell engulfment and ties the CED-1 pathway to cytoskeleton remodeling that is essential to pseudopod extension.
Strengths of this paper include the detail and extent of experiments performed. The amount of figures from experiments in the paper is very large, and only key experiments are discussed on this webpage. Many control experiments were performed and results were confirmed with multiple experimental designs. Additionally, the paper was organized well. Results were presented in a logical flow, with the paper explaining why each step was a logical next avenue for investigation. However, this paper did exhibit some weaknesses. Many of the figures were complex, and had very many subsections. This made analysis of the figures difficult, especially because the paper had somewhat unclear explanations in the figure legends. Because many of the experiments involved time-lapse fluorescence, the amount of images was very large. The researchers chose to include images from each minute of each time lapse experiment – it may have been easier for the reader to process if only the key images were shown in the paper and the others were included in the supplemental. Additionally, the conclusion the paper draws from the results of Figure 8 seems to be a bit broad from the results obtained – while the phenotypes caused by mutant components of the CED-1 pathway may be similar to those in epn-1(RNAi) and chc-1(RNAi) embryos, it does not necessarily mean that the CED-1 pathway regulates actin assembly through EPN-1 and CHC-1. The defects cannot definitively be said to be a specifically a result of non-functioning EPN-1 and CHC-1 and not some other compound in the CED-1 pathway.
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