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A caspase–RhoGEF axis contributes to the cell size threshold for apoptotic death in developing Caenorhabditis elegans [1]

['Aditya Sethi', 'Faculty Of Biology', 'Center For Integrative Protein Sciences Munich', 'Cipsm', 'Ludwig-Maximilians-University Munich', 'Planegg-Martinsried', 'Department Of Cell', 'Developmental Biology', 'Division Of Biosciences', 'University College London']

Date: 2022-10

A cell’s size affects the likelihood that it will die. But how is cell size controlled in this context and how does cell size impact commitment to the cell death fate? We present evidence that the caspase CED-3 interacts with the RhoGEF ECT-2 in Caenorhabditis elegans neuroblasts that generate “unwanted” cells. We propose that this interaction promotes polar actomyosin contractility, which leads to unequal neuroblast division and the generation of a daughter cell that is below the critical “lethal” size threshold. Furthermore, we find that hyperactivation of ECT-2 RhoGEF reduces the sizes of unwanted cells. Importantly, this suppresses the “cell death abnormal” phenotype caused by the partial loss of ced-3 caspase and therefore increases the likelihood that unwanted cells die. A putative null mutation of ced-3 caspase, however, is not suppressed, which indicates that cell size affects CED-3 caspase activation and/or activity. Therefore, we have uncovered novel sequential and reciprocal interactions between the apoptosis pathway and cell size that impact a cell’s commitment to the cell death fate.

Funding: Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs ( https://orip.nih.gov/ ) (P40 OD010440). This work was supported by UCL (Capital Equipment Fund, CEF2), a predoctoral fellowship from the China Scholarship Council ( https://www.csc.edu.cn/ ) to HW, a predoctoral fellowship from the Studienstiftung des Deutschen Volkes ( https://www.studienstiftung.de/ ) to NM, a Wolfson Fellowship from the Royal Society ( https://royalsociety.org/ ) to BC (RSWF\R1\180008), the Deutsche Forschungsgemeinschaft ( https://www.dfg.de/en/index.jsp ) (ZA619/3-1 and ZA619/3-2 to EZ; C0204/10-1 and EXC114 to BC), and the Biotechnology and Biological Sciences Research Council ( https://bbsrc.ukri.org/ ) (BB/V007572/1 to BC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We are studying the unequal division of mothers of cells “programmed to die” in C. elegans, including the unequal division of the NSMnb. Previously, we proposed that unequal NSMnb division is the result of transient polar cortical contractility of the actomyosin network in the NSMnb prior to its division and local extension of the plasma membrane during NSMnb division [ 14 ]. Furthermore, we obtained evidence that the apoptosis pathway is active at a low, nonlethal level in mothers of cells programmed to die [ 15 – 17 ]. Surprisingly, we also found that loss-of-function (lf) mutations of the BH3-only gene egl-1 (egl, egg-laying defective), the Apaf-1-like gene ced-4 (ced, cell-death abnormal), or the caspase gene ced-3 compromise the ability of mothers to divide unequally and to generate a daughter cell with a size below the critical lethal threshold [ 16 ]. To elucidate the mechanism(s) through which the C. elegans apoptosis pathway affects the unequal division of mothers, we used a deep-sequencing coupled yeast 2 hybrid screen to search for physical interactors of its downstream effector, the caspase CED-3. In this screen, we identified ECT-2, a guanine nucleotide exchange factor (GEF) of RhoA-type GTPases [ 18 – 20 ]. Through molecular and genetic studies of the interactions between CED-3 caspase and ECT-2 RhoGEF, we have uncovered a novel role of ced-3 caspase in the control of the actomyosin network in the context of unequal cell division. Our findings also provide in vivo evidence for the existence of an inverse correlation between a cell’s size and its likelihood to undergo apoptosis and suggest that cell size affects the activation and/or the activity of CED-3 caspase.

Interestingly, within populations of animal cells grown in culture, smaller cells have a higher likelihood to undergo apoptosis [ 6 ]. Furthermore, decreasing the sizes of tissue culture cells by treating them with hypertonic solutions can cause these cells to die through apoptosis [ 4 ]. In addition, HeLa cells sometimes divide unequally and generate a smaller and a larger daughter cell. A certain proportion of the smaller daughter cells subsequently undergoes apoptosis [ 7 ]. These observations suggest that at least in vitro, a decrease in cell size can also trigger the apoptotic death of a cell. The following observations support the view that cell size can trigger apoptosis also in vivo. In some mutant backgrounds, Drosophila melanogaster germline stem cells divide unequally and generate a smaller and a larger daughter cell. As in HeLa cells, a certain proportion of the smaller daughter cells subsequently undergoes apoptosis [ 8 ]. And, many of the unwanted cells that reproducibly die through apoptosis during C. elegans development are the smaller daughter of a blast cell that divides unequally by size [ 9 , 10 ]. Importantly, mutations that cause such a “mother” to divide equally, thereby causing an increase in the size of the smaller daughter cell, compromise the ability of the unwanted cell to die [ 11 , 12 ]. To give an example, approximately 410 min after the first cleavage of the C. elegans 1-cell embryo, the neurosecretory motor neuron (NSM) neuroblast (NSM neuroblast; referred to as “NSMnb”) divides unequally. Its larger daughter cell survives and differentiates into the NSM neuron, whereas its smaller daughter cell, the “NSM sister cell” (NSMsc), dies. The loss of the pig-1 (pig, par-1 like-gene) gene, which encodes a kinase similar to mammalian MELK (maternal embryonic leucine zipper kinase), causes the NSMnb to divide equally resulting in daughter cells of almost identical sizes [ 11 , 13 ]. As a result, the now larger NSMsc sometimes fails to die. Hence, across animal species, there appears to be a critical “lethal size” threshold. Below this threshold, apoptosis can be triggered in cells that normally live. Conversely, above this threshold, apoptosis can be blocked in cells that are programmed to die. How cell size is controlled in this context and how cell size impacts a cell’s commitment to the apoptotic fate remains unclear.

Apoptosis is a type of programmed cell death that is conserved throughout the animal kingdom. The pathway that triggers apoptosis in “unwanted” cells includes pro- and anti-apoptotic members of the Bcl-2 family of proteins (BH3-only and Bcl-2-like proteins, respectively), Apaf-1-like adaptor proteins (which form the apoptosome) and members of the caspase family of cysteine proteases [ 1 , 2 ]. In unwanted cells, BH3-only proteins become active, and this leads to apoptosome assembly and the activation of caspases. Once caspase activity has reached a critical “lethal” threshold, apoptosis is triggered. A hallmark of cells undergoing apoptosis is a decrease in cell size [ 3 ]. This decrease is likely induced by the opening of potassium and chloride channels in the plasma membrane, causing an efflux of potassium and chloride ions followed by water [ 4 ]. Blocking this shrinkage in apoptotic cells in Caenorhabditis elegans embryos compromises their ability to die, which suggests that cell shrinkage facilitates the execution of apoptosis [ 5 ].

Results

CED-3 caspase physically interacts with ECT-2 RhoGEF To identify physical interactors of the caspase CED-3 that might link its function to unequal cell division, we performed a yeast 2 hybrid screen using a next-generation sequencing based screening procedure (https://nextinteractions.com/). As a bait construct, we used the full-length ced-3 open reading frame harboring a missense mutation that results in the production of proCED-3 zymogen lacking the critical active site cysteine (proCED-3(C358S)); this protein will be unable to mature into the fully active enzyme (Fig 1A). The proCED-3(C358S) bait was tested for interactions with proteins produced from a C. elegans cDNA library that was generated from mRNAs isolated from 8 different developmental stages and that represents approximately 13,000 genes. Among the proteins found to interact with proCED-3(C358S) is ECT-2 (ECT, epithelial cell transforming 2), the C. elegans orthologue of the mammalian proto-oncoprotein Ect2, a GEF of the RhoA family of small GTPases [18–20]. The proCED-3(C358S)–ECT-2 interaction is at least 16-fold stronger than the interaction of proCED-3(C358S) with either of 2 negative controls (empty bait-vector control and cDNA control). PPT PowerPoint slide

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TIFF original image Download: Fig 1. CED-3caspase physically interacts with ECT-2RhoGEF in vitro. (A) Schematic representation of the yeast 2-hybrid screen performed by Next Interactions (https://nextinteractions.com/) to identify physical interactors of proCED-3(C358S). (B) Schematic representation of the various constructs used in the GST pull-down assay along with their expected molecular weight in kilodalton (kDa) on the left. (C and D) Autoradiographs and Coomassie-stained gels of representative GST pull-down experiments. Black asterisks indicate potential breakdown products resulting from growing recombinant proteins in bacterial cultures. The different lanes shown in the figure are from the same gel. (E) Autoradiographs of representative in vitro cleavage experiments with CED-9, ECT-2, and ECT-2’s PH domain. Red asterisks indicate CED-9 cleavage products. Black asterisks indicate potential ECT-2 cleavage products resulting from incubation with bacterial lysate. https://doi.org/10.1371/journal.pbio.3001786.g001 To confirm that proCED-3(C358S) interacts with ECT-2, we produced GST-tagged proCED-3(C358S) in E. coli (GST::proCED-3(C358S)), purified it and incubated it with in vitro translated, 35S-methinonine-labeled and tagged (S·TAG) full-length ECT-2 protein (35S-S·TAG::ECT-2). We found that 35S-S·TAG::ECT-2 co-purifies with GST::proCED-3(C358S) but not GST alone (Fig 1B and 1C). Furthermore, we found that 35S-S·TAG::ECT-2 also co-purifies with recombinant GST::proCED-3 and GST::CED-3, each of which presumably can mature into the fully active CED-3 caspase (Fig 1B and 1C). ECT-2 has an N-terminal domain with 2 “BRCA1 C Terminus” (BRCT) motifs, a C-terminal “Pleckstrin Homology” (PH) domain and a central “Dbl-Homology” (DH) domain, which includes the catalytic GEF (http://www.uniprot.org/uniprot/Q9U364) [21] (Fig 1B). To determine which of these ECT-2 domains interact(s) with CED-3, we tested each domain for binding to GST::proCED-3. We found that only the C-terminal PH domain co-purifies with GST::proCED-3 (Fig 1D). In summary, our results demonstrate that both proCED-3 zymogen and active CED-3 caspase can physically interact with ECT-2 RhoGEF in yeast and in vitro, and that these interactions are mediated by ECT-2’s PH domain. Therefore, proCED-3 and CED-3 caspase may interact with ECT-2 RhoGEF in vivo. ECT-2 RhoGEF has 6 predicted caspase cleavage sites, including a cleavage site in the PH domain that is conserved across Caenorhabditis species (S1 Fig). To determine whether ECT-2 RhoGEF is a proteolytic substrate of CED-3 caspase, we used an in vitro cleavage assay based on bacterially expressed FLAG-tagged CED-3 protein (CED-3::8xFLAG). As a positive control, we used in vitro translated, 35S-methionine-labeled and tagged (S·TAG) CED-9 protein (35S-S·TAG::CED-9), which was previously shown to be a proteolytic substrate of CED-3 [22]. We found that 35S-S·TAG::CED-9 is efficiently cleaved by CED-3 (Fig 1E). In contrast, in vitro translated, 35S-methinonine-labeled full-length ECT-2 (35S-S·TAG::ECT-2) or the ECT-2 PH domain (35S-S·TAG::PH) are not cleaved by CED-3 using this in vitro assay. These results do not support the idea that an interaction between CED-3 caspase and ECT-2 RhoGEF in vivo results in CED-3 caspase-dependent cleavage of ECT-2 RhoGEF.

ect-2 RhoGEF cooperates with ced-3 caspase to control the size of the NSM sister cell The ect-2 RhoGEF gene has been shown to play an important role in the division of the C. elegans 1-cell embryo, which like the division of mothers of cells programmed to die is unequal by size and generates 2 daughter cells with different fates. Specifically, ect-2 RhoGEF promotes polarization of the cortical actomyosin network, which is required for the establishment and maintenance of anterior-posterior PAR protein asymmetry prior to the 1-cell embryo’s first division [23–25]. ect-2 also plays a critical role in cytokinesis and, hence, is an essential gene [20,26]. To determine whether ect-2 RhoGEF activity also impacts the divisions of mothers of cells programmed to die, we analyzed the effects of a temperature-sensitive (ts) partial lf mutation of ect-2, ax751 [27], on the division of the NSM neuroblast (NSMnb). (The ax751 mutation causes a single amino acid change C-terminal to the PH domain (G738R) [S2A Fig].) The NSMnb was identified in comma to 1 ½ fold stage embryos based on position and cell shape using a transgene that labels cell boundaries (P pie-1 mCherry::PHPLCΔ [ltIs44]) [28] (Fig 2A). We followed the division of individual NSMnb, estimated the sizes of the NSM and NSM sister cell (NSMsc) immediately post-cytokinesis, and divided the size of the NSMsc by the size of the NSM to acquire the “daughter cell size ratio” (Fig 2A). (There are 2 NSMnb, the left and the right NSMnb. Since these 2 neuroblasts are functionally identical, for simplicity, we will refer to them as “NSMnb.”) In wild-type animals, we found that daughter cell size ratios range from 0.61 to 0.70 with a mean ratio of 0.66; i.e., the NSMsc is approximately 0.66 times the size of the NSM (Fig 2B). We found that in ect-2(ax751ts) animals that were shifted to the nonpermissive temperature (25°C) approximately 2 hours prior to the division of the NSMnb, the mean ratio is 0.72 (range 0.59 to 0.90). Next, we tested 3 different ced-3 lf mutations for interactions with ect-2(ax751ts): the weak lf mutation n2427, the intermediate lf mutation n2436, and the putative null mutation n717 (S2B Fig) [29,30]. We found that all 3 mutations increase the range of ratios observed in ect-2(ax751ts) animals (0.62 to 1.00 in the case of n2427, 0.55 to 1.05 in the case of n2426, and 0.54 to 1.00 in the case of n717) (Fig 2C). Importantly, in each of the double mutants, we observed cases in which the NSMnb divided equally, generating 2 daughter cells of essentially identical sizes. In addition, ced-3(n2436) and ced-3(n717) both increase the mean ratio of ect-2(ax751ts) from 0.72 to 0.78, which is significantly different from the mean ratio of 0.66 observed in wild type. These results demonstrate that decreasing ect-2 RhoGEF function impacts the unequal division of the NSMnb, resulting in larger NSMsc. Furthermore, reduction of ced-3 caspase function enhances the ect-2 lf phenotype. Based on these findings, we conclude that ced-3 caspase and ect-2 RhoGEF cooperate during unequal NSMnb division to ensure that the size of the NSMsc is below the critical lethal threshold. PPT PowerPoint slide

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TIFF original image Download: Fig 2. ced-3 caspase cooperates with ect-2 RhoGEF in the control of daughter cell sizes in the NSM lineage. (A) Schematic representation of the NSM lineage. The NSMsc and NSM can be identified in comma stage embryos using the transgene P pie-1 ::mCherry::PHPLCΔ (ltIs44), which labels the plasma membrane of cells (orange arrow indicates the NSMsc and blue arrow indicates the NSM). Using confocal imaging, a Z-stack of the NSMsc and NSM can be obtained immediately post-division and the size ratio of the NSMsc:NSM can be estimated. The Z-stack of a pair of NSMsc (orange) and NSM (blue) in +/+, ect-2(ax751ts) and ect-2(xs111gf) mutants is shown. The corresponding mean daughter cell size ratios (NSMsc:NSM) are given below. Scale bars: 10 μm and 2 μm. (B–D) Daughter cell size ratios in +/+ and various ect-2 and ced-3 single and double mutants measured using ltIs44 (n = 10–20). Each gray dot represents the daughter cell size ratio of 1 pair of daughter cells. Horizontal red lines represent mean values, which are also indicated on top. The horizontal red dotted line represents the mean daughter cell size ratio of wild-type (+/+) embryos for comparison. The horizontal black dotted line in Fig 2B and 2C represents a daughter cell size ratio of 1.0 indicating equal division. The horizontal black dotted line in Fig 2D represents a daughter cell size ratio of 0.5 indicating that the smaller daughter is twice as small as the larger daughter. Statistical significance was determined using the Dunnett’s T3 multiple comparisons test (**** = P < 0.0001, ** = P < 0.01, * = P < 0.05, ns = P > 0.05). NSM, neurosecretory motor neuron. https://doi.org/10.1371/journal.pbio.3001786.g002

ect-2 RhoGEF acts downstream of or in parallel to ced-3 caspase to control the size of the NSM sister cell To test whether increasing ect-2 RhoGEF function also impacts unequal NSMnb division, we took advantage of 3 gain-of-function (gf) mutations of ect-2, xs111gf, xs129gf, and zh8gf, which cause single amino acid changes in residues (conserved in Caenorhabditis species) in the first BRCT motif (E129K), the second BRCT motif (E225K), or the PH domain (G707D), respectively (S2A Fig) [19,31]. We found that all 3 mutations affect the range of daughter cell size ratios. For example, in ect-2(zh8gf) animals, the ratios range from 0.42 to 0.60, which indicates that some divisions generated an NSMsc that is less than half the volume of the NSM (Fig 2B). (Note, unc-4(e120) has no effect on daughter cell size ratio [S3 Fig].) Furthermore, ect-2(xs111gf) and ect-2(zh8gf) significantly decrease the mean daughter cell size ratio from 0.66 to 0.52 and 0.55, respectively. We also tested the 3 ced-3 lf mutations for interactions with the ect-2 gf mutations and found that overall, reducing ced-3 function has no significant effect on daughter cell size ratios in ect-2 gf mutants (Fig 2D). These results demonstrate that increasing ect-2 RhoGEF function impacts unequal NSMnb division, resulting in smaller NSMsc. Reduction in ced-3 caspase function, however, has no effect on the ect-2 gf phenotype. This suggests that ect-2 RhoGEF acts in parallel to, or downstream of, ced-3 caspase to ensure that the size of the NSMsc is below the critical lethal threshold.

The ced-3 caspase, ect-2 RhoGEF-dependent pathway acts in parallel to the pig-1 MELK, nmy-2 nonmuscle myosin II-dependent pathway to control the size of the NSM sister cell The cortical enrichment of nonmuscle myosin II NMY-2 protein approximately 5 minutes prior to NSM neuroblast division is dependent on pig-1 MELK [14] but not ced-3 caspase (Fig 4A). In addition, we previously found that the loss of ced-3 caspase in animals homozygous for the strong pig-1 MELK lf mutation, gm344 [11], increases the mean daughter cell size ratio in the NSM lineage from 1.0 to 1.25, which indicates that the NSMsc is now larger than the NSM [16]. In contrast, the loss of the gene strd-1 STRADα, which acts in a par-4 LKB-dependent pathway required for the activation of PIG-1 MELK kinase activity [32–34], does not increase the daughter cell size ratio in the NSM lineage in a pig-1(gm344) background (1.01 compared to 1.02; [14]). These observations suggest that ced-3 caspase acts in parallel to the pig-1 MELK, nmy-2 nonmuscle myosin II-dependent pathway to ensure that the size of the NSMsc is below the critical lethal threshold. To determine whether reducing ect-2 RhoGEF function also increases the daughter cell size ratio in pig-1(gm344) animals, we attempted to generate animals homozygous for both ect-2(ax751ts) and pig-1(gm344). Unfortunately, we were unable to obtain such a strain, suggesting that animals lacking both genes are not viable. However, we found that animals homozygous for ect-2(ax751ts) and ok2283, a strong lf mutation of strd-1 STRADα [35], are viable. To determine whether reducing ect-2 RhoGEF function increases the daughter cell size ratio in animals defective in the pig-1 MELK, nmy-2 nonmuscle myosin II-dependent pathway, we therefore analyzed ect-2(ax751ts); strd-1(ok2283) animals. We found that ect-2(ax751ts) increases the mean daughter cell size ratio in strd-1(ok2283) animals from 0.95 to 1.11, which is statistically significant (S7 Fig). Furthermore, we found that the ect-2 gf mutation xs111 reduces the mean daughter cell size ratio in strd-1(ok2283) animals from 0.95 to 0.83 (S7 Fig). (Of note, we were also unable to generate the strain ect-2(xs111gf); pig-1(gm344), suggesting that it is also not viable.) Based on these observations, we propose that the ced-3 caspase, ect-2 RhoGEF-dependent pathway acts in parallel to the pig-1 MELK, nmy-2 nonmuscle myosin II-dependent pathway to control the size of the NSMsc and to ensure that its size is below the critical lethal threshold.

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