The active role of corpse engulfment pathways during cell competition

Cell competition was first described in imaginal discs of genetically-mosaic Drosophila. In extreme cases, cell competition can replace entire compartments with the descendents of a single cell. We recently identified five genes that are required by wild-type epithelial cells to kill neighboring Minute cells during cell competition. These draper, wasp, phosphatidyl-serine receptor, MBC/DOCK180 and Rac1 genes, were each previously implicated in the engulfment of apoptotic corpses. The results draw attention to the active, killing role of engulfing cells during cell competition. Here we discuss the contributions of these engulfment genes to Minute competition in more detail, and compare Minute competition with competition between cells expressing different levels of Myc, or of Warts pathway genes. We also speculate about how cell interactions at clone boundaries may initiate cell competition.     

The Active Role of Corpse Engulfment Pathways During Cell Competition

Cell competition was first described in imaginal discs of genetically-mosaic Drosophila. In extreme cases, cell competition can replace entire compartments with the descendants of a single cell. We recently identified five genes that are required by wild type epithelial cells to kill neighboring Minute cells during cell competition. These draper, wasp, phosphatidyl-serine receptor, MBC/DOCK180 and Rac1 genes, were each previously implicated in the engulfment of apoptotic corpses. The results draw attention to the active, killing role of engulfing cells during cell competition. Here we discuss the contributions of these engulfment genes to Minute competition in more detail, and compare Minute competition with competition between cells expressing different levels of Myc, or of Warts pathway genes. We also speculate about how cell interactions at clone boundaries may initiate cell competition.     

Generation of Minute phenotypes by a transformed antisense ribosomal protein gene.

Antisense RNAs have been used for gene interference experiments in many cell types and organisms. However, relatively few experiments have been conducted with antisense genes integrated into the germ line. In Drosophila reduced ribosomal protein (r-protein) gene function has been hypothesized to result in a Minute phenotype. In this report we examine the effects of antisense r-protein 49 expression, a gene known to correspond to a Minute mutation An antisense rp49 gene driven by a strong and inducible promoter was transformed into the Drosophila germ line. Induction of this gene led to the development of flies with weak Minute phenotypes and to the transient arrest of oogenesis. Parameters that may affect the success of antisense gene inactivation are discussed.     

Cardiomyopathy is associated with ribosomal protein gene haplo-insufficiency in Drosophila melanogaster

The Minute syndrome in Drosophila melanogaster is characterized by delayed development, poor fertility, and short slender bristles. Many Minute loci correspond to disruptions of genes for cytoplasmic ribosomal proteins, and therefore the phenotype has been attributed to alterations in translational processes. Although protein translation is crucial for all cells in an organism, it is unclear why Minute mutations cause effects in specific tissues. To determine whether the heart is sensitive to haplo-insufficiency of genes encoding ribosomal proteins, we measured heart function of Minute mutants using optical coherence tomography. We found that cardiomyopathy is associated with the Minute syndrome caused by haplo-insufficiency of genes encoding cytoplasmic ribosomal proteins. While mutations of genes encoding non-Minute cytoplasmic ribosomal proteins are homozygous lethal, heterozygous deficiencies spanning these non-Minute genes did not cause a change in cardiac function. Deficiencies of genes for non-Minute mitochondrial ribosomal proteins also did not show abnormal cardiac function, with the exception of a heterozygous disruption of mRpS33. We demonstrate that cardiomyopathy is a common trait of the Minute syndrome caused by haplo-insufficiency of genes encoding cytoplasmic ribosomal proteins. In contrast, most cases of heterozygous deficiencies of genes encoding non-Minute ribosomal proteins have normal heart function in adult Drosophila.     

Elimination of oncogenic neighbors by JNK-mediated engulfment in Drosophila

A newly emerged oncogenic cell in the epithelial population has to confront antitumor selective pressures in the host tissue. However, the mechanisms by which surrounding normal tissue exerts antitumor effects against oncogenically transformed cells are poorly understood. In Drosophila imaginal epithelia, clones of cells mutant for evolutionarily conserved tumor suppressor genes such as scrib or dlg lose their epithelial integrity and are eliminated from epithelia when surrounded by wild-type tissue. Here, we show that surrounding normal cells activate nonapoptotic JNK signaling in response to the emergence of oncogenic mutant cells. This JNK activation leads to upregulation of PVR, the Drosophila PDGF/VEGF receptor. Genetic and time-lapse imaging analyses reveal that PVR expression in surrounding cells activates the ELMO/Mbc-mediated phagocytic pathway, thereby eliminating oncogenic neighbors by engulfment. Our data indicate that JNK-mediated cell engulfment could be an evolutionarily conserved intrinsic tumor-suppression mechanism that eliminates premalignant cells from epithelia.     

Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute

Ribosomal protein mutations, termed Minutes, have been instrumental in studying the coordination of cell and tissue growth in Drosophila. Although abundant in flies, equivalent defects in mammals are relatively unknown. Belly spot and tail (Bst) is a semidominant mouse mutation that disrupts pigmentation, somitogenesis and retinal cell fate determination. Here, we identify Bst as a deletion within the Rpl24 riboprotein gene. Bst significantly impairs Rpl24 splicing and ribosome biogenesis. Bst/+ cells have decreased rates of protein synthesis and proliferation, and are outcompeted by wild-type cells in C57BLKS<-->ROSA26 chimeras. Bacterial artificial chromosome (BAC) and cDNA transgenes correct the mutant phenotypes. Our findings establish Bst as a mouse Minute and provide the first detailed characterization of a mammalian ribosomal protein mutation.     

Ribosomal protein insufficiency and the minute syndrome in Drosophila: a dose-response relationship.

Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5'' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different.     

Genetic analysis of RpL38 and RpL5, two minute genes located in the centric heterochromatin of chromosome 2 of Drosophila melanogaster

The Minute mutations of Drosophila melanogaster are thought to disrupt genes that encode ribosomal proteins (RPs) and thus impair ribosome function and protein synthesis. However, relatively few Minutes have been tied to distinct RP genes and more Minute loci are likely to be discovered. We have identified point mutations in RpL38 and RpL5 in a screen for factors limiting for growth of the D. melanogaster wing. Here, we present the first genetic characterization of these loci. RpL38 is located in the centric heterochromatin of chromosome arm 2R and is identical to a previously identified Minute, M(2)41A, and also l(2)41Af. RpL5 is located in the 2L centric heterochromatin and defines a novel Minute gene. Both genes are haplo-insufficient, as heterozygous mutations cause the classic Minute phenotypes of small bristles and delayed development. Surprisingly, we find that RpL38(-)/+ and RpL5(-)/+ adult flies have abnormally large wings as a result of increased cell size, emphasizing the importance of translational regulation in the control of growth. Taken together, our data provide new molecular and genetic information on two previously uncharacterized Minute/RP genes, the heterochromatic regions in which they reside, and the role of their protein products in the control of organ growth.     

Differences in levels of the transmembrane protein Crumbs can influence cell survival at clonal boundaries

The survival and growth of individual cells in a tissue can be nonautonomously regulated by the properties of adjacent cells. In mosaic Drosophila imaginal discs, for example, wild-type cells induce the elimination of adjacent slow-growing Minute cells by apoptosis, while, conversely, certain types of faster-growing cells are able to eliminate adjacent wild-type cells. This process, known as cell competition, represents one example of a diverse group of phenomena in which short-range heterotypic interactions result in the selective elimination of one type of cell by another. The mechanisms that designate "winner" and "loser" genotypes in these processes are not known. Here we show that apoptosis is observed preferentially at boundaries that separate populations of cells that express different levels of the transmembrane protein Crumbs (Crb). Cells that express higher levels of Crb tend to be eliminated when they are near cells that express lower levels of Crb. We also observe distortions in the structure of epithelia on either side of boundaries between populations of cells that differ in Crb expression. Thus, while previous studies have focused mostly on the cell autonomous functions of Crb, we show that Crb can regulate cell survival and tissue morphology nonautonomously. Moreover, we find that the extracellular domain (ECD) of Crb, which seems to be dispensable for some of the other characterised functions of Crb, is required to elicit the nonautonomous effects on cell survival. The ECD can also regulate the subcellular localisation of Hippo pathway components, and possibly other proteins, in adjacent cells and may therefore directly mediate these effects. Several genetic lesions alter Crb levels, including loss-of-function mutations in hyperplastic tumour suppressors in the Hippo-Salvador-Warts pathway and in neoplastic tumour suppressor genes, such as scribble. Thus, Crb may be part of a "surveillance mechanism" that is responsible for the cell death that is observed at the boundaries of mutant clones in these cases.     

Induction of entosis by epithelial cadherin expression

Cell engulfment typically targets dead or dying cells for clearance from metazoan tissues. However, recent evidence demonstrates that live cells can also be targeted and that engulfment can cause cell death. Entosis is one mechanism proposed to mediate the engulfment and killing of live tumor cells by their neighbors, an activity often referred to as cell cannibalism. Here we report that the expression of exogenous epithelial cadherin proteins (E- or P-cadherin) in human breast tumor cells lacking endogenous expression of epithelial cadherins induces entosis and inhibits transformed growth. Entosis induced by cadherin expression is associated with the polarized distribution of Rho and Rho-kinase (ROCK) activity within entotic cells, which is dependent on p190A RhoGAP activity. ROCK inhibition or downregulation of p190A RhoGAP expression reduces entosis and increases the transformed growth of epithelial cadherin-expressing tumor cells. These data define new cell systems for the study of entosis, and identify entosis as a mechanism of cell cannibalism that is induced by the establishment of epithelial adhesion and inhibits transformed growth.     

The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre

Micrococcin-resistant mutants of Bacillus megaterium that carry mutations affecting ribosomal protein L11 have been characterised. The mutants fall into two groups. "L11-minus" strains containing an L11 gene with deletions, insertions or nonsense mutations which grow 2.5-fold slower than the wild-type strain, whereas other mutants carrying single-site substitutions within an 11 amino acid residue segment of the N-terminal domain of L11 grow normally. Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which regulates GTP hydrolysis on ribosomal factors. Micrococcin binding within the rRNA component of this centre was probed on wild-type and mutant ribosomes, in vivo, using dimethyl sulphate where it generated an rRNA footprint indistinguishable from that produced in vitro, even after the cell growth had been arrested by treatment with either kirromycin or fusidic acid. No drug-rRNA binding was detected in vivo for the L11-minus mutants, while reduced binding (approximately 30-fold) was observed for two single-site mutants P23L and P26L. For the latter, the reduced drug affinity alone did not account for the resistance-phenotype because rapid cell growth occurred even at drug concentrations that would saturate the ribosomes. Micrococcin was also bound to complexes containing an rRNA fragment and wild-type or mutant L11, expressed as fusion proteins, and they were probed with proteinases. The drug produced strong protection effects on the wild-type protein and weak effects on the P23L and P26L mutant proteins. We infer that inhibition of cell growth by micrococcin, as for thiostrepton, results from the imposition of a conformational constraint on protein L11 which, in turn, perturbs the function(s) of the ribosomal factor-guanosine nucleotide complexes.     

How the worm removes corpses: the nematode C. elegans as a model system to study engulfment

Apoptotic cell death in the nematode C. elegans culminates with the removal of the dying cells from the organism. This removal is brought forth through a rapid and specific engulfment of the doomed cell by one of its neighbors. Over half a dozen genes have been identified that function in this process in the worm. Many of these engulfment genes have functional homologs in Drosophila and higher vertebrates. Indeed, there is growing evidence supporting the hypothesis that the pathways that mediate the removal of apoptotic cells might be, at least in part, conserved through evolution.     

Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics.

We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method. Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E. coli chromosome. An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality. In this way, Ffh, the E. coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability. A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability. A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19. This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits. Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate. Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell. Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth.     

Cell cycle arrest determines the intensity of the global transcriptional response of Saccharomyces cerevisiae to ionizing radiation

Whole-genome analysis was performed using DNA microarrays to define the changes in the gene expression patterns occurring in Saccharomyces cerevisiae cells exposed to ionizing radiation. The effects of sublethal dose on wild-type, rad53 (enhanced sensitivity to radiation and impaired in a cell cycle damage checkpoint), and rad6 (enhanced sensitivity to radiation and functional cell cycle block by radiation) mutant backgrounds and of a higher dose on the wild-type and G(2)-phase-arrested cells were analyzed. Several gene pathways were identified as being implicated in the response to radiation. In particular, the cell cycle blockage that occurred in the wild-type strain after a high radiation dose and in the rad6 mutant after a lower dose entailed modifications of defined gene expression patterns, which are described here and are compared with the gene modulation patterns observed in the rad53 strain in the absence of efficient blockage. Loss of the RAD53 function caused a major increase in the number of genes modulated by radiation. Given that Rad53-Sad1p, the protein encoded by RAD53, has functions other than those directly connected to cell cycle arrest, we determined the gene patterns that were modulated upon irradiation of rad53 cells that had been forced to arrest in G(2) phase by nocodazole treatment. These differential whole-genome analyses shed light on the multiplicity of functions of the pivotal Rad53-Sad1p protein. The results obtained describe how the cells respond to different irradiation conditions by modulating important gene classes, including those associated with stress defense, ribosomal proteins, histones, ergosterol and GCR1-controlled sugar metabolism.