Kutscher, Lena et al. (2011) International Worm Meeting "lin-61 encodes a histone tail binding protein and acts as a transcriptional repressor."

Kutscher, Lena, Koester-Eiserfunke, Nora, Fischle, Wolfgang

[International Worm Meeting, 2011]

Malignant brain tumor (MBT) domain-containing proteins are involved in epigenetic regulation of the eukaryotic genome. These proteins have been implicated in a variety of cellular processes, including gene repression, germ cell formation and organism development. They localize to the nucleus and bind chromatin. MBT proteins in mammals and flies bind monomethylated and dimethylated lysines on histone tail peptides, with little discrimination towards a specific lysine residue. Key amino acid residues and binding mechanisms have been determined in these organisms in vitro, but many studies have struggled to relate MBT protein structure to its function in vivo. In C. elegans, however, one MBT protein, LIN-61, is unique in that it preferentially binds dimethylated and trimethylated lysines, and only in the context of histone H3 N-terminal tail methylated at lysine 9, a post-translational modification indicative of silenced heterochromatin. lin-61 is involved in vulva development, among other developmental pathways, in the nematode, and binding to H3K9me3 appears essential for this process. However, many open questions remain about its biology, and the binding mechanism of LIN-61 to chromatin has not yet been determined. To gain a complete understanding of LIN-61, we investigated its molecular function by implementing a multifaceted approach, including microarray analysis of lin-61 mutant worms to see how gene expression changes in its absence, its effect on the binding of HPL-2, a HP-1 homolog, to histone tail peptides, and a genetic assay to determine the role of H3K9me3 binding in genome instability. We found that lin-61 is a repressor of gene expression, with over 60% of the genes identified in the microarray as being upregulated in the absence of lin-61. Many of the genes that are upregulated include vulva development-related genes, genes involved in RNAi silencing in the germline, and genes involved in germ cell establishment. Additionally, we found that binding of HPL-2 to H3K9me3 histone tails is dependent on the presence of LIN-61, and that HPL-2 and LIN-61 seem to physically interact. Finally, we found that genome stability depends on the physical presence of LIN-61, and H3K9me3 binding appears to have a minimal role in maintaining genome stability. These findings provide essential information in determining the role of chromatin and HPL-2 interaction in regards the function of LIN-61 in C. elegans. L.K. was supported by the German Academic Exchange Service (DAAD).

Kutscher, Lena et al. (2017) International Worm Meeting "Competitive phagocytosis: a novel mechanism for clearance of the non-apoptotically dying linker cell."

Kutscher, Lena, Keil, Wolfgang, Shaham, Shai

[International Worm Meeting, 2017]

Programmed cell death and subsequent cell clearance are important in the development and maintenance of organisms and tissues. Defects in cell clearance can result in inappropriate inflammatory responses and autoimmune disorders. Although much is known about apoptotic cell corpse removal, our understanding of mechanisms driving the clearance of cells dying by non-apoptotic means is rudimentary. The C. elegans linker cell dies non-apoptotically through a death process termed LCD (Linker Cell-type Death), which has been molecularly characterized in our lab. This male-specific cell is born in the L2 larval stage, leads elongation of the developing gonad, and then dies in a programmed manner independently of known apoptosis, autophagy, or necrosis genes. Importantly, linker cell corpses are robustly engulfed in mutants carrying lesions in genes required for engulfment of apoptotic bodies, suggesting a novel engulfment program. Recently, we imaged the migration, death, engulfment, and degradation of the linker cell over a 20h period, through the L4-to-adult transition, using a new three-channel microfluidic imaging device we built, which allows for long-term imaging of C. elegans larval development. Using this device, we uncovered the precise sequence of events leading to linker cell demise and degradation. The linker cell leads the gonad to the cloaca, and plugs the distal gonad to prevent inappropriate leakage of gonadal contents. Near the cloaca, the cell is ensheathed, but not yet engulfed, by the two U.l/rp cells. Nuclear crenellation, a LCD hallmark, then become evident. Both U.l/rp cells next actively attempt to engulf the dying linker cell, eventually breaking it into two parts of unequal volume. This competitive phagocytosis occurs in 60/64 wild-type animals imaged. Once the cell is engulfed, the caspase ced-3 and its upstream activator ced-4, which are not required for LCD, act in the linker cell to facilitate its degradation. The small GTPases RAB-35 and ARF-6 function antagonistically in the engulfing cells for efficient linker cell degradation. Together, our results demonstrate the utility of long-term imaging of C. elegans larva at high spatiotemporal resolution, uncover components of a novel cell degradation mechanism, and reveal previously unknown roles for caspases in cell corpse degradation during caspase-independent cell death.

Kutscher, Lena M. et al. (2013) International Worm Meeting "Identification of interacting partners of a poly-glutamine protein involved in non-apoptotic cell death."

Shaham, Shai, Kutscher, Lena M.

[International Worm Meeting, 2013]

Cell death is an important cellular process in development and disease. While caspase-dependent apoptosis has been extensively studied, cell death still occurs in animals lacking these proteases, suggesting that cells must be able to engage non-apoptotic cell death pathways. One such example is the non-apoptotic cell death of the C. elegans linker cell, which leads the elongation of the developing male gonad during larval development. At the L4-to-adult transition, when the linker cell reaches the cloaca, it dies. Linker cell death is not apoptotic, as assessed by mechanism and morphology, and relies in part on a poly-glutamine protein, PQN-41C. We are interested in identifying binding partners of PQN-41C to determine its functions during linker cell death and outside the linker cell. We used a yeast two-hybrid screen to isolate 33 putative PQN-41C interactors. Gene ontology and protein domains identify five subclasses of interactors affecting proteolysis (asp-3, F32A5.3, K10C2.1, F57F5.1, nep-17), or possessing coiled-coil domains (pqn-41, pqn-59, R11A8.7, pqn-85, npp-4), ubiquitin-related domains (tag-214, pqn-59, F52G3.1, sao-1), and RNA-related (etr-1, asd-1, asd-2, pes-4), or DNA-associated domains (chd-7, zfp-1, tag-153, eef-2, T19D12.2, pqn-85). We tested the functions of the interacting proteins in linker cell death by examining whether RNAi against each candidate promotes linker cell survival. Of the genes we tested thus far, seven appear to have a function in linker cell death, with linker cell survival rates ranging from 12% to 33% upon RNAi knockdown. Two candidates may cause enhanced linker cell death or cell clearance upon knockdown. Our results therefore suggest that PQN-41C may act in complex with other proteins to promote linker cell death.

Kutscher, Lena et al. (2015) International Worm Meeting "Degradation and clearance of the linker cell is coordinated by a novel caspase function and the GTPases rab-35 and arf-6."

Shaham, Shai, Wang, Ying, Kutscher, Lena, Haley, Ryan, Zhou, Zheng

[International Worm Meeting, 2015]

Programmed cell death and cell corpse removal are important in the development and homeostasis of tissues and organisms. While much is known about apoptotic corpse clearance, clearance of cells that die by non-apoptotic programs is not well understood. The death of the linker cell, a male-specific cell that leads the elongation of the developing gonad, is independent of the apoptotic caspase CED-3. ced-3 mutations do not block linker cell death or engulfment, and the fraction of surviving cells in known linker cell death mutants is not enhanced by ced-3 lesions. However, we found that in animals carrying mutations in ced-3 or its regulator, ced-4/Apaf1, engulfed linker cells are degraded inefficiently. Mutations in the apoptotic regulators egl-1/BH3-only or ced-9/Bcl2 do not affect linker cell clearance. ced-3 is expressed in the linker cell as it dies but not during migration. While many linker cell-containing phagosomes are eventually degraded in ced-3 mutants, clearance is delayed by hours or even days. From a genetic screen in which artificial insemination was used to propagate relevant mutants, we recovered several in which linker cell degradation is affected. We found that two small GTPases, RAB-35 and ARF-6, act antagonistically within the U.l/rp cells, which engulf the dying linker cell, to degrade the linker cell phagosome. Genetic interaction studies suggest that RAB-35 inhibits ARF-6, which, in turn, blocks linker cell degradation. We identified RME-4 and TBC-10 as the relevant guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) for RAB-35, and EFA-6 and CNT-1 as the relevant GEF and GAP for ARF-6. Persistent linker cell-containing phagosomes in rab-35 mutants appear to have degraded linker cell contents, and this degradation is delayed by mutations in ced-3. Importantly, the frequency of persistent phagosomes is similar in rab-35(0) and rab-35(0); ced-3(0) animals. Thus, our observations suggest that ced-3 may function upstream of rab-35, which may then promote linker cell phagosome maturation. We also show that rab-35, but not arf-6, is involved in phagosome maturation of apoptotic corpses in the embryo and germline, and is localized to extending pseudopods and to phagosomal surfaces. Rab35 has been implicated in phagosome maturation in mammals, although not in the context of cell death. Caspases have been suggested to promote degradation but not initiation of cell dismantling during spermatogenesis in Drosophila and during neurite pruning in vertebrates. We propose therefore that the cell degradation pathway we uncovered may be conserved.

Kutscher, Lena M et al. (2013) International Worm Meeting "Using artificial insemination to identify genes involved in linker cell death and corpse removal."

Tishbi, Nima, Kutscher, Lena M, Shaham, Shai

[International Worm Meeting, 2013]

The male-specific C. elegans linker cell dies post-embryonically after leading male gonad elongation. Linker cell death occurs in the absence of all known apoptotic cell death genes, and dying linker cells are ultrastructurally distinct from apoptotic cells. Previously, a genome-wide RNAi screen revealed six genes involved in linker cell death, including one encoding a polyglutamine-containing protein. Subsequently, we also identified genes whose inactivation by mutation, but not by RNAi, prevents linker cell death. These results suggest that a forward genetic screen may reveal novel genes required for linker cell death and clearance. Our initial attempts at such a screen proved unsuccessful because of the possibility of male sterility, and the low recovery of males from clonal isolates of mutagenized Him strains. To circumvent these limitations, we artificially inseminated wild-type hermaphrodites with sperm derived from mutagenized F2 males with a surviving linker cell. We performed 66 successful artificial inseminations (from 181 attempts), resulting in 44 independent strains with linker cell death defects. The mutants are varied; they seem to block linker cell death, delay it, affect aspects of the morphology of the dying linker cell, or prevent engulfment. Linker cell engulfment mutants are of particular interest as this process is independent of all known engulfment genes. Whole genome sequencing of several of our mutants suggests that we have indeed identified novel cell death genes. Progress towards cloning some of these will be discussed.