The story behind the emergence of different forms of cell death.

Various types of cell death program are needed for cells to respond to changes in physiological conditions. In this collection of Voices, we asked scientists to tell the story behind their contributions to the identification and mechanistic dissection of cell death pathways and to discuss future directions for such research.

ULP-2 SUMO protease regulates UPR and mitochondrial homeostasis in Caenorhabditis elegans.

Mitochondria are the powerhouses of cells, responsible for energy production and regulation of cellular homeostasis. When mitochondrial function is impaired, a stress response termed mitochondrial unfolded protein response (UPRmt) is initiated to restore mitochondrial function. Since mitochondria and UPRmt are implicated in many diseases, it is important to understand UPRmt regulation.

PUF-8, a C. elegans ortholog of the RNA-binding proteins PUM1 and PUM2, is required for robustness of the cell death fate.

During C. elegans development, 1090 somatic cells are generated, of which 959 survive and 131 die, many through apoptosis. We present evidence that PUF-8, a C.

labeling of endogenous genomic loci in using CRISPR/dCas9.

Visualization of genomic loci with open chromatin state has been reported in mammalian tissue culture cells using a CRISPR/Cas9-based system that utilizes an EGFP-tagged endonuclease-deficient Cas9 protein (dCas9::EGFP) (Chen et al. 2013). Here, we adapted this approach for use in .

The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions.

Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear.

A caspase-RhoGEF axis contributes to the cell size threshold for apoptotic death in developing Caenorhabditis elegans.

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.

Genetic screen identifies non-mitochondrial proteins involved in the maintenance of mitochondrial homeostasis.

The mitochondrial unfolded protein response (UPR ) is an important stress response that ensures the maintenance of mitochondrial homeostasis in response to various types of cellular stress. We previously described a genetic screen for genes, which when inactivated cause UPR activation, and reported genes identified that encode mitochondrial proteins. We now report additional genes identified in the screen.

Methods to Study the Mitochondrial Unfolded Protein Response (UPR) in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is a powerful model to study cellular stress responses. Due to its transparency and ease of genetic manipulation, C. elegans is especially suitable for fluorescence microscopy.

Genome-wide RNAi screen for regulators of UPRmt in Caenorhabditis elegans mutants with defects in mitochondrial fusion.

Mitochondrial dynamics plays an important role in mitochondrial quality control and the adaptation of metabolic activity in response to environmental changes. The disruption of mitochondrial dynamics has detrimental consequences for mitochondrial and cellular homeostasis and leads to the activation of the mitochondrial unfolded protein response (UPRmt), a quality control mechanism that adjusts cellular metabolism and restores homeostasis. To identify genes involved in the induction of UPRmt in response to a block in mitochondrial fusion, we performed a genome-wide RNAi screen in Caenorhabditis elegans mutants lacking the gene fzo-1, which encodes the ortholog of mammalian Mitofusin, and identified 299 suppressors and 86 enhancers.

MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology.

While the analysis of mitochondrial morphology has emerged as a key tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a challenging task and bottleneck for statistically robust conclusions. Here, we present Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik.

PIG-1 MELK-dependent phosphorylation of nonmuscle myosin II promotes apoptosis through CES-1 Snail partitioning.

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that the C. elegans orthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor.

Autophagy compensates for defects in mitochondrial dynamics.

Compromising mitochondrial fusion or fission disrupts cellular homeostasis; however, the underlying mechanism(s) are not fully understood. The loss of C. elegans fzo-1MFN results in mitochondrial fragmentation, decreased mitochondrial membrane potential and the induction of the mitochondrial unfolded protein response (UPRmt).

Tunable light and drug induced depletion of target proteins.

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development.

Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and .

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of -methylcytosine (mC) in RNA- and single-stranded DNA oligonucleotides, demethylation of -methyladenosine (mA) in tRNA or formation of 5-formyl cytosine (fC) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria.

PCMD-1 Organizes Centrosome Matrix Assembly in C. elegans.

Centrosomes, the major microtubule-organizing centers of animal cells, are essential for the assembly of a bipolar spindle during mitosis. Spindle defective-5 (SPD-5), the main scaffold protein of the centrosome matrix in Caenorhabditis elegans, forms a thin core around non-mitotic centrioles. Upon mitotic entry, the SPD-5-containing centrosome matrix expands in a Polo-like-kinase 1 (PLK-1)-dependent manner and this enables an enhanced microtubule nucleation activity during mitosis.

Twenty million years of evolution: The embryogenesis of four Caenorhabditis species are indistinguishable despite extensive genome divergence.

The four Caenorhabditis species C. elegans, C. briggsae, C.

Caspase Is Required for Asymmetric Divisions That Generate Cells Programmed To Die.

Caspases have functions other than in apoptosis. Here, we report that CED-3 caspase regulates asymmetric cell division. Many of the 131 cells that are "programmed" to die during development are the smaller daughter of a neuroblast that divides asymmetrically by size and fate.

Overlapping expression patterns and functions of three paralogous P5B ATPases in Caenorhabditis elegans.

P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson's disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity; however, the identity of the transport substrate(s) remains unknown.

CES-1 Snail Represses MELK Expression To Control Asymmetric Cell Division.

Snail-like transcription factors affect stem cell function through mechanisms that are incompletely understood. In the neurosecretory motor neuron (NSM) neuroblast lineage, CES-1 Snail coordinates cell cycle progression and cell polarity to ensure the asymmetric division of the NSM neuroblast and the generation of two daughter cells of different sizes and fates. We have previously shown that CES-1 Snail controls cell cycle progression by repressing the expression of CDC25.

miRNAs cooperate in apoptosis regulation during development.

Programmed cell death occurs in a highly reproducible manner during development. We demonstrate that, during embryogenesis, miR-35 and miR-58 family microRNAs (miRNAs) cooperate to prevent the precocious death of mothers of cells programmed to die by repressing the gene , which encodes a proapoptotic BH3-only protein. In addition, we present evidence that repression of is dependent on binding sites for miR-35 and miR-58 family miRNAs within the 3' untranslated region (UTR), which affect both mRNA copy number and translation.