Breckenridge DG et al. (2009) Curr Biol "Bcl-2 proteins EGL-1 and CED-9 do not regulate mitochondrial fission or fusion in Caenorhabditis elegans."

Xue D, Kang BH, Breckenridge DG

[Curr Biol, 2009]

The Bcl-2 family proteins are critical apoptosis regulators that associate with mitochondria and control the activation of caspases. Recently, both mammalian and C. elegans Bcl-2 proteins have been implicated in controlling mitochondrial fusion and fission processes in both living and apoptotic cells. To better understand the potential roles of Bcl-2 family proteins in regulating mitochondrial dynamics, we carried out a detailed analysis of mitochondria in animals that either lose or have increased activity of egl-1 and ced-9, two Bcl-2 family genes that induce and inhibit apoptosis in C. elegans, respectively. Unexpectedly, we found that loss of egl-1 or ced-9, or overexpression of their gene products, had no apparent effect on mitochondrial connectivity or mitochondrial size. Moreover, loss of ced-9 did not affect the mitochondrial morphology observed in a drp-1 mutant, in which mitochondrial fusion occurs but mitochondrial fission is defective, or in a fzo-1 mutant, in which mitochondrial fission occurs but mitochondrial fusion is restricted, suggesting that ced-9 is not required for either the mitochondrial fission or fusion process in C. elegans. Taken together, our results argue against an evolutionarily conserved role for Bcl-2 proteins in regulating mitochondrial fission and fusion.

Wu, Zheng et al. (2021) International Worm Meeting "Investigating non-apoptotic roles for egl-1"

Wu, Zheng, Pierce, Jonathan

[International Worm Meeting, 2021]

The Bcl-2 Homology (BH) 3-only protein EGL-1 is a key regulator of apoptosis pathway in Caenorhabditis elegans and is conserved across metazoans. Generally, decades of research have shown that expression of egl-1 is necessary and sufficient to trigger apoptosis in C. elegans. However, transcripts of BH3-only proteins are also detected in living neurons of healthy mammalian brains. Non-apoptotic roles and their underlying mechanisms for BH-3-only domain proteins are not well understood. Our lab discovered that egl-1 is expressed throughout the life of the worm in the URX pair of sensory neurons without inducing apoptosis. This expression depends cell autonomously on neuronal activity and calcium. URX is well known for its function as an oxygen sensor, but it also projects a process into the body cavity neuron where it senses signals from coelomic fluid. We are investigating non-apoptotic roles for egl-1 expression in URX. Our preliminary results show that both egl-1 mutation and URX-specific RNAi knock down of egl-1 impaired URX-related behaviors in worms. Further study of how egl-1 functions in URX without inducing apoptosis may reveal insight regarding the functions of BH-3-only domain proteins in the mammalian brain.

Mapes J et al. (2012) Curr Biol "CED-1, CED-7, and TTR-52 regulate surface phosphatidylserine expression on apoptotic and phagocytic cells."

Chen YZ, Xue D, Kim A, Kang BH, Mitani S, Mapes J

[Curr Biol, 2012]

BACKGROUND: Phosphatidylserine (PS) normally confined to the cytoplasmic leaflet of plasma membrane (PM) is externalized to the exoplasmic leaflet (exPS) during apoptosis, where it serves as an "eat-me" signal to phagocytes. In addition, some living cells such as macrophages also express exPS. RESULTS: A secreted Annexin V (sAnxV::GFP) PS sensor reveals that exPS appears early on apoptotic cells in C. elegans embryos and decreases in older or unengulfed apoptotic cells. This decrease in exPS expression is blocked by loss of CED-7, an ATP binding cassette (ABC) transporter, or TTR-52, a secreted PS binding protein. Phagocytic cells also express exPS, which is dependent on the activity of CED-7, TTR-52, and TTR-52-interacting phagocyte receptor CED-1. Interestingly, a secreted lactadherin PS sensor (sGFP::Lact(C1C2)) labels apoptotic cells but not phagocytes, prevents sAnxV::GFP from labeling phagocytes, and compromises phagocytosis. Immuno-electron micrographs of embryos expressing sAnxV::GFP or sGFP::Lact(C1C2) reveal the presence of extracellular PS-containing vesicles between the apoptotic cell and neighboring cells, which are absent or greatly reduced in the ced-7 and ttr-52 mutants, respectively, indicating that CED-7 and TTR-52 promote the generation of extracellular PS vesicles. Loss of the tat-1 gene, which maintains PS asymmetry in the PM, restores phagocyte exPS expression in ced-1, ced-7, and ttr-52 mutants and partially rescues their engulfment defects. CONCLUSIONS: CED-7 and TTR-52 may promote the efflux of PS from apoptotic cells through the generation of extracellular PS vesicles, which lead to exPS expression on phagocytes via TTR-52 and CED-1 to facilitate cell corpse clearance.

Breckenridge DG et al. (2008) Mol Cell "Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell-death execution pathways downstream of ced-3 and independent of ced-9."

Mitani S, Kang BH, Breckenridge DG, Kokel D, Staehelin LA, Xue D

[Mol Cell, 2008]

The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3 or the profission gene drp-1 are not required for apoptosis activation in C. elegans. However, minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3 caspase to promote elimination of mitochondria in dying cells, an event that could facilitate cell-death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1''s proapoptotic function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell-death execution downstream of caspase activation.

Deak P et al. (1980) Worm Breeder's Gazette "bal-X-1 chromosome."

Deak P, Fodor A

[Worm Breeder's Gazette, 1980]

The isolation and first characterization about a balancer chromosome temporarily called Bal-X-1 was presented at the Cold Spring Harbor meeting. This chromosome when heterozygous with an X chromosome marked by dpy-8 and unc-3 suppress crossover on this region. Characterizing the Bal-X-1 further on, we found, that Bal-X-1 viable only in heterozygous or hemizygous form. Bal-X-1 has been marked with lon-2(e678) allele. Bal-X-1 (lon) males are perfectly normal and fertile. Hermaphrodites heterozygous for Bal-X-1 always segregate males about 8-20% frequencies. The males are XO. Bal-X animals homozygous for her-1(e1520) (kindly provided by Jonathan Hodgin) are viable but almost completely sterile. Even if they lay some eggs, their oldest progeny has never been larger than L1, which die soon. Bal-X-1 can balance the following mutants: mn-110 (BH); let-2(e1470) (A.F.); let-15(e1471) (A.F.). We are going on to learn, whether Bal-X-1 is a translocation. In these experiments, we use stocks of double homozygous for two markers ( one in the medium and one in one of the ends the autosome) of the different autosomes and measure the recombination between them. If Bal-X-1 is a translocation like Dp-1 (BH) we expect, that the recombination frequency will be changed on the autosome being involved.

Wang Y et al. (2016) Nat Commun "Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans."

Liang Q, Kang BH, Yin XM, Wang Y, Zhang Y, Chen L, Miao L, Xue D

[Nat Commun, 2016]

In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME.

Lama D et al. (2010) Biochemistry "Identification of core structural residues in the sequentially diverse and structurally homologous Bcl-2 family of proteins."

Lama D, Sankararamakrishnan R

[Biochemistry, 2010]

The Bcl-2 family of proteins regulates the intrinsic pathway of apoptosis and plays a significant role in mitochondrial outer membrane permeabilization. Bcl-2 homologues belonging to both anti- and pro-apoptotic classes have been identified in diverse organisms. While anti-apoptotic Bcl-2 proteins possess up to four BH sequence domains (BH1-BH4), the pro-apoptotic counterparts have either three BH (BH1-BH3) domains or only the BH3 domain. Many anti-apoptotic viral homologues do not seem to have any detectable BH homology regions and exhibit a very low level of sequence identity with other Bcl-2 family members. However, structures determined for several Bcl-2 anti- and pro-apoptotic proteins and their viral homologues show a remarkably conserved helical fold characterized by a central hydrophobic helix surrounded by five or six amphipathic helices. In this study, we have analyzed 16 nonredundant Bcl-2 structures from human, mouse, Caenorhabditis elegans, and five different viral species. While the length of the central hydrophobic helix is preserved in all the Bcl-2 structures, variations in length are observed for other helices. We performed multiple-structure alignment of all 16 structures. Eighty structurally equivalent positions, the bulk of them in the helical regions, constituted the ungapped blocks in the structure-based sequence alignment. Analysis of helix bundle geometry indicates that helix-helix packing differed in different Bcl-2 structures. This is presumably to accommodate disparate residue substitutions. Residue properties such as solvent accessibility, conservation of chemical nature, and/or size and involvement in interhelical interactions were analyzed in each position of the ungapped alignment regions. A sequence motif made up of small amino acids has been detected in the central helix that is proposed to be important for helix-helix association. We have found that residues in 22 positions in the helical regions are buried, exhibit conservation in hydrophobicity and/or size, and participate in interhelical interactions in at least 12 of the 16 structures studied. We also found 15 additional positions in which residues exhibit two of the three properties investigated. We suggest that these positions constitute the important structural core in the diverse Bcl-2 family members and could play a significant role in the folding of the protein. Results of our studies have been used in the identification of three putative Bcl-2 homologues from three different viral organisms. This study will help in the genome-wide identification of hitherto unrecognized Bcl-2 family members, especially in viral genomes.

Strasser A et al. (2018) Cell Death Differ "Viewing BCL2 and cell death control from an evolutionary perspective."

Vaux DL, Strasser A

[Cell Death Differ, 2018]

The last 30 years of studying BCL2 have brought cell death research into the molecular era, and revealed its relevance to human pathophysiology. Most, if not all metazoans use an evolutionarily conserved process for cellular self destruction that is controlled and implemented by proteins related to BCL2. We propose the anti-apoptotic BCL2-like and pro-apoptotic BH3-only members of the family arose through duplication and modification of genes for the pro-apoptotic multi-BH domain family members, such as BAX and BAK1. In that way, a cell suicide process that initially evolved as a mechanism for defense against intracellular parasites was then also used in multicellular organisms for morphogenesis and to maintain the correct number of cells in adults by balancing cell production by mitosis.

Sood P et al. (2017) Traffic "Cargo crowding at actin-rich regions along axons causes local traffic jams."

Kumar TV, Koushika SP, Nonet ML, Murthy K, Menon GI, Sood P

[Traffic, 2017]

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes (Kang, Tian et al. 2008, Tang, Scott et al. 2012, Tang, Scott et al. 2013, Iacobucci, Rahman et al. 2014). We examine the transport of precursors of synaptic vesicles (pre-SVs), endosomes and mitochondria in C. elegans touch receptor neurons (TRNs), showing that stationary cargo are predominantly present at actin-rich regions along the neuronal process. Stationary vesicles at actin-rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre-SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin-rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron.

Erin Peden et al. (2005) International Worm Meeting "Identification of additional pro-apoptotic BH3 only proteins in C. elegans."

Ding Xue, David Breckenridge, Erin Peden

[International Worm Meeting, 2005]

The Bcl-2 family of proteins is a group of conserved cell death regulators, first defined by the human proto-oncogene bcl-2, which promotes cell survival and was identified by virtue of its overexpression in a number of B-cell lymphomas. They are characterized by the presence of at least one of four conserved Bcl-2 homology (BH) domains and are found in organisms as distantly related as C. elegans and humans. Interestingly, members of this family can be either anti-apoptotic or pro-apoptotic and can form heterodimers with selected family members. The pro-apoptotic family members include proteins with multiple BH domains such as Bax and Bak and proteins with a single BH3 motif, which are named BH3-only proteins. BH3-only Bcl-2 proteins have been found to play important roles in activating the apoptotic program. However, due to the low overall sequence conservation and small region of homology (about 9 amino acids), BH3-only proteins are difficult to identify. In C. elegans, two BH3-only proteins, EGL-1 and CED-13, have been found and there are likely additional BH3-only proteins involved in regulating programmed cell death in nematodes. Current bioinformatics tools are not designed to identify short sequence motifs in the large sequence databases. A simple BLAST search for the consensus BH3 domain does not yield the known BH3 containing proteins. In order to identify BH3 containing proteins in silico, we have developed a BH3 search algorithm, which correctly identifies known BH3-containing proteins in C. elegans along with promising candidate genes. We are in the process of determining the potential roles of these candidate genes in C. elegans apoptosis using the RNAi approach or available deletion mutations in candidate genes. Our BH3 search algorithm may provide a promising tool to identify unknown BH3 only proteins that may affect apoptosis in C. elegans and in other organisms such as Drosophila where BH3-only proteins have yet to be found.