Anbing Shi et al. (2007) International Worm Meeting "A novel function for ALX-1/Alix in endocytic recycling."

Barth D Grant, Anbing Shi, Vanessa Figueroa, Carlos Chih-Hsiung Chen, Saumya Pant

[International Worm Meeting, 2007]

The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. We have previously shown that RME-1 functions as a critical regulator of endocytic recycling of cargo proteins that are internalized by clathrin-mediated and clathrin-independent endocytosis events. RME-1 is a conserved EHD family protein harboring a C-terminal EH domain. In a yeast 2-hybrid screen using the RME-1-EH domain as bait we identified a novel binding partner called ALX-1. ALX-1 is the C.elegans homolog of mammalian Alix and yeast Bro1p. Published reports indicate that Alix/Bro1p family proteins function in the multivesicular body(MVB) pathway. Oligomeric protein complexes containing ubiquitin binding protein Hrs and ESCRT complexes mediate sorting of cargo at the MVBs limiting membranes and membrane invagination. Studies in yeast have shown that Bro1 functions in concert with components of the ESCRT to regulate MVB formation. Alix is also required for the budding of retroviruses like HIV from the plasma membrane, and the actin-dependent intracellular positioning of endosomes in tissue culture cells. Although Alix has been suggested to function in multiple cellular processes, its precise functions still remain somewhat mysterious. The interaction with RME-1 suggested that ALX-1 might play a role in recycling, or that RME-1 plays a role in the MVB. Our studies did not find any role for RME-1 in membrane protein degradation, but revealed a requirement for ALX-1 in the recycling of clathrin-independent membrane proteins. In this study we define protein domains required for RME-1 and ALX-1 interaction, and have taken a functional approach to determine ALX-1 function in vivo taking advantage of the consortium knockout allele alx-1(gk275). We show a distinct functional involvement for ALX-1 in the RME-1-associated, clathrin-independent, endocytic recycling pathway of polarized epithelial cells of the C.elegans intestine. Loss of ALX-1 results in intracellular accumulation of both RME-1 and a clathrin independent endocytic recycling cargo protein, which suggests that the recycling of clathrin-independent cargo requires ALX-1. No changes were observed in the localization of the clathrin-dependent cargo protein hTfR, indicating that clathrin dependent cargo recycles normally in the absence of ALX-1. We also show that ALX-1 localizes to RME-1-positive recycling endosomes, and that mutant forms of ALX-1 that cannot bind RME-1 fail to rescue recycling phenotypes. Finally we also show functional association of ALX-1 with the membrane-protein degradation pathway of the C.elegans embryo, a function that is independent of RME-1.

Anbing Shi et al. (2006) Development & Evolution Meeting "Alx-1 Function in C. elegans Membrane Traffic"

Anbing Shi, Vanessa Figueroa, Saumya Pant, Carlos Chih-Hsiung Chen, Barth Grant

[Development & Evolution Meeting, 2006]

The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. We have previously shown that RME-1 functions as a critical regulator of endocytic recycling events in both clathrin medicated and clathrin independent endocytosis events. RME-1 is a conserved EHD family protein harboring a C-terminal EH domain. In a yeast 2-hybrid screen with the RME-1-EH domain we identified a novel binding partner called Alx-1. Alx-1 is the C. elegans homolog of mammalian Alix and yeast Bro1p. Published reports indicate that Alix/Bro1p functions in the multivesicular body (MVB) pathway. MVBs are endosomes containing lumenal vesicles formed by inward invagination of the limiting membrane of endosomes. MVBs are believed to mediate transport between early endosomes and late endosomes/lysosomes. Membrane proteins sorted into the lumenal vesicles of MVBs are degraded upon fusion of the MVBs with lysosomes. Oligomeric protein complexes containing ubiquitin binding protein Hrs and ESCRT (endosomal sorting complexes required for transport) complexes mediate sorting of cargo at the limiting membranes of the MVBs and invagination of the endosome membrane. Studies in yeast have shown that Bro1 functions in concert with components of the ESCRT machinery to regulate MVB formation (Odorizzi et al., 2003). Alix also associates with structural proteins of the cytoskeleton, including actin and tubulins (Schmidt et al., 2003). Recently, Alix has been reported to function in the actin-dependent intracellular positioning of endosomes in tissue culture cells (Cabezas et al., 2005). Although Alix has been suggested to function in multiple cellular processes, its precise functions still remain somewhat mysterious. The interaction with RME-1 suggests either that ALX-1 plays a role in recycling or that RME-1 plays a role in the MVB. We have taken a functional approach to determine ALX-1 function in vivo taking advantage of the consortium knockout allele alx-1 (gk275) and by colocalization analysis of ALX-1 with various endosome makers. Our progress in this analysis will be presented at the meeting.

Shi Y et al. (2000) East Coast Worm Meeting "CGH-1, an essential and conserved germline RNA helicase"

Shim EY, Kohara Y, Blackwell TK, Shi Y, Navarro RE

[East Coast Worm Meeting, 2000]

RCK is a conserved DEAD-box type RNA helicase that is present in the germline. In Drosophila and Xenopus, RCK is expressed specifically during oogenesis and early embryonic stages, and in Xenopus it is associated with maternal mRNA. S. pombe RCK (ste13) is required for entry into either G0 or meiosis, however, and in mice RCK is expressed in oocytes and also in some adult tissues, suggesting the possibility of a conserved role in self-renewing cells. In a screen for proteins that bind the germline protein PIE-1, we identified the C. elegans RCK homolog, which we refer to as conserved germline helicase, cgh-1. In adults, cgh-1 mRNA is present predominately in the germline. CGH-1 protein is detected within germline P granules beginning at the L1 stage. It is present in mitotic germline cells but its levels increase dramatically in both males and hermaphrodites during meiotic entry, then it disappears during sperm development but persists at high levels in oocytes and the early embryo. CGH-1 is present in P granules until the 1-200 cell embryonic stage, but is also found in other cytoplasmic granules that are present in somatic cells. Somatic CGH-1 staining declines after the 4 cell stage until it is undetectable after 50 cells. This embryonic pattern approximates the previously described distribution of maternal mRNA. It also suggests that CGH-1 is unlikely to be involved directly in germline silencing by PIE-1, but might contribute to other aspects of PIE-1 function. RNAi experiments indicate that cgh-1 is essential for fertility in both males and hermaphrodites. In cgh-1 RNAi F1 males, the gonad appears superficially normal. Cgh-1 RNAi F1 hermaphrodites also appear normal through the early L4 stage and generate sperm, but then produce grossly abnormal oocytes. Multiple P granule components are localized appropriately in cgh-1 RNAi animals, and their germline cells express appropriate markers of mitotic, pachytene, and developing oocyte stages. Apparently, CGH-1 is not essential for P granule structure or germline fate. The data suggest, however, that regulatory mechanisms required for fission yeast to become quiescent and self-renewing are linked to P granules, and required for germ cell function in a metazoan.

Yanjun, Shi et al. (2021) International Worm Meeting "A gut neuroendocrine signal regulates synaptic assembly in the brain"

Yanjun, Shi, Zhiyong , Shao, Lu, Qin

[International Worm Meeting, 2021]

The gut-brain axis plays an essential role in regulating neural development in response to internal environmental stimuli, such as microbes or nutrients. Defects in gut-brain communication can lead to various neurological disorders. However, it remains unknown whether gut plays an intrinsic role in regulating neuronal development. Through a genetic screen in C. elegans, we uncovered that an intrinsic Wnt-endocrine pathway in gut regulates synaptic development and neuronal activity in brain. Specifically, the Wnt signaling upregulates the expression of the neuropeptide NLP-40 in the gut and facilitates presynaptic assembly through the neuronal expressed GPCR AEX-2 receptor during development. The NLP-40 acts most likely through modulating neuronal activity and promoting synaptic protein trafficking. Our study reveals a novel role of gut in synaptic development in the brain and provides additional molecular basis for gut-brain interactions. Key words: Gut-brain axis; Synaptic development; Wnts; Endocrine; Neuropeptide/NLP-40; GPCR/AEX-2; Neuronal activity

Shi, Zhen et al. (2011) International Worm Meeting "The mevalonate pathway has a role in microRNA repression of target genes."

Ruvkun, Gary, Shi, Zhen

[International Worm Meeting, 2011]

The mevalonate pathway is present in all higher eukaryotes and many bacteria and mediates the production of isoprenoids. The isoprenoids feed into a wide range of biosynthetic pathway: sterols, dolichol, ubiquinone, heme A, and the lipid moiety for protein prenylation. C. elegans possesses a functional mevalonate pathway but lacks enzymes for the cholesterol synthesis, suggesting that mevalonate generates precursors for the other biosynthetic pathways. Inactivation of genes function in the synthetic steps in the mevalonate pathway as well as drugs that inhibit this pathway (the statins) strongly enhance the retarded phenotypes of the hypomorphic mutation in the let-7 miRNA. These phenotypes are suppressed by the inactivation of let-7 targets, suggesting that mevalonate is needed for normal let-7 repression of target genes. We found that the non-cholesterol biosynthetic outputs of the mevalonate pathway are required for the repression of these let-7 targets. In addition, inactivation of the mevalonate pathway also results in the de-silencing of lin-14, the target of lin-4 miRNA, compared to stage-matched control worms. To address which downstream branch(s) of the mevalonate pathway regulate(s) the developmental timing, we are performing a candidate-based RNAi screen for gene inactivations that cause retarded developmental timing. The cherry-picked RNAi library we are screening includes genes that function in protein prenylation, dolichol, ubiquinone and heme A biosynthesis.

Weiwei Shi et al. (2008) "High-throughput single-C. elegans analysis using a droplet based microfluidic system"

Weiwei Shi, Bingcheng Lin, Jianhua Qin, Nannan Ye, Hui Ma

[2008]

"Interest in C. elegans as a target for drug discovery has grown considerably, particularly in the pharmaceutical industries. Several features of C. elegans make it a model system for investigating numerous neurodegenerative diseases and physiological processes. However, conventional method is manual, laborious, and time-consuming, which is not amendable to high throughput screening. Microfluidics (or lab-on-a-chip) is a newly developed technology, which is characterized by devices containing networks of enclosed, micron-dimension channels. Recently, it has aroused increasing interest in biological and medical sciences and has become an attractive platform for high throughput drug screening.In this study, a droplet-based microfluidic system was investigated for the high throughput analysis of individual C. elegans in micro-droplets. The system is composed of two functional regions for droplets generation and droplets trapping, respectively. The C. elegans were continuously encapsulated in the micro-droplets by two liquid phases mixing and the micro-droplets were immobilized in a parallel of trappers in a single operation. The change of mobility behavior of C. elegans in response to neurotoxin was investigated. The system is able to generate the nanoliter-size micro-droplets with C. elegans in array format and the phenotype characteristics of C. elegans in response to neurotoxin can be characterized at single animal resolution, demonstrating the potential of this miniaturized platform for high throughput drug screening at organism level. "

Shi, Shujie et al. (2015) International Worm Meeting "The ENaC/degenerin Protein MEC-10 Regulates the Channel's Response to Mechanical Forces."

Kleyman, Thomas R., Shi, Shujie

[International Worm Meeting, 2015]

The epithelial Na+ channel (ENaC)/degenerin family encodes a group of structurally related ion channels that are highly selective for Na+ and sensitive to amiloride and its derivatives. Located within kidney tubules, ENaC is constantly exposed to laminar shear stress (LSS), a mechanical force. Previous studies have shown that LSS activates ENaC by increasing channel open probability, and multiple sites of ENaC subunits are involved in conformational changes during the channel's gating in response to LSS. MEC-4 and MEC-10 of C. elegans are the pore-forming subunits of the mechanosensitive ion channel complex required for the worm's gentle touch response. Worms bearing null mutations in either gene show deficient response to the gentle touch. However, the gating mechanism has remained elusive, largely due to inherent limitations of studying ion channels in nematodes.To gain insights regarding the structural features of the degenerin channel that are important for worm's mechanosensing, we have begun to explore the response of these channels to LSS in Xenopus oocytes, an overexpression system. We discovered that, similar to ENaCs, LSS evoked a rapid increase in the whole cell Na+ current in oocytes expressing MEC-4d/10/2/6. The change in channel activity was reversible and relatively stable to repetitive LSS stimulations. MEC-10 was required for a robust LSS response, whereas MEC-2 and MEC-6 were dispensable. The LSS response was substantially diminished by a putative mec-10 null allele or selected touch-insensitive MEC-10 mutations, suggesting a correlation between the channel's responses to two different mechanical forces. Our results provide a novel experimental system to examine the mechanosensitivity of the C. elegans degenerin channel and essential information regarding the role of MEC-10 in the channel's gating mechanism. .

Shi, C. et al. (2017) International Worm Meeting "Mating and male pheromone kill Caenorhabditis males through distinct mechanisms."

Shi, C., Runnels, A., Murphy, C.

[International Worm Meeting, 2017]

The difference in longevity between sexes is a mysterious yet general phenomenon across great evolutionary distances. C. elegans males live significantly shorter when maintained in groups, whereas the longevity of hermaphrodites is not influenced by population density. Previous investigations into the factors that affect the lifespan of C. elegans have primarily focused on hermaphrodites, thus how C. elegans male lifespan is regulated remains poorly understood. We find that male-specific population density-dependent death in C. elegans is due to the perception of male pheromone as a toxin. Neurons and the germline are required for male pheromone-dependent male death. Hermaphrodites with a masculinized nervous system secrete male pheromone and are susceptible to male pheromone killing. Male pheromone-mediated killing is unique to androdioecious Caenorhabditis, and may reduce the number of males in hermaphroditic populations; neither males nor females of gonochoristic species are susceptible to male pheromone killing. The toxicity of male pheromone may explain the contradictory results from previous publications in which grouped males were used as the control in testing whether mating affects the lifespan of Caenorhabditis males. We previously showed that mating causes shrinking and shortens the lifespan of Caenorhabditis hermaphrodites and females. Using single worm lifespan assays, we discovered that Caenorhabditis males also experience post-mating changes and earlier death. Mating-induced death is characterized by germline-dependent shrinking, glycogen loss, and ectopic vitellogenin expression, utilizes distinct molecular pathways from pheromone-induced death, and is shared between the sexes and across species. The study of sex- and species-specific regulation of aging reveals deeply conserved mechanisms of longevity and population structure regulation.

Shi, Cheng et al. (2013) International Worm Meeting "Mating-induced somatic collapse reveals a novel soma-germline interaction."

Murphy, Coleen, Shi, Cheng

[International Worm Meeting, 2013]

Interactions between the germline and the soma are important to optimize reproduction and to influence somatic aging. The prevailing germline-soma model suggests that the lifespan shortening signal from the germline antagonizes the lifespan lengthening signal in the somatic gonad in C. elegans. Previous studies focused on removal of germline cells and its longevity-repressing activity to reveal the somatic gonad lifespan-extending signal. We have identified a new phenomenon linking reproductive status to longevity and have illustrated the elusive lifespan-shortening signal for the first time: in C. elegans, mating leads to "post-mating somatic collapse" (PMSC), a state in which mated worms shrink pronouncedly and die early, compressing the post-reproductive life span. The post-mating shrinking and lifespan phenomena are genetically separable, and the DAF-12 steroid receptor and DAF-16/FOXO transcription factor are involved in regulating different aspects of PMSC. Components from males contribute differently to PMSC. Likewise, hermaphrodites have germline-dependent and germline-independent responses that process the corresponding male-initiated signals. Post-mating somatic collapse may be an extreme version of the influence that males in many species exert on female behavior to maximize their own reproductive success. Our study provides new insight into the communication between males and the female germline and soma to regulate reproduction and longevity.

Shi, Yanjun et al. (2017) International Worm Meeting "The ROR/CAM-1 determines synaptic spatial specificity through opposite functions in synaptogenesis."

Shi, Yanjun, Shao, Zhiyong, Li, Qian, Wang, Kai

[International Worm Meeting, 2017]

Neurons form synapses with remarkable specificity both at the cellular and subcellular level. However, the molecular mechanisms underlying the synaptic specificity is not well understood. Here we use C. elegans AIY interneuron as a model to address this question. AIY forms presynapses with distinct and highly reproducible pattern: the ventral region proximal to soma without synapse (Zone 1), the turn region from the ventral to the dorsal fragment with enriched synapses (Zone 2) and the distal region with a few scattered synapses (Zone 3). We found that the receptor tyrosine kinase ROR/CAM-1 is required for AIY synaptic specificity. Cam-1 regulates AIY presynaptic spacial specificity through the opposite roles in synaptogenesis: it promoters synapse formation at the synaptic enriched region (Zone 2) and suppresses synaptogenesis at the asynaptic region (Zone 1). To determine the temporal role of cam-1, we monitored the dynamic change of the synaptic pattern during different development stages. We found that cam-1 act during both embryonic and larval stages. Further study indicated that the prosynaptogenesis role is mediated through wnt pathway: cwn-2/cfz-2/dsh-2/sys-1. While the antisynaptogenesis role of CAM-1 is independent of any known wnt receptors, Disheveled or beta -catenin. We conclude that ROR/CAM-1 regulates synaptic spacial specificity through opposite functions in synaptogenesis mediated by distinct signaling pathways.