Fang-Yen, Christopher et al. (2009) International Worm Meeting "The worms crawl in, the worms crawl out: An analysis of C. elegans locomotory gaits."

Wen, Quan, Samuel, Aravinthan, Kawai, Risa, Fang-Yen, Christopher, Wyart, Matthieu, Chen, Sway, Chklovskii, Dmitri

[International Worm Meeting, 2009]

C. elegans exhibits very different locomotory patterns when swimming in fluids and crawling on a solid substrate. Compared with crawling, swimming is characterized by a longer wavelength and higher frequency of undulations. We are interested in understanding how a single circuit composed of command neurons, motor neurons, and muscles is capable of supporting these two locomotory gaits. We estimate that when crawling on moist surfaces, worms are held by capillary forces up to 100,000 times greater than the viscous forces experienced during swimming. To investigate in more detail the effect of this mechanical loading on locomotion, we immerse worms in solutions of varying viscosity and measure their wavelength, frequency, and curvature by machine vision algorithms. We show that worm exhibit a continuous transition from swimming to crawling behavior as viscosity is increased from that of water to a value 5 orders of magnitude larger. Worms in fluids of intermediate viscosity exhibit locomotory patterns intermediate between swimming and crawling. Swimming and crawling can therefore be seen as low-load and high-load limits of a continuum of locomotory patterns. We show that our results can be understood in terms of a biomechanical model of C. elegans locomotion in which the swimming to crawling transition is associated with a transition between elastic-dominated and viscous-dominated dynamics. Next we analyze the behavior of worms undergoing spatial transitions between swimming and crawling behaviors, such that mechanical loading varies over the length of the worm. The analysis of such transitions gives insights into the generation and propagation of the sinusoidal bending wave, and the mechanisms of modulation of locomotory gaits. We show that the worm behavior follows neither strictly local nor strictly global modulation, but rather can be described by a set of rules governing the generation and propagation of undulatory waves.

Takahiro Tanji et al. (2010) East Asia Worm Meeting "Genetic interaction of HAF-4 and HAF-9, half-type ABC transporters required for the biogenesis of intestinal lysosome-related organelle in Caenorhabditis elegans"

Takahiro Tanji, Kenji Nishikori, Ayako Ohashi-Kobayashi, Hirohisa Shiraishi

[East Asia Worm Meeting, 2010]

C. elegans HAF-4 and HAF-9 are half-type ABC transporters highly homologous to human lysosomal peptide transporter TAPL (TAP-like; ABCB9). They show high identity at their amino acid sequences and are predicted to be paralogues. Previously, we reported that both HAF-4 and HAF-9 localize to the membrane of non-acidic but LAMP (lysosome-associated membrane protein) homolog LMP-1-positive lysosome-related organelles in intestinal cells, and that they are required for the biogenesis of the organelles and some other physiological aspects, such as normal brood size and growth rate (Kawai et al. (2009) Mol. Biol. Cell, 20, 2979-90). As HAF-4 and HAF-9 are half-type transporters, they should function as homodimers and/or a heterodimer. Here, we show that HAF-4::GFP and HAF-9::GFP do not rescue the intestinal granular defect in the haf-9 and haf-4 deletion mutants, respectively, and that haf-4 and haf-9 alleles are complementing, indicating that they are not functionally identical. On the other hand, the double mutants for haf-4 and haf-9 do not show severer phenotype than the single mutants. These results propose two possible models for the function of HAF-4 and HAF-9; they function as a heterodimer, and/or they function differentially but cooperatively as homodimers. Physical interaction analysis will lead to the identification of the functional units of HAF-4 and HAF-9.

Hirohisa Shiraishi et al. (2010) East Asia Worm Meeting "Characterization of HAF-2 as a lysosomal ABC transporter in Caenorhabditis elegans"

Keiko Gengyo-Ando, Takahiro Tanji, Ayako Ohashi-Kobayashi, Masatomo Maeda, Shohei MItani, Kenji Nishikori, Nau Wakimoto, Hirohisa Shiraishi

[East Asia Worm Meeting, 2010]

We have been studying on the physiological function of lysosomal ABC transporters (ATP-binding cassette transporters) in C. elegans. We already reported that HAF-4 and HAF-9, two nematode homologues of human lysosomal peptide transporter TAPL (TAP-like, ABCB9), are required for the normal formation of a subset of intestinal granules in which LMP-1, a nematode LAMP (lysosome-associated membrane protein) homologue, localizes [Kawai et al., Mol. Biol. Cell 2009]. Here, we report the expressional and functional analysis of HAF-2, another putative peptide transporter of ABCB subfamily.We have found that HAF-2 fused with GFP is observed in pharynx, muscle, coelomocytes, and the most anterior and posterior intestinal cells from late larval to adult stage. In addition to these tissues and cells, it turned out that HAF-2 is also expressed in neuronal cells including amphid and phasmid neurons. Although the subcellular localization of HAF-2 is still under investigation in most of these HAF-2-expressing cells, HAF-2 was on the periphery of granules with lysosomal characters at least in coelomocytes, suggesting that HAF-2 is a lysosomal ABC transporter as well as HAF-4 and HAF-9.Further expression analysis at the embryonic stage revealed that HAF-2 was expressed at the comma stage of embryogenesis at the latest and its expression pattern was not overlapped with intestinal expression of LMP-1 driven by the intestine-specific ges-1 gene promoter. These results raise a possibility that several intracellular ABC transporters feature the diversification of lysosomal organelles in various tissues in C. elegans. To clarify this point of view, we are now investigating the effect of HAF-2 ectopic expression in entire intestinal cells on the formation of intestinal lysosome-related organelles.

Nishikori, Kenji et al. (2015) International Worm Meeting "Genes involved in proton transport are required for the formation of HEBE granule, which is a novel aging- and fasting-responsive organelle."

Nishikori, Kenji, Tanji, Takahiro, Ohashi-Kobayashi, Ayako, Takahashi, Rei, Shiraishi, Hirohisa, Sato, Mami

[International Worm Meeting, 2015]

HEBE (HAF-4/HAF-9-Enriched Body Evanescent with age) is a novel intestinal granular organelle that emerges with larval development and disappears with aging or fasting in C. elegans [see also Hamada et al. and Tanji et al. posters]. "HEBE" is also named after the goddess of youth, who is the cupbearer for the gods and goddesses in Greek mythology. Besides HEBE granules, nematode intestine owns other granular organelles, such as acidic granules (or merely called gut granules) and lipid droplets. Given that HAF-4 and HAF-9 are homologous to a mammalian lysosomal peptide transporter (ABCB9), HEBE granules seemed to be lysosome-related. However, their lumen was non-acidic[1]. Mutant analyses for haf-4 and haf-9 revealed that they cooperatively function to form HEBE granules and are involved in the growth rate and the brood size[1][2]. Collectively, we speculated that the organelle plays a role in nutritional resource allocation in response to the physiologic change during young adult stage, although the regulatory mechanism of the organelle dynamics has yet to be determined. In this meeting, we report ongoing screening for genes involved in the formation of intestinal granules using C. elegans RNAi Feeding Library (Open Biosystems). So far more than a hundred of candidates with reduced number of intestinal granules have been identified from approximately 9,000 genes tested. Among the candidates, we performed a preceding secondary screening focusing on genes involved in proton transport from intestinal cytosol to intestinal lumen and/or acidic organelles, using a transgenic worm expressing HAF-4::GFP as a marker for HEBE granules. The RNAi of genes encoding V-ATPase subunits and an intestinal sodium/proton exchanger, NHX-2, induced loss or decrease of HEBE granules. Especially, most knocked-down worms for each V1 component of the V-ATPase (vha-8, vha-9, vha-11, vha-12, vha-13, vha-14 and vha-15) resulted in distorted shape of the HAF-4::GFP-positive organelles. These observations raise possibilities that loss of HEBE granules may be caused by deficiency in fatty acids and dipeptides, uptake of which is inhibited by high pH in intestinal lumen, and that activities of proteins essential for the granular formation may be attenuated by aberrant cellular pH.[1] Kawai et al., Mol. Biol. Cell 2009.[2] Tanji et al., Biochem. J. 2013.

Tanji, Takahiro et al. (2015) International Worm Meeting "Characterization of HEBE granules, novel organelles found in the intestine of well-fed young adult C. elegans."

Nishikori, Kenji, Tanji, Takahiro, Ohashi-Kobayashi, Ayako, Kanno, Yuki, Kobayashi, Yusuke, Haga, Syoko, Shiraishi, Hirohisa

[International Worm Meeting, 2015]

The C. elegans intestine is a multifunctional organ involved with the uptake, metabolism, storage and distribution of nutrients, detoxification, immune defense and so on. The intestinal cells are packed with granular organelles (intestinal granules); among them, acidified gut granules with autofluorescent contents and lipid droplets are capturing the attention of many worm researchers and have been extensively analyzed. We are focusing on non-acidified intestine-specific granules where HAF-4 and HAF-9, ATP-binding cassette transporter proteins highly homologous to mammalian lysosomal peptide transporter ABCB9 (TAP-like), localize (Kawai et al. 2009 MBC). Although these granules are prominent from late larval to young adult stage, they decrease with age (Nishikori et al. 2012 C. elegans Topics Meeting). Therefore we named them HEBE (HAF-4/HAF-9-Enriched Body Evanescent with age). We speculate about the role of HEBE granules in the storage of some nutrient because they dramatically reduce shortly after food deprivation; however, their exact physiological function and the relationship with other intestinal granules remain elusive.To illuminate the relationship among intestinal granules, we advanced the characterization of HEBE granules using organelle marker proteins and mutant worms defective in the biogenesis of specific organelles. Firstly, HAF-4::GFP- and HAF-9::GFP-localizing granules were not stained with Nile red following fixation, and the storage of neutral lipid was not impaired in the haf-4 and haf-9 mutants defective in the HEBE granule biogenesis. HAF-9::mCherry-localizing granules were negative for yolk protein VIT-2::GFP, and the distribution of VIT-2::GFP to embryos was also intact in the haf-4 haf-9 mutant. These results indicate that HEBE granules are distinct organelles from lipid droplets and yolk granules. Secondly, HAF-9::mCherry-localizing granules were not GLO-1::GFP-positive lysosome-related gut granules, and were not impaired in the glo-1 mutant defective in the gut granule biogenesis, suggesting that HEBE granules are also distinct from lysosome-related gut granules. Thirdly, HAF-9::mCherry-localizing granules were positive for late endosome- and lysosome-associated protein GFP::RAB-7, and the rab-7 mutant showed the defect in the formation of HEBE granules but neither in that of autofluorescent granules nor lipid droplets. These results suggest that HEBE granules are novel intestinal organelles relevant to endocytic pathway.