Golgi Bodies Size

[Cassaundra Marley]

Coordination of cell growth is essential for the development of the brain, but the molecular mechanisms underlying the regulation of glial and neuronal size are poorly understood. To investigate the mechanisms involved in glial size regulation, we used Caenorhabditis elegans amphid sheath (AMsh) glia as a model and show that a conserved cis-Golgi membrane protein eas-1/GOLT1B negatively regulates glial growth. We found that eas-1 inhibits a conserved E3 ubiquitin ligase rnf-145/RNF145, which, in turn, promotes nuclear activation of sbp-1/ SREBP, a key regulator of sterol and fatty acid synthesis, to restrict cell growth. At early developmental stages, rnf-145 in the cis-Golgi network inhibits sbp-1 activation to promote the growth of glia, and when animals reach the adult stage, this inhibition is released through an eas-1-dependent shuttling of rnf-145 from the cis-Golgi to the trans-Golgi network to stop glial growth. Furthermore, we identified long-chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA), as downstream products of the eas-1-rnf-145-sbp-1 pathway that functions to prevent the overgrowth of glia. Together, our findings reveal a novel and potentially conserved mechanism underlying glial size control.

Copyright: 2020 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This project is supported by the Holland Trice Awards. Z.W. was supported by the National Key Research and Development Program of China (2016YFA0501000) and the National Natural Science Foundation of China (31671039). A.Z., K.O and D.Y. are also supported by National Institutes of Health ( ) R01s (NS094171 and NS105638 to D.Y.). K.O. was supported by Duke University MGM summer undergraduate research engagement program (MGM SURE). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We show that eas-1 can negatively regulate the conserved E3 ubiquitin ligase rnf-145/RNF145 and in turn activate sbp-1/ SREBP to limit the growth of AMsh cells. Furthermore, we find that the long-chain polyunsaturated fatty acid (LC-PUFAs) eicosapentaenoic acid (EPA) is likely a downstream product of this pathway that helps to set the brakes on cell growth in AMsh glia. Our findings uncover a novel and potentially conserved pathway regulating glial size and represents the first reported function of eas-1/GOLT1B in animals.

To identify genes that may potentially regulate cell size, we conducted an unbiased forward genetic screen for mutants with abnormally sized AMsh cells and isolated a mutant yad70 with enlarged AMsh cells (Fig 1A and 1B). yad70 animals exhibited enlarged AMsh cells that increased in penetrance from larval stages to day 3 (D3) adults, where the phenotype penetrance reached 100% by the day 2 (D2) adult stage (Fig 1C). Furthermore, the volume of AMsh cell bodies progressively increased at a faster rate (Fig 1D), and AMsh cells of yad70 animals were roughly 4 times the size of their control counterparts by D3 (S1A Fig). The AMsh phenotypes observed were not due to an overall increase in animal size, as the length of yad70 animals were slightly shorter than that of their control counterparts at D2 (S1B Fig). We also noticed that the yad70 phenotype did not depend on the nutritional condition of animals, as starvation did not suppress the enlarged AMsh cells (S1C Fig). Similarly, phasmid sheath glia (PHsh) also had an enlarged cell size phenotype.

Unlike their AMsh counterparts, the 12 amphid neurons stained by DiI in yad70 animals appear to have no noticeable change in size or number (S1D and S1E Fig). As uptake and concentration of the lipophilic dye is believed to require exposed cilia, these neurons likely have at least partially functional cilia [25]. Interestingly, yad70 animals exhibited reduced chemotaxis toward benzaldehyde and pyrazine (S1F and S1G Fig), a behavior mediated by the AWC and AWA neuron, respectively [26]. Similarly, long-range avoidance of the repulsive odorant 1-octanol, a behavior that is partly mediated by ADL neurons [27], was affected in yad70 mutants (S1H Fig). This is consistent with previous findings that ablation of AMsh cells leads to reduced AWC-mediated chemotaxis toward benzaldehyde, AWA-mediated chemotaxis toward pyrazine, and ADL-mediated long-range repulsion from 1-octanol [11].

Next, to determine whether the carboxyl terminus that was affected in yad70 was essential in the regulation of AMsh size, we conducted rescue experiments using various eas-1 constructs with altered carboxyl termini. We found that AMsh-specific expression of eas-1 with the last 13 amino acids deleted (ΔC) as well as eas-1 with a highly conserved aspartate (D131) residue within these 13 amino acids replaced with an alanine (D131A) were unable to rescue the phenotype (Fig 2B; S3C and S3E Fig). We then generated an eas-1 allele, yad83, with a D131A mutation and showed eas-1(yad83) displayed the same phenotype as that of eas-1(yad70) (Fig 2A). This suggests that the carboxyl terminus and the D131 within it are essential for eas-1 function in regulating AMsh cell size. On the other hand, replacing the last 13 amino acids of eas-1 with the human version (human C) was able to rescue the phenotype, further suggesting functional conservation of the eas-1 carboxyl terminus (Fig 2B; S3C and S3E Fig).

eas-1 is an essential gene, and deletion of the eas-1 locus caused 100% embryonic lethality, which prevented further analysis of eas-1 null allele phenotypes. To confirm that the enlarged AMsh cells we observed in eas-1(yad70) and eas-1(yad83) animals were due to a loss of function of the gene, we conducted RNAi knockdown in control animals. We found that although knockdown of eas-1 caused strong lethality phenotypes, about 10% of animals were able to reach D2, and all of them recapitulated the phenotype observed in yad70 mutants (Fig 2C and 2D; S3F Fig), suggesting that yad70 represents a loss-of-function mutation.

Use of the functional GFP::EAS-1 reporter revealed that it fully colocalized with the cis-Golgi marker mRuby::MannII [49] (Fig 2E; S3G Fig) and not the COPII vesicle marker SEC-24::mCherry [50] (S3H Fig), unlike what was previously reported for its homolog in yeast [14] and rice [15]. Furthermore, coexpression of the mutant construct mCherry::EAS-1(D131) with the functional GFP::EAS-1 reporter in AMsh cells showed almost complete colocalization as well (Fig 2F), suggesting that the mutations in the carboxyl terminus did not affect eas-1 function by altering its localization.

To determine whether eas-1 was transiently or continuously required for AMsh cell size regulation, we generated a C. elegans strain expressing eas-1 under a heat shock promoter to temporally manipulate eas-1 expression. We heat-shocked the worms at 33C for 3 hours at the time periods indicated (Fig 2G) and found that induction of eas-1 expression from day 1 (D1) was enough to significantly reduce the eas-1(yad70) phenotype penetrance compared to the no heat shock control when examined at D2. Furthermore, transient heat shocks during embryonic or L1 stages were unable to affect the phenotype when examined at D2, suggesting that continuous eas-1 expression may be required to maintain proper AMsh cell size. Together, our results show that eas-1 is required for preventing the overgrowth of AMsh cells.

SREBPs are membrane-bound transcription factors that play essential roles in regulating lipid homeostasis, and cleavage of SREBPs release their N-terminal that could translocate to the nucleus and facilitate transcription [16,52]. The use of the translational fusion reporter Psbp-1::gfp::sbp-1 shows that sbp-1 is likely broadly expressed throughout the animal including many cells in the head (S4D Fig). Thus, we used nuclear localization as a marker to determine whether the activation of sbp-1 is associated with AMsh growth. By expressing a functional mCherry::SBP-1 fusion reporter in AMsh cells, we found that in wild-type animals, SBP-1 does not accumulate in the nucleus during the early larval stages (L1 to L3) when the AMsh grow rapidly, but has strong nuclear localization when the animal enters the adult stage (Fig 3E; S4E and S4F Fig). Based on these observations, we hypothesize that the activation of SBP-1 may prevent the overgrowth of AMsh cells, and the regulation of AMsh cell size by eas-1 and rnf-145 may be through affecting SBP-1 nuclear localization. Indeed, we found that loss of function of eas-1 abrogated the nuclear localization of SBP-1 in adult animals, and loss of function of rnf-145 caused early-onset nuclear localization of SBP-1 in L1 animals (Fig 3E; S4F Fig). As expected, SBP-1 nuclear localization was restored in adult eas-1(yad70) animals when combined with the rnf-145(yad110) mutation (Fig 3E; S4F Fig). These results suggest that the regulation of SBP-1 activation by eas-1 and rnf-145 are important in the control of AMsh cell size.

Next, we wanted to test whether the activation of SBP-1 was important for and sufficient to reduce AMsh growth. We first expressed the nucleus-localized transcription-activating region of SBP-1 (c) [16] in the AMsh cells of eas-1(yad70) animals and found that it was able to suppress the enlarged AMsh phenotype (Fig 5A and 5B; S6A Fig). As controls, expression of full-length SBP-1 or the membrane-tethered transcription activating region of SBP-1 (u) in AMsh cells did not affect eas-1(yad70) phenotypes (Fig 5A and 5B; S6A Fig). We then tested whether driving SBP-1 activity in the nucleus was sufficient to reduce cell growth and found that expression of SBP-1 (c) in the AMsh cells of control animals could suppress their growth, and as a result, the size of AMsh cells in D2 animals was on average, about 25% smaller than those in control animals (Fig 5C). Similar small-sized AMsh cells were observed in rnf-145 mutants, which have strong nuclear localization of SBP-1 in the L1 stage (Figs 3E and 5C). These results support the conclusion that SBP-1 activation is important to slow down AMsh growth, and the enlarged AMsh phenotypes in eas-1 mutants likely arise in part due to a lack of SBP-1 activity.

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