Identifying proteins and lipids enriched at Pex30 subdomains would shed light on mechanistic details of LD biogenesis.
Great findings on the origins of LDs. Do you expect Pex30 levels to change depending on lipid availability/organismal stress? It would be interesting to see if it's upregulated in response to high PA levels
Moreover, unlike the cells devoid of Pex30, cells expressing Pex30 TMD mutants exhibit a dominant negative effect on LD biogenesis as they show decreased LD formation at 5-hour timepoint
Do you expect these mutations are strong enough to create a dominant negative effect in a wild-type Pex30 background?
To do this, we overexpressed Pex30-GFP, Pex30 (TMD1)-GFP, Pex30 (TMD2)-GFP and Pex30 (TMD1.TMD2)-GFP in are1are2dga1pex30Δ with LRO1 expressed under GAL1 promoter and labelled with Erg6-mCherry
I would love to see some of these images in figure 5.
are1are2dga1pex30Δ
Could you introduce the significance of this background for this experiment?
Our data indicates that the short hairpin TMDs are important for Pex30 localization and ER shaping function.
It would help to add some quantification here to show the extent of this phenotype
(Figure 1D and 1E).
I am having trouble seeing the strength of the phenotype, especially for the TMD2 mutant. Could you add some arrows to highlight WT vs abnormal cortical ER morphology?
Firstly, synchronized worm suspension in the reservoir was loaded into microfluidic chip. During the experiment, culturing medium can be replenished and consistently loaded into the chip at a low flow rate, to provide sufficient nutrients for worms.
How labor intensive is this loading process? Are you able to just open the channel briefly and load a set number of animals into the chip or do you have to carefully monitor the flow of the liquid until you see the correct amount of worms and then turn it off? I'm wondering about this as in regards to making this more high-throughput. If the loading can be standardized, do you think it would be possible to keep the worms in a 96 cell plate, with an automatic sampler and bring worms into the chamber one well at a time for imaging, and then either dispose of them or return them to their well for later imaging? e.g. if you were going through a lot of mutants or drugs or doing a mutagenesis screen.
The worm cultivation module includes a microfluidic chip for worm culturing and observation, equipped with pressure-based delivery system. Based on this module, diets and test substances can be automatically added at regular intervals, and worms can be cultured in microfluidic chip until death. During chronic toxicity testing assays, the phenotypes of worms in bright filed can be monitored and collected regularly with the monitoring module, which includes a motion stage and a Pi camera. After capturing images or videos of worms, the body length and bend frequency can be calculated automatically with the image analysis module. Fig.1B shows the physical photograph of the entire platform, which is controlled through the control center.
I think this is a very powerful device with a lot of applications. This is a small issue, but the diagram is a bit confusing the first time you look at it. You show the chip between the waste and reagent, and then an image of the chip on the right side of the red monitoring module box. Is that second box what the camera is seeing from the chip and sending to the control center? It's a bit confusing as it looks like there are two different chips in the system.
In addition, fluorescence images also showed that the body length of nematodes exposed to L511A in the high-dose group was shorter than that in the control group, suggesting that L511A may have growth and developmental toxicity.
It would help to show this data in figure 6 to give more detail on how much body length is impacted.
We treated worms with two commercially available flavor mixtures (codenamed L511A and X6145A), two cigarette brands of smoke Cambridge filter collections (codenamed Cig-1 and Cig-7), and a single tobacco component (solanone, codenamed Fla-1) to investigate their effects on bend frequency of worms. The experimental design covered four dose groups of 1/100000, 1/10000, 1/1000 and 1/200 of the stock solution, and 0.5% DMSO was set up as the solvent control.
It would be helpful to discuss how these stock concentrations relate to the concentrations humans are exposed to in their environment. Also, a great addition to the paper would be to do longer term studies of these chemicals' impact on worms. This device is a really powerful way to measure impacts on worm health and behavior over long periods and the introduction to the paper is framed from the perspective of human cumulative exposure to toxins. I'd love to see longer time course assays on how these drugs affect the worms, especially at environmentally relevant concentrations.
The average lifespan (± SD) of the worms in multi-well plates and microfluidic chips were 12 (± 4) days and 11 (± 3) days, respectively, which revealed no significant difference between the survival curves of two groups (P > 0.05) (Fig. 2D).
These lifespans seem shorter than the average in the literature. Do you think the constrained space in the PDMS chip and on the 96-well plate, is reducing their longevity? Also did you check development rates on the chips and do they go through the larval stages at normal rates?
Since scav-5 and scav-6 are paralogs of scav-4, we analysed their functions in lipid accumulation using scav-5(ok1606) deletion mutants and scav-6 knockout alleles generated in this study through CRISPR/Cas9-mediated gene editing (Figure 4B). We found that when fed with JUb74, both scav-5(-) and scav-6(-) mutants had moderately reduced LD sizes, but not to the extent of scav-4(-) mutants (Figure 4E). Previous promoter reporter studies showed that scav-5 and scav-6 were expressed in the intestine.34 We constructed translational reporters for both genes and found weak or no signals for SCAV-5::TagRFP possibly due to low protein levels. The SCAV-6::TagRFP fusion protein was expressed in the intestine and was localized to the apical membrane (Figure 4C). From the fluorescent intensity, the scav-6 expression appeared to be weaker than the scav-4 expression. Moreover, scav-4(-) scav-6(-) double mutants had the same LD diameter as scav-4(-) single mutants (Figure 4F). The above results suggested that SCAV-4 may play a more significant role than the other two paralogs in intestinal lipid uptake.
I'm surprised that the scav-5 and scav-6 paralogs were both able to reduce the large LD phenotype to the same extent as scav-4 (there doesn't appear to be significant difference between the mutants). To me this suggests either they each contribute a third of the BCFA uptake, or that they operate together to internalize BCFAs. The scav-4;scav-6 double mutant suggests the first idea isn't correct as you don't see a stronger effect there. Do you think its possible these transporters are working as a complex? I would be interested to see if you can rescue each of these mutants with scav-4 expression, or if rescue requires all receptors to be present.
In a small-scale screen of a few thousand haploid genomes, we isolated an allele unk28, which led to the formation of supersized LDs under the JUb74 diet (Figure 4A).
It's amazing you were able to find a GOF mutation that exacerbated this phenotype! I think it would help accentuate this if you included a picture of wt animals on the JUb74 food, next to the L462F animals on JUB74 food in Fig 4A to contrast that shift in LD size.
Conversely, we also supplemented either specific BCFAs or a mixture of them to the OP50 culture but were not able to increase the BCFA levels above 30%, likely because OP50 could not take in large amount of BCFAs (Figure S2C). Feeding those BCFA-treated OP50 to wild-type C. elegans did not induce large LDs (Figure S2D). Thus, we concluded that, in addition to the dosage effect, a threshold existed for the minimal level of BCFAs that could induce large LDs.
Could you preform the experiment in C. elegans defined media supplemented with BCFAs? That would fully remove other variables from the Microbacterium and confirm this is solely due to BCFAs.