Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila

  1. Norin Chaudhry
  2. Margaux Sica
  3. Satya Surabhi
  4. David Sanchez Hernandez
  5. Ana Mesquita
  6. Adem Selimovic
  7. Ayesha Riaz
  8. Laury Lescat
  9. Hua Bai
  10. Gustavo C. MacIntosh
  11. Andreas Jenny

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Abstract

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7095710.

    Summary of the work:

    Authors found a novel role of LAMP1 in Drosophila which is not essential for autophagy or viability but rather required in lipid metabolism compared to its vertebrate homologs where its known to show autophagy defects and embryonic lethality. They found homozygous Lamp1 mutants in Drosophila to be viable and show no developmental delay. Nor do they show any macro/micro autophagy defects.But during starvation, they have seen increase in acidic vesicles in fat bodies suggesting its independent role in lipid homeostasis during stress conditions.

    Comments

    All the experiments and methods seem fine to address the problem. Few comments I think could be addressable.

    -Why is just starvation used as a stress condition, authors could have tried other methods of stress induction like ROS by H2O2 to validate their claims.

    -Secondly, in methods, the fly heads are homogenized and protein was estimated by lowry method. This could be error-prone as the eye pigments in the Drosophila can interfere with the absorbance of Lowry assay(660nm).

    Reference : CR Sharkey et.al, 2020 :The spectral sensitivity of Drosophila photoreceptors.

     

  2. Version published to 10.1080/15548627.2022.2038999
  3. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5562772.

    LAMP proteins localize at the lysosomal membrane, controlling autophagy-related processes but also lipid transport and metabolism. While it is well-demonstrated the existence of a link between LAMP misfunction and human diseases, the mechanisms underlying LAMP functions are still poorly understood. To better understand the different roles of LAMP proteins, authors aimed to characterize in this work the only LAMP member identified in Drosophila, Lamp1.

     

    Authors first developed new Lamp1 null mutant alleles, as well as an antibody against Lamp1. The validation and characterization of these molecular tools is very convincing, resulting in a valuable toolkit to explore Lamp1 in Drosophila. In this work, authors used the generated mutant alleles to assess the requirement of Lamp1 in autophagy-related processes and in lipid homeostasis. To do that, they combine genetics, confocal and electron microscopy, and biochemistry approaches. On one hand, the presented data indicates that unlike what previously reported in mammals, Lamp1 is not required, or at least is not essential, for autophagy. On the other hand, authors provide evidences supporting a role for Lamp1 in general lipid metabolism, likely including lipid transport. Therefore, the presented data suggests that the previously reported roles of LAMP proteins (autophagy and lipid metabolism) might be independent.

     

    Given the structural and functional evolutionary conservation of LAMP proteins, this work should be of wide interest, validating Drosophila as a useful model for further studies in the field. In general, authors provide solid data. However, I found a number of minor points in the manuscript that I would recommend addressing to fully support the conclusions reached and to make the data more clear:

     

    - Figures order does not correlate with the description order in the main text, what results confusing to follow the data. Some examples of this are: Supp. Fig. 1E-H mentioned before Supp. Fig. 1A-D, or Fig. 1D-E before Fig. 1 B-C (pages 4-5).

     

    - Authors justify the analysis of lysotracker under starvation since "barely any Lysotracker positive structures are found under fed conditions" (page 4). However, in this work lysotracker shows a clear signal under fed conditions, where in fact the most dramatic changes between control and Lamp1 mutants are observed (quantified in Fig. 2I). Therefore, the use of starvation should be explained further, or it should be dicussed at least the unexpected clear signal observed under fed conditions.

     

    - The current version of Figure 2 shows a clear lysotracker signal in control animals (Fig. 2A'), while in the rescue condition the signal it is barely detectable (Fig. 2D'). Given the similar signal levels showed in the quantification for both genotypes (Fig. 2I), it would be recommendable to use different images to illustrate representative examples of control and/or rescue animals.

     

    - By the end of page 6, the description of the expression levels of Atg5 and Atg8a should refer to Fig. 4I, instead of Fig. 4E.

     

    - In Fig. 6, to make the data easier to follow and more illustrative, it would be helpful to have first the WT panel(s), containing examples of APGLs, ALs and lysosomes, and later include the mutant panels.

     

    - The sentence "[Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]" appears repeatedly throughout the references list.

     

  4. Version published to 10.1101/2021.03.03.432938v1 on bioRxiv