The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans without its KASH or actin-binding domains

  1. Hongyan Hao
  2. Shilpi Kalra
  3. Laura E. Jameson
  4. Leslie A. Guerrero
  5. Natalie E. Cain
  6. Jessica Bolivar
  7. Daniel A. Starr

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Abstract

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER was unanchored, moving throughout the cytoplasm, and often fragmented. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

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  1. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on August 24 2020, follows.

    Summary

    The current and widely accepted model how nuclei are positioned in the cell proposes that their anchorage is mediated through nuclear membrane spanning SUN and KASH domain containing proteins which connect the cytoskeleton to the nuclear interior. This study presents evidence that this model needs reconsidering. The authors follow up on their previous observation that nuclear positioning differs between mutants in SUN (UNC-84) and KASH proteins (ANC-1), where anc-1 mutants display a more severe phenotype. The study nicely combines structure-function analysis of ANC-1 with the readout of nuclei positioned in the syncytial cell hyp7 in fixed samples and by live imaging, and the mutant analysis correlates well with the fitness of the whole organism. The authors also show that the expected regions (the KASH domain or the actin binding domain) play rather minor roles in nuclear anchorage. Instead, anchorage is mapped to a portion of the transmembrane domain and to spectrin-like repeats in the cytoplasmic localised region of the protein. Mutating those protein domains not only leads to irregularly positioned nuclei but also to disorganisation and fragmentation of the ER, which also appears unanchored as evidenced by in vivo imaging. Consistently, the authors find ANC-1 prominently associated with the ER and that this association is independent of the KASH domain.

    This paper will be very interesting to a wide readership as it challenges current models of nuclear positioning and shows that mis-positioning and loss of connectivity can have severe consequences to the development of the organism. However, the reviewers felt that some of the conclusions required additional data to support the statements made, in particular to strengthen the proposed the ER link, as well as some other points that require clarification as detailed below.

    Essential Revisions

    1. The data shown in Figure 6 suggesting ANC-1 targets to the ER was not convincing and was also very hard to contextualise as no other markers for other organelles or the nucleus were present, and no merged images provided. Given that the authors also repeatedly draw conclusions relating to mitochondria, including a mitotracker in the far-red channel would be important. The ANC1 signal appeared rather diffusely localised in all cases (WT and TK/KASH mutants; noting that the KDEL signal is very low in the TK mutant image compared to the others) and it could be argued any potential overlap might be coincidental rather than specific. No other evidence for ER targeting was provided but would be required to support the current conclusions. The authors should also at minimum calculate the co-localization index and provide examples of line scans to show a correlation between signal intensities. Also the sentence "The slight discrepancy between localization of mKate2::ANC-1b and GFP::KDEL could be explained by the distance between the ANC-1 N terminus and the ER membrane" will need to be modified. This is highly unlikely due to the resolution of the microscope used to acquire this image. The discrepancy might be due to differences in the focus plane of the two light channels. Please remove or edit this speculation.

    2. A further concern is the lack of any cytoskeletal network analysis in the paper; the authors (somewhat extrapolated) conclusions are focused on the concept that nuclei and the ER are detached from any sort of cytoplasmic structural network in anc-1 mutants, but this is not tested in the study. It would be very helpful to provide this evidence, but we recognise that analysing the cytoskeleton in detail may prove impractical given the current restrictions. However, if experimental data cannot be provided, it would be important to tone down such statements given the lack of formal evidence provided for this model.

    3. ANC1b-del6RPS and del5RPs appear to be expressed at much lower levels than other mutants (or WT protein) eg: Fig5; is this the case and if so, might the nuclear positioning defect just be due to this (ie: as the animals are more like anc-1 null)? It would be important to understand relative stability of these mutant proteins to interpret this data. Please provide more information on the relative intensity levels of each mutant and if they are similar, provide more representative example images.

    4. The authors put forward the claim that ANC-1 anchors other organelles besides the ER. There are a lot of markers available for different organelles, and it would be important for the authors to provide some staining in the most significant mutants (delta 6rps, delta tk), for mitochondria or other organelles. On page 11 the authors also write "Nuclear shape changes were observed during live imaging in anc-1 mutants consistent with a model where anc-1 mutant nuclei are susceptible to pressures from the cytoplasm, perhaps crashing into lipid droplets that corresponded with dents in nuclei.". It is not clear why the authors suggest "crashing into lipid droplets" and not any other organelles in the cell (mitochondria, endosomes, etc). Is there any evidence for this statement that can be provided?

    5. p. 14: ...was not enriched at the nuclear envelope...". it would very important to include pictures of the ANC-1 staining together with a marker for the nuclear envelope in Figure 5 to support this statement.

    6. Page 6, Figure 2A,B. it is not clear in the text that two of the four nonsense mutations that were analysed only disrupt isoform a and c (w427 and w621). If these mutations only affect isoform a and c, and not the shorter isoform b, then the authors could explain this better, so that this result goes together with the RNAi data.

    7. Figure 1c and Figure 3C - the dataset of WT and anc-1(e1873) seems to be the same in both these figures. Although duplication of the same data in multiple figures should not typically occur in publications, the duplication should be indicated clearly in the figure legend for transparency. Ideally, the duplicated data should also be shown in a different color/format in the figure so that they are immediately obvious. Other duplications of data in the manuscript should also be indicated. Why was unc-84(n369) data repeated three times on the graph in Fig 3C but with different values?

    8. The percentage of touching nuclei in unc84(n369), KASH mutants (fig 1C), and anc-1(dF1)(fig 3c), are around 20%. However, the authors classify the KASH mutants as "mild nuclear anchorage", whereas anc-1(dF1) on fig 3c was classified as "severe nuclear anchorage". The authors also used the term "intermediate nuclear anchorage" for other phenotypes. The authors should use the same classification (clearly described) throughout the manuscript.

    9. Figure 4A - the authors described the image of GFP:Kdel of hyp7 syncytia as a "branched network". It is very difficult to observe any branches and a network in these images. We recognise the authors want to use the same terminology that is used to characterize ER in other cells, but it is difficult to understand what the authors mean by a branch. Diffuse staining with dark round objects in it is visible, but branches are not clear. The authors should provide a better example, more similar to unc-84(n369) mutant, where branches and a network are more distinct and annotate these properly to support this statement.

    10. Figure 4C and D; movie 1 and 2- In these figures and the authors compare the dynamics of ER in WT and anc-1 mutant. The authors also provide representative movies. The WT animal appears to be crawling significantly less than the anc-1 mutant, making it difficult to directly compare the anchorage of ER in WT during crawling in the representative movies. Moreover, - Supp Movie 1 appears to be far shorter (fewer frames) than Supp movie 2 - ideally, equivalent time scales should be provided for WT animals to be comparable with the mutant.

    11. Figure 6A - in this scheme and through the manuscript, the authors refer to the c-term region of ANC-1, after the TM domain, as the KASH domain. However, by definition, the KASH domain includes part of the neck region, the TM domain and the c-term region after the TM domain (see e.g. Fig4 of Wihelmsen et al. JCS 2006, or PFAM definition of KASH - 10541). Therefore, the neck region of the KASH domain plays a role in nuclear positioning. Furthermore, labelling of Figure 6A should be changed accordingly to standard definition.

  2. Version published to 10.1101/2020.07.14.202838 on bioRxiv