LAP2alpha maintains a mobile and low assembly state of A-type lamins in the nuclear interior

  1. Nana Naetar
  2. Konstantina Georgiou
  3. Christian Knapp
  4. Irena Bronshtein
  5. Elisabeth Zier
  6. Petra Fichtinger
  7. Thomas Dechat
  8. Yuval Garini
  9. Roland Foisner

  • Curated by eLife

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    Summary: This work builds on prior studies by the Foisner group that investigated the function(s) of the soluble A-type lamin binding protein, LAP2a. One of their prior observations using antibody labeling was that there appeared to be a depletion of the nucleoplasmic pool of A-type lamins in cells lacking LAP2a. In this manuscript, the authors employ CRISPR-Cas9 editing to develop new tools to investigate the attributes specific to nucleoplasmic versus lamina-integrated A-type lamins. Using this new approach (and comparing it with their prior observations), the authors hit upon a new model in which LAP2a influences the conformational state of A-type lamins, which in turn influences its detection by a commonly used antibody. This technical detail explains the new realization that nucleoplasmic lamin A persists in LAP2a-null cells, albeit in a different state. The authors provide evidence that LAP2a antagonizes stable lamin A filament assembly, that is absence leads to stabilized intranuclear lamin A assemblies, and that telomere mobility is negatively influenced by loss of LAP2a in a manner depending on the presence of lamin A/C. The authors' work further identifies two pathways by which nucleoplasmic lamins emerge, namely by 1) initial localization to the lamina followed by relocalization to the nucleoplasm, and 2) from the pool of mitotic lamins which are not associated to the lamina.

    Overall there was enthusiasm for the study, with the reviewers stating their appreciation for the author's mechanistic approach to studying lamin assembly state and the use of complementary cell biology/microscopy and biochemical approaches. The rigor of the science was also lauded, including inclusion of, for example, genome editing quality control measures. Taken together the reviewers felt that the findings provided a new perspective on how LAP2a influences the state of A-type lamins. As the impact of lamins on nuclear organization is critical for nuclear functions and important for nuclear integrity, these results are fundamental for the understanding of both lamin A/C and LAP2a.

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Abstract

Lamins form stable filaments at the nuclear periphery in metazoans. Unlike B-type lamins, lamins A and C localize also in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). We show that lamin A in the nuclear interior is formed from newly expressed pre-lamin A during processing and from soluble mitotic mature lamins in a LAP2α-independent manner. Binding of LAP2α to lamins A/C in the nuclear interior during interphase inhibits formation of higher order structures of lamin A/C in vitro and in vivo , keeping lamin A/C in a mobile low assembly state independent of lamin A/C S22 phosphorylation. Loss of LAP2α causes formation of larger, less mobile and biochemically stable lamin A/C structures in the nuclear interior, which reduce the mobility of chromatin. We propose that LAP2α is essential to maintain a mobile lamin A/C pool in the nuclear interior, which is required for proper nuclear functions.

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  1. eLife

    Reviewer #3:

    In this manuscript, Naetar et al. investigate the role of LAP2α binding to A-type lamins in the nucleoplasm. LAP2α was already thought to be important for maintaining the nucleoplasmic pool of soluble A-type lamins, because knockout of LAP2α has previously been shown to reduce nucleoplasmic signal from an antibody that recognizes the lamin-A/C amino terminus. However, by directly tagging A-type lamins with fluorescent proteins and by using an alternative antibody to stain them, Naetar et al. find that the presence of LAP2α does not appreciably affect the pool of soluble lamins in the nucleoplasm. Instead, they find that LAP2α affects the assembly state of soluble lamins within the nucleoplasm, preventing formation of higher order A-type lamin structures that impede the mobility of telomeres within the nucleus.

    There is a lot to like about this paper. I admire the author's mechanistic approach to studying lamin assembly state. The complementary cell biology/microscopy approaches paired with the biochemical approaches in figure 5 lead to an overall convincing story. And finally, I appreciate the efforts the authors made to "show their work," including their genome editing quality control measures.

    Major comments:

    1. Although I appreciate the transparency of the authors in demonstrating their workflow and quality control measures (see above), some of the terminology makes the manuscript difficult to read. At times it feels more like reading a lab notebook than reading a manuscript. For example, The manuscript would be easier to understand if cell lines were given descriptive names (eg: LAP2α KO, or mEos3.2-lmna instead of "WT#21") rather than continuing to refer to them by the small guide RNA that was used to generate them. A second example: it is nice to show biological replicate data as in figure 1, but it took me a while to figure out that the second and third columns in panels A and B were biological replicates; I spent some time trying to determine which experimental condition was different. Perhaps one biological replicate could be displayed in the main text and the second could be moved to the supplement, especially considering that it appears that only one of the clones was used for the quantifications shown in the bottom panels.

    2. Why was the choice made to disrupt LAP2α at the beginning of exon 4? How large are exons 1 and 2, which are not shown in the schematic in the supplemental figures? What percentage of the LAP2α peptide primary sequence is affected by a frameshift mutation at the start of exon 4? Why was this approach preferable to introducing a frameshift mutation closer to the 5' end of the gene? I am concerned that the "LAP2α KO" cells used in the experiments may have some partially functional truncated LAP2α protein.

    3. On page 16, the authors describe a set of experiments that are meant to demonstrate that their failure to see a difference in nucleoplasmic A-type lamins in LAP2α mutants is not due to the fluorescent protein tag used, however, instead of looking at untagged lamins, they elect to look at a cell line that has all lmna alleles tagged. Wouldn't it be better to use the LAP2α KO cells from figure 1 and stain with both the 3A6 antibody and the N18 antibody to determine whether untagged lamins behave the same way as tagged lamins? Perhaps this experiment could be added along with the current data, as it would be nice to compare directly between a cell line with all lmna alleles tagged and a cell line with no lmna alleles tagged.

    This experiment would also give the authors a chance to compare morphology and overall fitness of cells with all untagged lmna with cells with all tagged lmna, to determine whether the tagged proteins are fully functional. Even if the tagged protein is fully functional, it would be appropriate to add a brief discussion of the possibility that fluorescent tags do perturb lamin-A/C function. After all, many lamin mutations do not cause obvious phenotypes in tissue culture cells, but defects can still emerge during development and aging in the context of an animal.

    1. The authors build a convincing case that binding to A-type lamins by LAP2α influences their ability to assemble. But how do cells leverage this relationship for biological functions? Do cells tune the amount of fully soluble vs. partially assembled A-type lamins in the nucleoplasm in order to control nuclear structure or function in response to certain stimuli? Have the A-type lamins in the nucleoplasm been found to be in a different assembly state in different cell types? As the study is currently written, it presents an interesting molecular mechanism but no biological mechanism.
  2. eLife

    Reviewer #2:

    Naetal et al. studied the effect of Lap2a on lamin A/C dynamics-of-assembly and mobility as well as telomere movements. This study indicated that lamin A/C are first assembled into the lamina, before some of the lamin A/C is re-localized to the nucleoplasm. Interestingly, the amount of nucleoplasmic lamins is independent of Lap2a although its physical properties are different. The results indicated that Lap2a contributes to the dynamics of lamin A/C in the nucleoplasm while its absence reduces nucleoplasmic lamin and telomere dynamics. These results reveal the function of Lap2a as regulator of lamin anchorage in the nucleoplasm but it has no major role in recruiting lamins into the nucleoplasm. Since the impact of lamins on the nuclear organization is critical for nuclear functions and important for nuclear integrity, these results are fundamental for the understanding of both lamin A/C and Lap2a.

    The authors also identified two pathways in which nucleoplasmic lamin emerged. First, lamin can be localized to the lamina and then relocated to the nucleoplasm, and second, from the pool of mitotic lamins which are not associated with the lamina.

    The authors may consider some textual changes, in particular regarding the state of nucleoplasmic lamin polymerization:

    1. The nuclear lamina filaments are typically 200-400 nm in length, but they are very flexible. A 200 nm filament would have a molecular weight of <1.4MDa ( ~50% of a ribosome) and can be bent and curved. That would mean that a single filament has a reasonably high diffusion coefficient. At the lamina, lamins are less mobile, however, it is likely to be due to binding partners that anchor lamins to the INM and chromatin (e.g. emerin is a membrane protein that binds lamin A) - the diffusion of 1.4 MDa protein complexes is quite fast. The above is mentioned because nucleoplasmic lamins may be polymerized but more mobile (less anchored) than their lamina-hosted lamins population.

    2. The authors show that nucleoplasmic lamins are first localized to the lamina, where they can polymerize. Isn't it possible that filaments can be released into the nucleoplasm?

    3. In vitro assembly assays of lamin A in the presence of Lap2a indicated that lamin A assembly is inhibited by Lap2a. Based on these results the authors suggest that Lap2a keeps lamin in a less polymerized state. Previous work by Zwerger et al. 2015, showed that inhibitors of in vitro lamin A assembly, have no impact on incorporation and localization of lamin A into the lamina, while incorporation of lamin A into the nuclear lamina was abolished when other lamin binders that have no effect on lamin assembly in vitro were used. That would suggest that either in vitro assembly is not representing the cellular lamin assembly or assembly of lamin into the lamina is independent of polymerization states of lamins. The authors may want to discuss these views.

  3. eLife

    Reviewer #1:

    Taken collectively, the findings described in the manuscript provide a new perspective on how LAP2alpha influences the state of A-type lamins. By extension, one impact of the findings is that they provide a mechanism by which A-type lamin state is distinct within the nucleoplasm and at the nuclear lamina. The authors also arrive at some additional insights that are valuable. For example, the data supporting the initial peripheral localization of what is argued to be pre-lamin A during processing rather than filament assembly was interesting and, although indirect, largely convincing. I would encourage the authors to address the fact that this work drives a reinterpretation of their prior findings early in the paper. I also have some concern that the impact of the findings is somewhat narrow.

    Major points:

    1. Given that a major focus of the paper is to explain conflicting results with (the same group's) prior published data on the effect of LAP2alpha depletion, it would have helped to lay this out more clearly from the outset of the paper. As written, the reader is confused until arriving at Figure 3. I appreciate that resolving this conflict leads to a new perspective - namely that LAP2alpha influences the state of the lamin assembly in a way that disrupts its detection by the N18 antibody, but structuring the manuscript to get to this point as quickly as possible would improve its accessibility.

    2. I found the plots in Fig. 1A and B confusing. Can the authors clarify how the measurements are achieved - through ROIs for the entire nucleoplasm/periphery? How do they capture the diffuse versus focal signal within the nucleoplasm? There is also some concern that the nucleoplasmic signal may simply be too low to detect robustly at early time points (leading to an increase at later time points as the protein accumulates). Line profiles (which are useful in Fig. 3) would be very helpful if used more broadly for assessing the data particularly for Figure 1.

    3. Related to Figure 1 - the results for the deltaK32 mutant is essential for the interpretation and should be included in the primary figures.

    4. The authors make no comment on the functionality of the mEos-tagged lamin A/C CRISPR lines. However, the comment suggesting that some clones could have altered nuclear morphology (line 225) raises some questions. How did the authors interpret this? Were these clones in which there were indels in some lmnA alleles affecting the levels? Or is this a consequence of the fusion? How do the authors explain the relatively low expression level of the mEos fusion relative to the untagged? If the MDFs are diploid, presumably we would expect this to be one allele tagged and one allele untagged. Given that the expression ratio is very different from this, could the tagged lamin A/C be targeted for degradation? As these cell lines are critical for the rest of the study, this information is important.

    5. How does the deltaK32 mutation affect the ability to detect lamin A/C with the N18 antibody? Could this provide further insight into the impact of LAP2alpha by extension?

    6. Greater explanation for the apparent paradox between the increase in immobile fraction by FRAP and the increased diffusion coefficient by FCS in the LAP2alpha-depleted condition is needed. The authors suggest that the latter is due to the loss of LAP2alpha binding (line 395), but some modeling would go a long way here. What form are the lamins thought to be in, and how does the bulk that LAP2 alpha would bring match the apparent changes in diffusivity?

    7. One prediction that arises from the proposed model is that regulation of LAP2alpha levels will modulate the relative pool of A-type lamins at the nuclear interior versus the nucleoplasm. Beyond the knock-out cells, is there any other evidence of this relationship?

    8. Much of the biochemical characterization seems confirmatory - e.g. the binding and gradients in Fig. 5A and B. Use of the assembly mutants of lamin here could be informative is essential to interpret the changes induced by addition of LAP2alpha.

    9. With regards to the effects on chromatin mobility - over what time interval was the volume of movement observed? This is important because more fluctuations in nuclear position, for example, could influence this measure. In addition, telomeres are a confusing choice, given abundant evidence that there is crosstalk between the state of the nuclear lamina and telomere biology (e.g. lamin mutants affecting telomere homeostasis, etc.). At a minimum, acknowledging that telomeres may not reflect the effect on chromatin globally is important. Examples of the raw mean squared displacements would be more informative. Is the difference between lmna KO and lmna/Lap2alpha DKO (Fig. 6 right panel) significant?

    10. How do the authors think the membrane integrated LAP2beta fits into the story?

  4. eLife

    Summary: This work builds on prior studies by the Foisner group that investigated the function(s) of the soluble A-type lamin binding protein, LAP2a. One of their prior observations using antibody labeling was that there appeared to be a depletion of the nucleoplasmic pool of A-type lamins in cells lacking LAP2a. In this manuscript, the authors employ CRISPR-Cas9 editing to develop new tools to investigate the attributes specific to nucleoplasmic versus lamina-integrated A-type lamins. Using this new approach (and comparing it with their prior observations), the authors hit upon a new model in which LAP2a influences the conformational state of A-type lamins, which in turn influences its detection by a commonly used antibody. This technical detail explains the new realization that nucleoplasmic lamin A persists in LAP2a-null cells, albeit in a different state. The authors provide evidence that LAP2a antagonizes stable lamin A filament assembly, that is absence leads to stabilized intranuclear lamin A assemblies, and that telomere mobility is negatively influenced by loss of LAP2a in a manner depending on the presence of lamin A/C. The authors' work further identifies two pathways by which nucleoplasmic lamins emerge, namely by 1) initial localization to the lamina followed by relocalization to the nucleoplasm, and 2) from the pool of mitotic lamins which are not associated to the lamina.

    Overall there was enthusiasm for the study, with the reviewers stating their appreciation for the author's mechanistic approach to studying lamin assembly state and the use of complementary cell biology/microscopy and biochemical approaches. The rigor of the science was also lauded, including inclusion of, for example, genome editing quality control measures. Taken together the reviewers felt that the findings provided a new perspective on how LAP2a influences the state of A-type lamins. As the impact of lamins on nuclear organization is critical for nuclear functions and important for nuclear integrity, these results are fundamental for the understanding of both lamin A/C and LAP2a.

  5. Version published to 10.1101/2020.09.25.313296 on bioRxiv