Lamins are intermediate filament proteins in metazoan nuclei that, together with numerous inner nuclear membrane proteins, form a filamentous protein meshwork at the nuclear periphery, called the nuclear lamina (Gruenbaum and Foisner, 2015a). Based on their biochemical properties and expression patterns, lamins are grouped into two subtypes, A-type and B-type lamins. B-type lamins are ubiquitously expressed in all cell types and throughout development (Yang et al., 2011), whereas A-type lamins are expressed at low levels in embryonic stem cells and undifferentiated cells, but are significantly upregulated during differentiation (Constantinescu et al., 2006; Eckersley-Maslin et al., 2013; Röber et al., 1989). In mammalian cells, the major A-type lamins are lamins A and C encoded by the Lmna gene, whereas the two major B-type lamins, lamins B1 and B2, are encoded by Lmnb1 and Lmnb2, respectively (Gruenbaum and Foisner, 2015a). Both lamin subtypes share a similar intermediate filament protein-type domain structure with a central rod domain, an N-terminal head and a globular C-terminal tail, which contains a nuclear localization signal, an Ig fold and, except for lamin C, a C-terminal CaaX motif (C: cysteine; a: aliphatic amino acid; X: any amino acid) (de Leeuw et al., 2018; Gruenbaum and Medalia, 2015b). The CaaX motif undergoes a series of post-translational modifications, including farnesylation of the cysteine, removal of the last three amino acids, followed by carboxymethylation (Rusiñol and Sinensky, 2006). Whereas B-type lamins remain farnesylated and carboxymethylated, pre-lamin A undergoes an additional processing step catalyzed by the metalloprotease Zmpste24, leading to the removal of 15 amino acids from its C-terminus, including the farnesylated cysteine residue. As a consequence, mature B-type lamins are tightly associated with the nuclear membrane via their farnesylated C-terminus and mainly localize at the nuclear periphery, whereas mature lamin A is found both in filaments within the peripheral nuclear lamina, and additionally in a soluble and dynamic pool in the nuclear interior (Naetar et al., 2017). Lamin C that lacks a CaaX motif contributes also to the peripheral and nucleoplasmic pool of A-type lamins.

Lamin filaments at the nuclear periphery were recently visualized by cryo-electron tomography (Turgay et al., 2017), but their structure and assembly are far from being well understood. Lamins form dimers via their central rod domains, which further assemble into head-to-tail polymers (de Leeuw et al., 2018). Recent work by the Medalia lab using cryo-electron tomography revealed that in mammalian nuclei two head-to-tail filaments assemble laterally into 3.5-nm-thick filaments in a staggered fashion (Turgay et al., 2017). Lamin filaments at the nuclear periphery are considered stable, resistant to biochemical extraction and highly immobile (Bronshtein et al., 2015; Moir et al., 2000; Shimi et al., 2008). Together with proteins of the inner nuclear membrane, they fulfill essential functions, defining the mechanical properties of nuclei (Buxboim et al., 2014; Cho et al., 2019; Davidson and Lammerding, 2014; Osmanagic-Myers et al., 2015; Swift et al., 2013) and regulating higher order chromatin organization through anchorage of peripheral heterochromatic genomic regions (Solovei et al., 2013; van Steensel and Belmont, 2017).

The regulation and properties of A-type lamins in the nuclear interior are far less understood, although recent studies have unraveled novel functions of lamins in the nuclear interior in chromatin regulation that are fundamentally different from those fulfilled by the nuclear lamina (Gesson et al., 2014; Naetar et al., 2017). Nucleoplasmic lamins A and C bind to and regulate euchromatic regions of the genome, globally affecting epigenetic modifications and possibly chromatin accessibility (Gesson et al., 2016; Naetar et al., 2017). They also provide chromatin ‘docking sites’ in the nucleoplasm slowing down chromatin movement in nuclear space (Bronshtein et al., 2015). Moreover, intranuclear A-type lamins are required for the proper assembly of repressive polycomb protein foci (Bianchi et al., 2020; Cesarini et al., 2015) and are involved in the regulation of telomere function (Chojnowski et al., 2015; Gonzalez-Suarez et al., 2009; Wood et al., 2014). Nucleoplasmic lamins were also found to affect gene expression directly by binding to gene regulatory sequences, such as promoters and enhancers (Briand et al., 2018; Ikegami et al., 2020; Oldenburg et al., 2017).

The structure and assembly state of nucleoplasmic lamins remain enigmatic. Fluorescence recovery after photobleaching (FRAP), continuous photobleaching (CP), and fluorescence correlation spectroscopy (FCS) studies of fluorescently tagged A-type lamins showed that the majority of nucleoplasmic lamin complexes are highly mobile compared to the stable peripheral lamina (Bronshtein et al., 2015; Moir et al., 2000; Shimi et al., 2008). Biochemical studies revealed that intranuclear lamins can be easily extracted in salt and detergent-containing buffers, suggesting that they have a low assembly state (Kolb et al., 2011; Naetar et al., 2008) and/or weaker interactions with nuclear protein complexes and chromatin compared to the lamins at the lamina. Taken together, the dynamic nucleoplasmic lamin pool has very distinct properties compared to the static and stable peripheral lamina, allowing them to fulfill a unique set of functions. However, why and how nucleoplasmic A-type lamins display such different properties in the nuclear interior compared to their counterparts at the nuclear lamina remains poorly understood. Possible mechanisms include post-translational modifications and/or interactions with specific nucleoplasmic binding partners. For example, phosphorylation of lamins influences the localization, solubility and mobility of lamins both during mitosis and interphase (Kochin et al., 2014; Machowska et al., 2015). Moreover, the specific interaction partner of nucleoplasmic lamins, lamin-associated polypeptide 2 alpha (LAP2α) was suggested to regulate the localization of lamin A/C in the nuclear interior, since the nucleoplasmic lamin pool was found significantly reduced in the absence of LAP2α (Dechat et al., 2000; Gesson et al., 2016; Gesson et al., 2014; Naetar et al., 2008). However, the mechanisms remain unknown.

Here, we address the open question if and how LAP2α regulates the formation, properties and functions of nucleoplasmic A-type lamins. Surprisingly, we find that LAP2α is not involved in the generation of the nucleoplasmic lamin A/C pool from newly synthesized lamin A/C in interphase cells and from mitotic lamins in G1 phase, but instead regulates lamin A/C properties in a lamin A/C phosphorylation-independent manner. These findings led to a new perspective of LAP2α function in lamin A/C regulation, suggesting that LAP2α affects the state of lamin A/C assembly and/or their interactions in the nucleoplasm. We show that in the absence of LAP2α, lamins A and C in the nuclear interior are more resistant toward biochemical extraction, possibly through the formation of higher order structures and/or stronger interactions with nuclear components, leading to an impaired detection by antibodies and decreased mobility. The impaired accessibility for antibodies against the lamin A/C N-terminus likely explains the previously observed reduction of nucleoplasmic lamins A/C in cells and tissues lacking LAP2α (Naetar et al., 2008). We propose that the interaction of soluble lamins with LAP2α in the nuclear interior maintains their mobile, low assembly state, which is important for proper lamin functions in nuclear organization and chromatin regulation.

Here, we show that LAP2α, a known binding partner of nucleoplasmic lamin A/C (Dechat et al., 2000; Naetar and Foisner, 2009) is essential to maintain lamins A and C in the nuclear interior in a mobile and low assembly state. This occurs in a lamin A/C S22 phosphorylation-independent manner, likely by inhibiting lamin assembly through direct interaction of LAP2α with lamin A/C and/or by changing interactions of nucleoplasmic lamin complexes with chromatin.

Our study confirms and extends the finding that lamins A and C in the nuclear interior have fundamentally different properties compared to their counterparts at the nuclear periphery. While lamins at the nuclear periphery form stable 3.5-nm-wide filaments (de Leeuw et al., 2018; Moir et al., 2000; Shimi et al., 2008; Turgay et al., 2017), lamins in the nuclear interior are highly mobile (Broers et al., 1999; Bronshtein et al., 2015; Shimi et al., 2008) and easily extractable in salt and detergent-containing buffers (Kolb et al., 2011; Naetar et al., 2008). However, very little is known about the mechanisms securing these unique properties of lamins in the nuclear interior, given that in solution they have a strong drive to assemble and tend to form higher order structures (Aebi et al., 1986; Moir et al., 1991). Immunofluorescence microscopy has previously shown that intranuclear lamin A/C staining is significantly reduced in cells and tissues from LAP2α knockout mice (Naetar et al., 2008), leading to a model, where LAP2α is required for the formation and/or maintenance of the nucleoplasmic lamin pool (Naetar and Foisner, 2009). Based on data shown in this study, we propose a modified and updated version of the model suggesting that LAP2α is essential to maintain the highly mobile and soluble state of lamins in the nuclear interior. In the absence of LAP2α, lamins in the nucleoplasm are not lost but they are transformed into more stable, immobile structures (Figure 7) that can no longer be recognized by lamin A/C antibodies directed to an N-terminal epitope, which – in wildtype cells – favors nucleoplasmic over peripheral lamin staining (Gesson et al., 2016). These findings can be explained by the formation of higher order lamin complexes in the nuclear interior, whose properties may resemble more those of lamins at the nuclear periphery. This is supported by the increased resistance of nucleoplasmic lamins to extraction with detergent and salt-containing buffers and their decreased mobility in the absence of LAP2α. Moreover, high-resolution microscopy revealed significantly more and, importantly, larger extraction-resistant structures in LAP2α knockout versus wildtype cells. As for potential mechanisms describing how LAP2α can maintain lamins in a soluble and dynamic state, we can envisage at least two different not mutually exclusive mechanisms. Our in vitro data using purified recombinant LAP2α and lamin A protein suggest that direct binding of LAP2α to lamins may prevent them from assembling into higher order structures. However, one has to keep in mind that the in vitro assembly of lamins may not fully represent lamin assembly pathways within a cell, as previously indicated in a study using lamin-binding designed ankyrin repeat proteins (DARPins), where DARPins inhibiting in vitro lamin A assembly had no impact on incorporation of lamin A into the lamina in vivo and, vice versa, DARPins affecting the incorporation of lamin A into the nuclear lamina in vivo had no effect on lamin assembly in vitro. Nevertheless, we showed in a previous study that, unlike wildtype lamin A/C staining (Naetar et al., 2008 and Figure 3), antibody-staining of the assembly-deficient lamin AΔK32 mutant in the nuclear interior is not affected in LAP2α knockout cells (Pilat et al., 2013). This suggests that nucleoplasmic lamin A assembly in the absence of LAP2α leads to the observed antibody epitope masking.

Besides affecting lamin assembly, LAP2α may also regulate the interaction of A-type lamin complexes with chromatin. In this model, the absence of LAP2α would lead to a tighter and more stable association of lamins with chromatin, which would in turn also lead to an increased extraction resistance and possibly decreased mobility. In support of this model, the post-fixation digestion of formaldehyde-fixed cells with DNase/RNase fully recovered the intranuclear lamin A/C staining using antibodies to the lamin A/C N-terminus, and chromatin motion was slowed down upon LAP2α knockout in a lamin A/C-dependent manner. However, the finding that the intranuclear lamin structures are resistant to 500 mM high salt, which is expected to extract chromatin-associated proteins (Herrmann et al., 2017), supports the notion that larger lamin A structures can be formed independently of chromatin. As these different scenarios are not mutually exclusive, we favor a model in which the absence of LAP2α induces the formation of higher order lamin A assemblies, which in turn may bind more tightly and stably to chromatin (Figure 7).

As phosphorylation of lamins provides another means to regulate lamin filament (dis)assembly in mitosis (Heald and McKeon, 1990; Ward and Kirschner, 1990) and to influence their assembly state, solubility and mobility in interphase (Buxboim et al., 2014; Kochin et al., 2014; Moiseeva et al., 2016), we tested the relationship between lamin phosphorylation and interaction with LAP2α. We found that LAP2α loss does neither affect total levels of lamins phosphorylated at serines 22 and 392 nor does it change the localization and solubility of phosphorylated lamins. Vice versa, LAP2α can interact with both phosphorylated and un(der)phosphorylated lamins, but it affects only the solubility and assembly state of un(der)phosphorylated lamins. Thus, interaction with LAP2α and lamin phosphorylation seem to be two independent, non-mutually exclusive mechanisms for keeping lamins in a low assembly state.

Surprisingly, the initial formation of the nucleoplasmic lamin A/C pool does not require LAP2α, as it can form normally from newly synthesized pre-lamin A and from soluble mitotic lamins in LAP2α knockout cells. Thus, we propose that these processes may primarily depend on other mechanisms, such as lamin A/C phosphorylation. At the onset of mitosis, lamins are targeted by cyclin-dependent kinase 1 (Cdk1) particularly at serines 22 and 392 in the N-terminal head and proximal to the C-terminal end of the rod, respectively (Ward and Kirschner, 1990), leading to a complete disassembly of the lamina (Dechat et al., 2004; Heald and McKeon, 1990; Moir et al., 2000). During post-mitotic nuclear envelope reassembly, lamins A and C are imported into the nucleoplasm of newly formed nuclei, followed by gradual dephosphorylation and re-assembly at the nuclear periphery (Moir et al., 2000; Steen et al., 2003; Steen and Collas, 2001). It is tempting to speculate that the majority of lamins A and C is still phosphorylated at S22 and possibly other residues when they are re-imported into the nucleoplasm after post-mitotic nuclear membrane reassembly, preventing their incorporation into the peripheral lamina. In the nucleoplasm, a fraction of the phosphorylated lamins may then bind to LAP2α. When lamins are gradually dephosphorylated during G1 (Steen et al., 2003; Steen and Collas, 2001), LAP2α-bound lamin A/C may be maintained in a low assembly state, while free lamin A/C can assemble into the lamina at the periphery. Thus, in the absence of LAP2α, the nucleoplasmic lamin A/C pool may still form, but, whereas phosphorylated lamins stay soluble independently of LAP2α, hypophosphorylated lamins tend to form higher-order structures at the ‘wrong’ position, that is within the nucleoplasm as opposed to the nuclear periphery in LAP2α knockout cells. This model suggests that in wild-type cells, there are three pools of intranuclear A-type lamins (Figure 7): (1) lamins phosphorylated at S22 (and possibly other residues) that are soluble independent of LAP2α; (2) hypophosphorylated lamins bound to LAP2α that are soluble independent of their phosphorylation status; and (3) hypophosphorylated lamins not bound to LAP2α that tend to form immobile and extraction-resistant structures in the nuclear interior. Previous reports and our own data showing that a small fraction of nucleoplasmic A-type lamins displayed low mobility (Bronshtein et al., 2015; Shimi et al., 2008) in wildtype cells supports this model. In the absence of LAP2α, the balance between these different intranuclear lamin structures is disturbed toward increased levels of immobile, higher order lamin A/C structures.

What is the physiological relevance of a dynamic lamin A/C pool in the nucleoplasm and how is the nucleoplasmic lamin A/C pool regulated during various cellular processes? We propose that the unique dynamic properties allow intranuclear A-type lamins to fulfill a set of functions that is different from those covered by the peripheral lamina. One of the most prominent examples for different functions of the peripheral versus intranuclear lamins is their different role in chromatin organization. While the peripheral lamina is well known to mediate stable interaction with heterochromatic genomic regions, termed lamina-associated domains (LADs) (Kind et al., 2013; van Steensel and Belmont, 2017), thereby contributing to gene silencing in general (Leemans et al., 2019) or during differentiation (Robson et al., 2016), lamins in the nuclear interior have a much more complex role in regulating open chromatin and gene expression. Lamin A/C in the nuclear interior interact primarily with open euchromatic regions of the genome (Gesson et al., 2016; Lund et al., 2015) and affect their epigenetic landscape (Bianchi et al., 2020; Briand et al., 2018; Cesarini et al., 2015; Oldenburg et al., 2017). Furthermore, lamin A/C in the nucleoplasm has also been shown to directly interact with promoters and enhancers and seems to be involved in both positive and negative gene regulations (Ikegami et al., 2020; Lund and Collas, 2013; Lund et al., 2013). It is conceivable that gene regulatory pathways have to be highly flexible and dynamic to respond efficiently to internal and external cues and thus require a dynamic lamin complex that can dynamically associate with and regulate chromatin. Indeed, loss of LAP2α (Gesson et al., 2016) or disease-linked mutations in lamins (Bianchi et al., 2020; Briand et al., 2018; Ikegami et al., 2020; Oldenburg et al., 2017) affect epigenetic regulation and/or gene expression.

Another function of nucleoplasmic lamins is to regulate the mobility of chromatin in the three-dimensional nuclear space. Dynamic interactions of lamin complexes with telomeres and other genomic loci has been shown to limit free diffusion of chromatin inside the nucleus (Bronshtein et al., 2015). We demonstrate here that knockout of LAP2α reduces telomere motion mostly in a lamin A/C-dependent manner, most likely through the formation of more stable lamin A/C structures that bind chromatin more tightly and stably. It is reasonable to assume that a tightly regulated movement of genes inside the nucleus is important for controlling coordinated regulation of gene expression during various cellular processes. Notably, LAP2α may have another lamin A-independent role in the regulation of chromatin diffusion. By direct binding to chromatin, LAP2α may additionally slow down chromatin diffusion. However, the major contribution to restricted, anomalous chromatin diffusion in the nuclear interior seems to stem from nucleoplasmic lamin A/C, as its depletion has the highest impact on chromatin movement.

Overall, we speculate that a dynamic nucleoplasmic lamin A/C complex may be required to maintain genomic plasticity, allowing the cell to efficiently respond to external and internal cues, such as during cell differentiation. In line with this hypothesis, we found nucleoplasmic lamins and LAP2α in proliferating tissue progenitor cells in the epidermis, colon, skeletal muscle and in preadipocytes, while upon their differentiation (Dorner et al., 2006; Gotic et al., 2010; Markiewicz et al., 2005; Naetar et al., 2008) or cell cycle exit into a quiescence or senescence state (Naetar et al., 2007) LAP2α was downregulated at transcriptional and protein level leading to loss of the dynamic nucleoplasmic lamin A/C pool. Thus, when genome plasticity is no longer required, such es in terminally differentiated cells, dynamic lamin A/C levels may be reduced through downregulation of LAP2α. Accordingly, the absence of the dynamic nucleoplasmic lamin A/C pool in the LAP2α knockout progenitor cells leads to impaired or delayed differentiation (Gotic et al., 2010; Naetar et al., 2008). This regulatory pathway may also be impaired in lamin-linked diseases, such as the premature aging disease Hutchinson Gilford Progeria Syndrome (HGPS), where LAP2α expression is lost, leading to premature senescence, while re-expression of LAP2α in HGPS cells rescues cell proliferation (Vidak et al., 2015).

In summary, we propose that lamin A/ C complexes in the nuclear interior are in a dynamic state, established by LAP2α, which is a prerequisite for their multiple roles in chromatin regulation both on a genome-wide scale and in a gene-specific manner during cell differentiation.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2G-H, Figure 4A-B and Figure 6A.

1. 1. U Aebi
   2. J Cohn
   3. L Buhle
   4. L Gerace

   (1986) The nuclear Lamina is a meshwork of intermediate-type filaments

   Nature 323:560–564.

   https://doi.org/10.1038/323560a0

   • PubMed
   • Google Scholar
2. 1. EM Bank
   2. K Ben-Harush
   3. N Wiesel-Motiuk
   4. R Barkan
   5. N Feinstein
   6. O Lotan
   7. O Medalia
   8. Y Gruenbaum

   (2011) A laminopathic mutation disrupting lamin filament assembly causes disease-like phenotypes in Caenorhabditis elegans

   Molecular Biology of the Cell 22:2716–2728.

   https://doi.org/10.1091/mbc.e11-01-0064

   • PubMed
   • Google Scholar
3. 1. AT Bertrand
   2. L Renou
   3. A Papadopoulos
   4. M Beuvin
   5. E Lacène
   6. C Massart
   7. C Ottolenghi
   8. V Decostre
   9. S Maron
   10. S Schlossarek
   11. ME Cattin
   12. L Carrier
   13. M Malissen
   14. T Arimura
   15. G Bonne

   (2012) DelK32-lamin A/C has abnormal location and induces incomplete tissue maturation and severe metabolic defects leading to premature death

   Human Molecular Genetics 21:1037–1048.

   https://doi.org/10.1093/hmg/ddr534

   • PubMed
   • Google Scholar
4. 1. A Bianchi
   2. C Mozzetta
   3. G Pegoli
   4. F Lucini
   5. S Valsoni
   6. V Rosti
   7. C Petrini
   8. A Cortesi
   9. F Gregoretti
   10. L Antonelli
   11. G Oliva
   12. M De Bardi
   13. R Rizzi
   14. B Bodega
   15. D Pasini
   16. F Ferrari
   17. C Bearzi
   18. C Lanzuolo

   (2020) Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy

   Journal of Clinical Investigation 130:2408–2421.

   https://doi.org/10.1172/JCI128161

   • PubMed
   • Google Scholar
5. 1. N Briand
   2. AC Guénantin
   3. D Jeziorowska
   4. A Shah
   5. M Mantecon
   6. E Capel
   7. M Garcia
   8. A Oldenburg
   9. J Paulsen
   10. JS Hulot
   11. C Vigouroux
   12. P Collas

   (2018) The lipodystrophic hotspot lamin A p.r482w mutation deregulates the mesodermal inducer T/Brachyury and early vascular differentiation gene networks

   Human Molecular Genetics 27:1447–1459.

   https://doi.org/10.1093/hmg/ddy055

   • PubMed
   • Google Scholar
6. 1. EK Brinkman
   2. T Chen
   3. M Amendola
   4. B van Steensel

   (2014) Easy quantitative assessment of genome editing by sequence trace decomposition

   Nucleic Acids Research 42:e168.

   https://doi.org/10.1093/nar/gku936

   • PubMed
   • Google Scholar
7. 1. JL Broers
   2. BM Machiels
   3. GJ van Eys
   4. HJ Kuijpers
   5. EM Manders
   6. R van Driel
   7. FC Ramaekers

   (1999)

   Dynamics of the nuclear Lamina as monitored by GFP-tagged A-type lamins

   Journal of Cell Science 112 (Pt 20):3463–3475.

   • PubMed
   • Google Scholar
8. 1. I Bronshtein
   2. E Kepten
   3. I Kanter
   4. S Berezin
   5. M Lindner
   6. AB Redwood
   7. S Mai
   8. S Gonzalo
   9. R Foisner
   10. Y Shav-Tal
   11. Y Garini

   (2015) Loss of lamin A function increases chromatin dynamics in the nuclear interior

   Nature Communications 6:8044.

   https://doi.org/10.1038/ncomms9044

   • PubMed
   • Google Scholar
9. 1. A Buxboim
   2. J Swift
   3. J Irianto
   4. KR Spinler
   5. PC Dingal
   6. A Athirasala
   7. YR Kao
   8. S Cho
   9. T Harada
   10. JW Shin
   11. DE Discher

   (2014) Matrix elasticity regulates lamin-A,C phosphorylation and turnover with feedback to actomyosin

   Current Biology 24:1909–1917.

   https://doi.org/10.1016/j.cub.2014.07.001

   • PubMed
   • Google Scholar
10. 1. E Cesarini
    2. C Mozzetta
    3. F Marullo
    4. F Gregoretti
    5. A Gargiulo
    6. M Columbaro
    7. A Cortesi
    8. L Antonelli
    9. S Di Pelino
    10. S Squarzoni
    11. D Palacios
    12. A Zippo
    13. B Bodega
    14. G Oliva
    15. C Lanzuolo

    (2015) Lamin A/C sustains PcG protein architecture, maintaining transcriptional repression at target genes

    Journal of Cell Biology 211:533–551.

    https://doi.org/10.1083/jcb.201504035

    • PubMed
    • Google Scholar
11. 1. S Cho
    2. M Vashisth
    3. A Abbas
    4. S Majkut
    5. K Vogel
    6. Y Xia
    7. IL Ivanovska
    8. J Irianto
    9. M Tewari
    10. K Zhu
    11. ED Tichy
    12. F Mourkioti
    13. HY Tang
    14. RA Greenberg
    15. BL Prosser
    16. DE Discher

    (2019) Mechanosensing by the Lamina protects against nuclear rupture, DNA damage, and Cell-Cycle arrest

    Developmental Cell 49:920–935.

    https://doi.org/10.1016/j.devcel.2019.04.020

    • PubMed
    • Google Scholar
12. 1. A Chojnowski
    2. PF Ong
    3. ES Wong
    4. JS Lim
    5. RA Mutalif
    6. R Navasankari
    7. B Dutta
    8. H Yang
    9. YY Liow
    10. SK Sze
    11. T Boudier
    12. GD Wright
    13. A Colman
    14. B Burke
    15. CL Stewart
    16. O Dreesen

    (2015) Progerin reduces LAP2α-telomere association in Hutchinson-Gilford progeria

    eLife 4:e07759.

    https://doi.org/10.7554/eLife.07759

    • PubMed
    • Google Scholar
13. 1. D Constantinescu
    2. HL Gray
    3. PJ Sammak
    4. GP Schatten
    5. AB Csoka

    (2006) Lamin A/C expression is a marker of mouse and human embryonic stem cell differentiation

    Stem Cells 24:177–185.

    https://doi.org/10.1634/stemcells.2004-0159

    • PubMed
    • Google Scholar
14. 1. PM Davidson
    2. J Lammerding

    (2014) Broken nuclei--lamins, nuclear mechanics, and disease

    Trends in Cell Biology 24:247–256.

    https://doi.org/10.1016/j.tcb.2013.11.004

    • PubMed
    • Google Scholar
15. 1. R de Leeuw
    2. Y Gruenbaum
    3. O Medalia

    (2018) Nuclear lamins: thin filaments with major functions

    Trends in Cell Biology 28:34–45.

    https://doi.org/10.1016/j.tcb.2017.08.004

    • PubMed
    • Google Scholar
16. 1. T Dechat
    2. B Korbei
    3. OA Vaughan
    4. S Vlcek
    5. CJ Hutchison
    6. R Foisner

    (2000)

    Lamina-associated polypeptide 2alpha binds intranuclear A-type lamins

    Journal of Cell Science 113 Pt 19:3473–3484.

    • PubMed
    • Google Scholar
17. 1. T Dechat
    2. A Gajewski
    3. B Korbei
    4. D Gerlich
    5. N Daigle
    6. T Haraguchi
    7. K Furukawa
    8. J Ellenberg
    9. R Foisner

    (2004) LAP2alpha and BAF transiently localize to telomeres and specific regions on chromatin during nuclear assembly

    Journal of Cell Science 117:6117–6128.

    https://doi.org/10.1242/jcs.01529

    • PubMed
    • Google Scholar
18. 1. D Dorner
    2. S Vlcek
    3. N Foeger
    4. A Gajewski
    5. C Makolm
    6. J Gotzmann
    7. CJ Hutchison
    8. R Foisner

    (2006) Lamina-associated polypeptide 2alpha regulates cell cycle progression and differentiation via the retinoblastoma-E2F pathway

    Journal of Cell Biology 173:83–93.

    https://doi.org/10.1083/jcb.200511149

    • PubMed
    • Google Scholar
19. 1. MA Eckersley-Maslin
    2. JH Bergmann
    3. Z Lazar
    4. DL Spector

    (2013) Lamin A/C is expressed in pluripotent mouse embryonic stem cells

    Nucleus 4:53–60.

    https://doi.org/10.4161/nucl.23384

    • PubMed
    • Google Scholar
20. 1. HP Erickson

    (2009) Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy

    Biological Procedures Online 11:32–51.

    https://doi.org/10.1007/s12575-009-9008-x

    • PubMed
    • Google Scholar
21. 1. N Foeger
    2. N Wiesel
    3. D Lotsch
    4. N Mücke
    5. L Kreplak
    6. U Aebi
    7. Y Gruenbaum
    8. H Herrmann

    (2006) Solubility properties and specific assembly pathways of the B-type lamin from Caenorhabditis elegans

    Journal of Structural Biology 155:340–350.

    https://doi.org/10.1016/j.jsb.2006.03.026

    • PubMed
    • Google Scholar
22. 1. K Gesson
    2. S Vidak
    3. R Foisner

    (2014) Lamina-associated polypeptide (LAP)2α and nucleoplasmic lamins in adult stem cell regulation and disease

    Seminars in Cell & Developmental Biology 29:116–124.

    https://doi.org/10.1016/j.semcdb.2013.12.009

    • PubMed
    • Google Scholar
23. 1. K Gesson
    2. P Rescheneder
    3. MP Skoruppa
    4. A von Haeseler
    5. T Dechat
    6. R Foisner

    (2016) A-type lamins bind both hetero- and Euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha

    Genome Research 26:462–473.

    https://doi.org/10.1101/gr.196220.115

    • PubMed
    • Google Scholar
24. 1. I Gonzalez-Suarez
    2. AB Redwood
    3. SM Perkins
    4. B Vermolen
    5. D Lichtensztejin
    6. DA Grotsky
    7. L Morgado-Palacin
    8. EJ Gapud
    9. BP Sleckman
    10. T Sullivan
    11. J Sage
    12. CL Stewart
    13. S Mai
    14. S Gonzalo

    (2009) Novel roles for A-type lamins in telomere biology and the DNA damage response pathway

    The EMBO Journal 28:2414–2427.

    https://doi.org/10.1038/emboj.2009.196

    • PubMed
    • Google Scholar
25. 1. I Gotic
    2. WM Schmidt
    3. K Biadasiewicz
    4. M Leschnik
    5. R Spilka
    6. J Braun
    7. CL Stewart
    8. R Foisner

    (2010) Loss of LAP2 alpha delays satellite cell differentiation and affects postnatal fiber-type determination

    Stem Cells 28:480–488.

    https://doi.org/10.1002/stem.292

    • PubMed
    • Google Scholar
26. 1. Y Gruenbaum
    2. R Foisner

    (2015a) Lamins: nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation

    Annual Review of Biochemistry 84:131–164.

    https://doi.org/10.1146/annurev-biochem-060614-034115

    • PubMed
    • Google Scholar
27. 1. Y Gruenbaum
    2. O Medalia

    (2015b) Lamins: the structure and protein complexes

    Current Opinion in Cell Biology 32:7–12.

    https://doi.org/10.1016/j.ceb.2014.09.009

    • PubMed
    • Google Scholar
28. 1. R Heald
    2. F McKeon

    (1990) Mutations of phosphorylation sites in lamin A that prevent nuclear Lamina disassembly in mitosis

    Cell 61:579–589.

    https://doi.org/10.1016/0092-8674(90)90470-Y

    • PubMed
    • Google Scholar
29. 1. C Herrmann
    2. DC Avgousti
    3. MD Weitzman

    (2017) Differential salt fractionation of nuclei to analyze Chromatin-associated proteins from cultured mammalian cells

    Bio-Protocol 7:e2175.

    https://doi.org/10.21769/BioProtoc.2175

    • PubMed
    • Google Scholar
30. 1. K Ikegami
    2. S Secchia
    3. O Almakki
    4. JD Lieb
    5. IP Moskowitz

    (2020) Phosphorylated lamin A/C in the nuclear interior binds active enhancers associated with abnormal transcription in progeria

    Developmental Cell 52:699–713.

    https://doi.org/10.1016/j.devcel.2020.02.011

    • PubMed
    • Google Scholar
31. 1. J Kind
    2. L Pagie
    3. H Ortabozkoyun
    4. S Boyle
    5. SS de Vries
    6. H Janssen
    7. M Amendola
    8. LD Nolen
    9. WA Bickmore
    10. B van Steensel

    (2013) Single-cell dynamics of genome-nuclear Lamina interactions

    Cell 153:178–192.

    https://doi.org/10.1016/j.cell.2013.02.028

    • PubMed
    • Google Scholar
32. 1. V Kochin
    2. T Shimi
    3. E Torvaldson
    4. SA Adam
    5. A Goldman
    6. CG Pack
    7. J Melo-Cardenas
    8. SY Imanishi
    9. RD Goldman
    10. JE Eriksson

    (2014) Interphase phosphorylation of lamin A

    Journal of Cell Science 127:2683–2696.

    https://doi.org/10.1242/jcs.141820

    • PubMed
    • Google Scholar
33. 1. T Kolb
    2. K Maass
    3. M Hergt
    4. U Aebi
    5. H Herrmann

    (2011) Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the Lamina of cultured human cells

    Nucleus 2:425–433.

    https://doi.org/10.4161/nucl.2.5.17765

    • PubMed
    • Google Scholar
34. 1. C Leemans
    2. MCH van der Zwalm
    3. L Brueckner
    4. F Comoglio
    5. T van Schaik
    6. L Pagie
    7. J van Arensbergen
    8. B van Steensel

    (2019) Promoter-Intrinsic and local chromatin features determine gene repression in LADs

    Cell 177:852–864.

    https://doi.org/10.1016/j.cell.2019.03.009

    • PubMed
    • Google Scholar
35. 1. E Lund
    2. AR Oldenburg
    3. E Delbarre
    4. CT Freberg
    5. I Duband-Goulet
    6. R Eskeland
    7. B Buendia
    8. P Collas

    (2013) Lamin A/C-promoter interactions specify chromatin state-dependent transcription outcomes

    Genome Research 23:1580–1589.

    https://doi.org/10.1101/gr.159400.113

    • PubMed
    • Google Scholar
36. 1. EG Lund
    2. I Duband-Goulet
    3. A Oldenburg
    4. B Buendia
    5. P Collas

    (2015) Distinct features of lamin A-interacting chromatin domains mapped by ChIP-sequencing from sonicated or micrococcal nuclease-digested chromatin

    Nucleus 6:30–39.

    https://doi.org/10.4161/19491034.2014.990855

    • PubMed
    • Google Scholar
37. 1. E Lund
    2. P Collas

    (2013) Nuclear lamins: making contacts with promoters

    Nucleus 4:424–430.

    https://doi.org/10.4161/nucl.26865

    • PubMed
    • Google Scholar
38. 1. M Machowska
    2. K Piekarowicz
    3. R Rzepecki

    (2015) Regulation of lamin properties and functions: does phosphorylation do it all?

    Open Biology 5:150094.

    https://doi.org/10.1098/rsob.150094

    • PubMed
    • Google Scholar
39. 1. E Markiewicz
    2. M Ledran
    3. CJ Hutchison

    (2005) Remodelling of the nuclear Lamina and nucleoskeleton is required for skeletal muscle differentiation in vitro

    Journal of Cell Science 118:409–420.

    https://doi.org/10.1242/jcs.01630

    • PubMed
    • Google Scholar
40. 1. RD Moir
    2. AD Donaldson
    3. M Stewart

    (1991)

    Expression in Escherichia coli of human lamins A and C: influence of head and tail domains on assembly properties and paracrystal formation

    Journal of Cell Science 99 (Pt 2):363–372.

    • PubMed
    • Google Scholar
41. 1. RD Moir
    2. M Yoon
    3. S Khuon
    4. RD Goldman

    (2000) Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells

    The Journal of Cell Biology 151:1155–1168.

    https://doi.org/10.1083/jcb.151.6.1155

    • PubMed
    • Google Scholar
42. 1. O Moiseeva
    2. S Lopes-Paciencia
    3. G Huot
    4. F Lessard
    5. G Ferbeyre

    (2016) Permanent farnesylation of lamin A mutants linked to progeria impairs its phosphorylation at serine 22 during interphase

    Aging 8:366–381.

    https://doi.org/10.18632/aging.100903

    • PubMed
    • Google Scholar
43. 1. N Naetar
    2. S Hutter
    3. D Dorner
    4. T Dechat
    5. B Korbei
    6. J Gotzmann
    7. H Beug
    8. R Foisner

    (2007) LAP2alpha-binding protein LINT-25 is a novel chromatin-associated protein involved in cell cycle exit

    Journal of Cell Science 120:737–747.

    https://doi.org/10.1242/jcs.03390

    • PubMed
    • Google Scholar
44. 1. N Naetar
    2. B Korbei
    3. S Kozlov
    4. MA Kerenyi
    5. D Dorner
    6. R Kral
    7. I Gotic
    8. P Fuchs
    9. TV Cohen
    10. R Bittner
    11. CL Stewart
    12. R Foisner

    (2008) Loss of nucleoplasmic LAP2alpha-lamin A complexes causes erythroid and epidermal progenitor hyperproliferation

    Nature Cell Biology 10:1341–1348.

    https://doi.org/10.1038/ncb1793

    • PubMed
    • Google Scholar
45. 1. N Naetar
    2. S Ferraioli
    3. R Foisner

    (2017) Lamins in the nuclear interior - life outside the Lamina

    Journal of Cell Science 130:2087–2096.

    https://doi.org/10.1242/jcs.203430

    • PubMed
    • Google Scholar
46. 1. N Naetar
    2. R Foisner

    (2009) Lamin complexes in the nuclear interior control progenitor cell proliferation and tissue homeostasis

    Cell Cycle 8:1488–1493.

    https://doi.org/10.4161/cc.8.10.8499

    • PubMed
    • Google Scholar
47. 1. J Norrander
    2. T Kempe
    3. J Messing

    (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis

    Gene 26:101–106.

    https://doi.org/10.1016/0378-1119(83)90040-9

    • PubMed
    • Google Scholar
48. 1. A Oldenburg
    2. N Briand
    3. AL Sørensen
    4. I Cahyani
    5. A Shah
    6. JØ Moskaug
    7. P Collas

    (2017) A lipodystrophy-causing lamin A mutant alters conformation and epigenetic regulation of the anti-adipogenic MIR335 locus

    Journal of Cell Biology 216:2731–2743.

    https://doi.org/10.1083/jcb.201701043

    • PubMed
    • Google Scholar
49. 1. S Osmanagic-Myers
    2. T Dechat
    3. R Foisner

    (2015) Lamins at the crossroads of mechanosignaling

    Genes & Development 29:225–237.

    https://doi.org/10.1101/gad.255968.114

    • PubMed
    • Google Scholar
50. 1. U Pilat
    2. T Dechat
    3. AT Bertrand
    4. N Woisetschläger
    5. I Gotic
    6. R Spilka
    7. K Biadasiewicz
    8. G Bonne
    9. R Foisner

    (2013) The muscle dystrophy-causing δk32 lamin A/C mutant does not impair the functions of the nucleoplasmic lamin-A/C-LAP2α complex in mice

    Journal of Cell Science 126:1753–1762.

    https://doi.org/10.1242/jcs.115246

    • PubMed
    • Google Scholar
51. 1. FA Ran
    2. PD Hsu
    3. J Wright
    4. V Agarwala
    5. DA Scott
    6. F Zhang

    (2013) Genome engineering using the CRISPR-Cas9 system

    Nature Protocols 8:2281–2308.

    https://doi.org/10.1038/nprot.2013.143

    • PubMed
    • Google Scholar
52. 1. RA Röber
    2. K Weber
    3. M Osborn

    (1989)

    Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study

    Development 105:365–378.

    • PubMed
    • Google Scholar
53. 1. MI Robson
    2. JI de Las Heras
    3. R Czapiewski
    4. P Lê Thành
    5. DG Booth
    6. DA Kelly
    7. S Webb
    8. ARW Kerr
    9. EC Schirmer

    (2016) Tissue-Specific gene repositioning by muscle nuclear membrane proteins enhances repression of critical developmental genes during myogenesis

    Molecular Cell 62:834–847.

    https://doi.org/10.1016/j.molcel.2016.04.035

    • PubMed
    • Google Scholar
54. 1. V Roukos
    2. G Pegoraro
    3. TC Voss
    4. T Misteli

    (2015) Cell cycle staging of individual cells by fluorescence microscopy

    Nature Protocols 10:334–348.

    https://doi.org/10.1038/nprot.2015.016

    • PubMed
    • Google Scholar
55. 1. AE Rusiñol
    2. MS Sinensky

    (2006) Farnesylated lamins, progeroid syndromes and farnesyl transferase inhibitors

    Journal of Cell Science 119:3265–3272.

    https://doi.org/10.1242/jcs.03156

    • PubMed
    • Google Scholar
56. 1. T Shimi
    2. K Pfleghaar
    3. S Kojima
    4. CG Pack
    5. I Solovei
    6. AE Goldman
    7. SA Adam
    8. DK Shumaker
    9. M Kinjo
    10. T Cremer
    11. RD Goldman

    (2008) The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription

    Genes & Development 22:3409–3421.

    https://doi.org/10.1101/gad.1735208

    • PubMed
    • Google Scholar
57. 1. I Solovei
    2. AS Wang
    3. K Thanisch
    4. CS Schmidt
    5. S Krebs
    6. M Zwerger
    7. TV Cohen
    8. D Devys
    9. R Foisner
    10. L Peichl
    11. H Herrmann
    12. H Blum
    13. D Engelkamp
    14. CL Stewart
    15. H Leonhardt
    16. B Joffe

    (2013) LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation

    Cell 152:584–598.

    https://doi.org/10.1016/j.cell.2013.01.009

    • PubMed
    • Google Scholar
58. 1. RL Steen
    2. M Beullens
    3. HB Landsverk
    4. M Bollen
    5. P Collas

    (2003) AKAP149 is a novel PP1 specifier required to maintain nuclear envelope integrity in G1 phase

    Journal of Cell Science 116:2237–2246.

    https://doi.org/10.1242/jcs.00432

    • PubMed
    • Google Scholar
59. 1. RL Steen
    2. P Collas

    (2001) Mistargeting of B-type lamins at the end of mitosis: implications on cell survival and regulation of lamins A/C expression

    The Journal of Cell Biology 153:621–626.

    https://doi.org/10.1083/jcb.153.3.621

    • PubMed
    • Google Scholar
60. 1. J Swift
    2. IL Ivanovska
    3. A Buxboim
    4. T Harada
    5. PC Dingal
    6. J Pinter
    7. JD Pajerowski
    8. KR Spinler
    9. JW Shin
    10. M Tewari
    11. F Rehfeldt
    12. DW Speicher
    13. DE Discher

    (2013) Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation

    Science 341:1240104.

    https://doi.org/10.1126/science.1240104

    • PubMed
    • Google Scholar
61. 1. Y Turgay
    2. M Eibauer
    3. AE Goldman
    4. T Shimi
    5. M Khayat
    6. K Ben-Harush
    7. A Dubrovsky-Gaupp
    8. KT Sapra
    9. RD Goldman
    10. O Medalia

    (2017) The molecular architecture of lamins in somatic cells

    Nature 543:261–264.

    https://doi.org/10.1038/nature21382

    • PubMed
    • Google Scholar
62. 1. B van Steensel
    2. AS Belmont

    (2017) Lamina-Associated domains: links with chromosome architecture, Heterochromatin, and gene repression

    Cell 169:780–791.

    https://doi.org/10.1016/j.cell.2017.04.022

    • PubMed
    • Google Scholar
63. 1. S Vidak
    2. N Kubben
    3. T Dechat
    4. R Foisner

    (2015) Proliferation of progeria cells is enhanced by lamina-associated polypeptide 2α (LAP2α) through expression of extracellular matrix proteins

    Genes & Development 29:2022–2036.

    https://doi.org/10.1101/gad.263939.115

    • PubMed
    • Google Scholar
64. 1. A Vivante
    2. E Brozgol
    3. I Bronshtein
    4. V Levi
    5. Y Garini

    (2019) Chromatin dynamics governed by a set of nuclear structural proteins

    Genes, Chromosomes and Cancer 58:437–451.

    https://doi.org/10.1002/gcc.22719

    • PubMed
    • Google Scholar
65. 1. S Vlcek
    2. H Just
    3. T Dechat
    4. R Foisner

    (1999) Functional diversity of LAP2alpha and LAP2beta in postmitotic chromosome association is caused by an alpha-specific nuclear targeting domain

    The EMBO Journal 18:6370–6384.

    https://doi.org/10.1093/emboj/18.22.6370

    • PubMed
    • Google Scholar
66. 1. GE Ward
    2. MW Kirschner

    (1990) Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C

    Cell 61:561–577.

    https://doi.org/10.1016/0092-8674(90)90469-U

    • PubMed
    • Google Scholar
67. 1. AM Wood
    2. JM Rendtlew Danielsen
    3. CA Lucas
    4. EL Rice
    5. D Scalzo
    6. T Shimi
    7. RD Goldman
    8. ED Smith
    9. MM Le Beau
    10. ST Kosak

    (2014) TRF2 and lamin A/C interact to facilitate the functional organization of chromosome ends

    Nature Communications 5:5467.

    https://doi.org/10.1038/ncomms6467

    • PubMed
    • Google Scholar
68. 1. SH Yang
    2. HJ Jung
    3. C Coffinier
    4. LG Fong
    5. SG Young

    (2011) Are B-type lamins essential in all mammalian cells?

    Nucleus 2:562–569.

    https://doi.org/10.4161/nucl.2.6.18085

    • PubMed
    • Google Scholar
69. 1. M Zhang
    2. H Chang
    3. Y Zhang
    4. J Yu
    5. L Wu
    6. W Ji
    7. J Chen
    8. B Liu
    9. J Lu
    10. Y Liu
    11. J Zhang
    12. P Xu
    13. T Xu

    (2012) Rational design of true monomeric and bright photoactivatable fluorescent proteins

    Nature Methods 9:727–729.

    https://doi.org/10.1038/nmeth.2021

    • PubMed
    • Google Scholar

Acceptance summary:

This manuscript describes new tools to investigate the attributes specific to nucleoplasmic versus lamina-integrated A-type lamins. Based on their findings, the authors develop a new model in which LAP2α influences the conformational state of A-type lamins, antagonizing stable lamin A filament assembly with ramifications for chromatin mobility.

Decision letter after peer review:

Thank you for submitting your article "LAP2α maintains a mobile and low assembly state of A-type lamins in the nuclear interior" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Megan C King as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Suzanne Pfeffer as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

This work builds on prior studies by the Foisner group that investigated the function(s) of the soluble A-type lamin binding protein, LAP2α. 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 LAP2α. 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 LAP2α 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 LAP2α-null cells, albeit in a different state. The authors provide evidence that LAP2α 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 LAP2α 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 LAP2α 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 LAP2α. The reviewers also made specific suggestions for a small number of experimental revisions as well as further editing of the text to improve clarity and to provide greater context.

Essential revisions:

1) Structure of the Results. 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 LAP2α 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. While the reviewers appreciate that resolving this conflict leads to a new perspective – namely that LAP2α influences the state of the lamin assembly in a way that disrupts its detection by the N18 antibody, structuring the manuscript to get to this point as quickly as possible would improve its accessibility.

2) Suggested edits to clarify the manuscript. Although the reviewers appreciated the transparency of the authors in demonstrating their workflow and quality control measures, some of the terminology made the manuscript difficult to read. For example, the manuscript would be easier to understand if cell lines were given descriptive names (e.g.: 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: while presenting biological replicate data as in Figure 1 is appreciated, it was challenging to ascertain that the second and third columns in panels A and B were biological replicates. The authors could improve clarity by presenting one biological replicate in the main text with additional replicate(s) 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.

3) Alternative explanations. Further comment and/or clarification is necessary when discussing the state of nucleoplasmic lamin polymerization and how the dynamic measurements reflect lamin assembly state versus anchorage to other lamin binding proteins. For example, although the nuclear lamina filaments are typically 200-400 nm in length, they are also very flexible. A 200 nm filament would have a molecular weight of <1.4MDa ( ~50% of a ribosome) and can be bent and curved. As a consequence, a single filament would be expected to have 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 rather than assembly state as the diffusion of even a 1.4 MDa protein complexes is quite fast. Thus, the authors need to be careful in their interpretation as nucleoplasmic lamins may be polymerized but more mobile (less anchored) than their lamina-bound lamin population without a change in assembly state. Along these lines, greater explanation for the apparent paradox between the increase in immobile fraction by FRAP and the increased diffusion coefficient by FCS in the LAP2α-depleted condition is needed. The authors suggest that the latter is due to the loss of LAP2α binding (subsection “LAP2α loss changes properties of nucleoplasmic lamin A/C rendering them less mobile and more resistant to biochemical extraction”), but some modeling would go a long way here. What form are the lamins thought to be in, and how does the bulk that LAP2α would bring match the apparent changes in diffusivity?

4) Edited clones. It was noted that the authors chose to disrupt LAP2α at the beginning of exon 4, but the reason for this was unclear. For the readers' clarity, can the authors include the size of exons 1 and 2 in the schematic in the supplementary figures? Can the authors provide evidence that the "LAP2α KO" cells used in the experiments do not express a truncated LAP2α protein that could influence the results and interpretation?

5) Concerns over whether lamin tagging may influence the results. 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 (subsection “LAP2α-deficiency does neither affect the formation nor the steady state level of endogenous lamin A in the nuclear interior”) 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? One approach we suggest is 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. 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, particularly as 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. Further, in the subsection “LAP2α loss changes properties of nucleoplasmic lamin A/C rendering them less mobile and more resistant to biochemical extraction”, 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. The authors' statements would be strengthened if the LAP2α KO cells from Figure 1 were stained with both the 3A6 antibody and the N18 antibody to determine whether untagged lamins behave the same way as tagged lamins. More generally, including experiments that allow the reader to compare directly between a cell line with all lmna alleles tagged and a cell line with no lmna alleles tagged is important.

6) Implications for regulation of A-type lamin assembly. 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 (e.g. cyclical stretch, for example)? Have the A-type lamins in the nucleoplasm been found to be in a different assembly state in different cell types? Similarly, one prediction that arises from the proposed model is that regulation of LAP2α 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? How do the authors think the membrane integrated LAP2β fits into the story? Any further insight provided would greatly help to address biological mechanism.

7) Lamin assembly mechanism. 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? The authors suggest that Lap2α keeps lamin in a less polymerized state based in part on the findings that Lap2α inhibits lamin A assembly in vitro. However, 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. This suggests 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 should discuss these views in the revised manuscript. Greater use of the ΔK32 mutant was also suggested, specifically as related to Figure 1, where the data on this assembly mutant should be included in the primary figures and with regards to the sensitivity of the N18 antibody – does this detect the ΔK32 mutated lamin A? Could this provide further insight into the impact of LAP2α by extension?

8) Chromatin dynamics. Over what time interval was the volume of movement for the telomeres 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/Lap2α DKO (Figure 6 right panel) significant?

Essential revisions:

1) Structure of the Results. 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 LAP2α 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. While the reviewers appreciate that resolving this conflict leads to a new perspective – namely that LAP2α influences the state of the lamin assembly in a way that disrupts its detection by the N18 antibody, structuring the manuscript to get to this point as quickly as possible would improve its accessibility.

We understand the concerns of the reviewers and have now rewritten the Abstract and the last paragraph in the Introduction to more clearly point out the aims of this study and the surprising finding that led to a new adapted model of LAP2α function in the regulation of nucleoplasmic lamins. In addition, we streamlined the text describing the formation of the nucleoplasmic lamin pool in the Results (Figures 1 and 2) and changed the focus more towards the effect of LAP2α rather than the pathways involved in the formation of lamins A/C in the nuclear interior.

We also considered rearranging the figures, but after extensive discussion we prefer to keep the current order of figures, as rearrangements would make the structure of the manuscript less clear and disturb the logical flow of the text and experimental set up (e.g. ectopic proteins versus endogenously labeled lamin A/C). We are confident that with the included text changes we can best address the reviewer’s comments, while maintaining the order of the figures.

2) Suggested edits to clarify the manuscript. Although the reviewers appreciated the transparency of the authors in demonstrating their workflow and quality control measures, some of the terminology made the manuscript difficult to read. For example, the manuscript would be easier to understand if cell lines were given descriptive names (e.g.: 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: while presenting biological replicate data as in Figure 1 is appreciated, it was challenging to ascertain that the second and third columns in panels A and B were biological replicates. The authors could improve clarity by presenting one biological replicate in the main text with additional replicate(s) 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.

We now use descriptive names for the cell lines throughout the text and in most figures. In figures, where different LAP2α knockout clones generated by different sgRNAs were used, as well as cell clones treated with the same sgRNAs, but still expressing LAP2α, we designated the cells LAP2α KO sgxx and WT sgxx ctrl (see for example Figure 2F and G). These designations are clearly explained in the figure legend of Figure 2. We agree that these changes make it much easier for a broad readership to follow the logical flow of the manuscript.

In order to address the point of the biological replicate, we removed replicate panels in Figure 1A and B. Instead of showing replicate panels in Figure 1, we now include these data in additional video files (Videos 3 and 7).

3) Alternative explanations. Further comment and/or clarification is necessary when discussing the state of nucleoplasmic lamin polymerization and how the dynamic measurements reflect lamin assembly state versus anchorage to other lamin binding proteins. For example, although the nuclear lamina filaments are typically 200-400 nm in length, they are also very flexible. A 200 nm filament would have a molecular weight of <1.4MDa ( ~50% of a ribosome) and can be bent and curved. As a consequence, a single filament would be expected to have 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 rather than assembly state as the diffusion of even a 1.4 MDa protein complexes is quite fast. Thus, the authors need to be careful in their interpretation as nucleoplasmic lamins may be polymerized but more mobile (less anchored) than their lamina-bound lamin population without a change in assembly state. Along these lines, greater explanation for the apparent paradox between the increase in immobile fraction by FRAP and the increased diffusion coefficient by FCS in the LAP2α-depleted condition is needed. The authors suggest that the latter is due to the loss of LAP2α binding (subsection “LAP2α loss changes properties of nucleoplasmic lamin A/C rendering them less mobile and more resistant to biochemical extraction”), but some modeling would go a long way here. What form are the lamins thought to be in, and how does the bulk that LAP2α would bring match the apparent changes in diffusivity?

We toned down the conclusions on lamin polymerization and included alternative explanations based on lamin filament anchorage to explain the observed differences in lamin A mobility (i.e. movement) in LAP2α KO versus WT cells throughout the text and added a paragraph on this topic in the Discussion.

The increase in the immobile fraction in the absence of LAP2α as measured by constant photobleaching suggests that a larger fraction of intranuclear lamins are immobile, likely because they are bound to an entity that prevents its motion (e.g. chromatin or larger lamin filament assemblies in the nucleoplasm). These immobile structures cannot be measured by FCS, as their fluorescence intensity is not fluctuating. Hence, FCS data refer exclusively to the unbound, mobile fraction of lamin A, which is reduced (but still present) in LAP2α knockout cells. This mobile lamin A/C fraction may be diffusing faster as the complexes without LAP2α are smaller than in WT cells. In the manuscript we explain the results more clearly and give an estimation of a potential correlation of diffusion coefficient changes with changes in the molecular mass of the remaining dynamic lamin complex in the absence of LAP2α. The exact form these mobile lamin-LAP2α complexes are in is unknown. For the calculations, we are assuming the simplest form with one molecule each present in the complexes. Notably, our unpublished data suggest a stoichiometry of 1:1 or 1:2 for soluble lamin A/C-LAP2α complexes.

4) Edited clones. It was noted that the authors chose to disrupt LAP2α at the beginning of exon 4, but the reason for this was unclear. For the readers' clarity, can the authors include the size of exons 1 and 2 in the schematic in the supplementary figures? Can the authors provide evidence that the "LAP2α KO" cells used in the experiments do not express a truncated LAP2α protein that could influence the results and interpretation?

We now clarify in the figure legend that exon 4 is the first and only LAPα-specific exon in the LAP2 gene, while exons 1-3 are common to all LAP2 isoforms. Thus, generating a LAP2α-specific knockout without disturbing expression of the other isoforms requires targeting the beginning of exon 4. To address the reviewer’s specific point, we also added exons 1 to 3 in the schematic drawings of Figure 1—figure supplement 1 and Figure 2—figure supplement 2.

As for a truncated LAP2α fragment, one LAP2α KO clone (sg3) indeed expresses low levels of a small LAP2α fragment (see Figure 2—figure supplement 2D), but we mostly used sg1 clones for the analyses, which do not contain a fragment (Figure 2—figure supplement 2D). In experiments, where sg3 was used (data in Figure 2E-G, Figure 3—figure supplement 1A), the respective data are clearly labeled and explained in the legend, and we included a heterozygous clone that was treated with sg3 (former clone sg3-2, now labeled with WT sg3 ctrl) as control, expressing LAP2α from 1 allele and a short truncated LAP2α fragment from another targeted allele (see Figure 2—figure supplement 2C and D). WT sg3 ctrl expresses full length LAP2α at WT levels (Figure 2B).

5) Concerns over whether lamin tagging may influence the results. 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 (subsection “LAP2α-deficiency does neither affect the formation nor the steady state level of endogenous lamin A in the nuclear interior”) 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? One approach we suggest is 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. 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, particularly as 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. Further, in the subsection “LAP2α loss changes properties of nucleoplasmic lamin A/C rendering them less mobile and more resistant to biochemical extraction”, 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. The authors' statements would be strengthened if the LAP2α KO cells from Figure 1 were stained with both the 3A6 antibody and the N18 antibody to determine whether untagged lamins behave the same way as tagged lamins. More generally, including experiments that allow the reader to compare directly between a cell line with all lmna alleles tagged and a cell line with no lmna alleles tagged is important.

The clone used in most experiments (mEos3.2-Lmna WT clone #21, see Figure 2—figure supplement 1D and F) is tetraploid (as determined from sequencing data and TIDE analysis, see Figure 2—figure supplement 2B and C) and expresses tagged lamin A/C from 1 allele and untagged lamin A/C from 3 alleles (see Figure 2—figure supplement 1G for ratio of tagged versus untagged lamin A mRNA and protein levels in newly added protein quantification).

There may be a certain influence of the tag on protein stability, as the ratio of tagged to untagged lamin A/C protein is shifted towards approx. 1:8 on the protein level (Figure 2—figure supplement 1G), but functions and properties of tagged lamin A and C seem to be unaffected. In the manuscript, we refer to the correct localization of tagged lamin A (Figure 2C), to the stable integration into the lamina by FRAP (former Figure 2—figure supplement 1H, now moved to Figure 2 as new panel D), normal post-mitotic lamina assembly (shown in Figure 2) and similar biochemical properties, such as solubility in salt/detergent buffer (newly added quantification of solubility in Figure 2—figure supplement 1G, right panel) (subsection “LAP2α-deficiency does neither affect the formation nor the steady state level of endogenous lamin A in the nuclear interior”).

However, while some mEos3.2-Lmna clones (independent of whether WT lamin A/C was still present or not) had altered nuclear morphology, this often correlated with very low total lamin A/C levels due to indels in some Lmna alleles as also pointed out by the reviewers. We chose two clones expressing only mEos-tagged lamin A/C, one WT and one KO for LAP2α (WT#5 and LAP2α KO#17, see Figure 2—figure supplement 1D and F) for further control experiments, as suggested by the reviewers. The WT clone expressing only tagged lamin A/C has lower total levels of lamin A/C (likely expressed only from 1 allele while other alleles contain indels), but still displays normal nuclear morphology (see Figure 2—figure supplement 1I). Following the suggestion of the reviewers to test the fitness of these cell clones and to confirm that tagged lamin A/C behaves like untagged lamins, we added additional data comparing the parental WT and LAP2α KO cells (no tagged lamins) with the cell clones expressing only tagged lamin A/C regarding cell viability, nuclear morphology, lamin localization and lamina structure (Figure 2—figure supplement 1H and I). Notably, WT#5 cells expressing lower total levels of mEos-tagged lamins A/C displays a slight reduction in cell viability (pointed out in the figure legend), which is likely caused by reduced total lamin levels rather than the tagging of lamins, as LAP2α KO#17 cells expressing higher mEos-lamin A/C levels display normal viability, when compared to parental cells with untagged Lmna alleles (Figure 2—figure supplement 1H).

Additionally, we performed immunofluorescence microscopic analyses of these cells using anti-lamin A/C N18 and 3A6 antibody and confirmed the lack of intranuclear lamin staining in the absence of LAP2α in both, cells without tagged lamins and cell clones expressing only tagged lamins (Figure 3—figure supplement 1C and D). Thus, by all these means cells with only tagged lamins behave as cells with untagged lamins.

We also discuss functionality of tagged lamin A/C now in more detail in the Results section.

6) Implications for regulation of A-type lamin assembly. 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 (e.g. cyclical stretch, for example)? Have the A-type lamins in the nucleoplasm been found to be in a different assembly state in different cell types? Similarly, one prediction that arises from the proposed model is that regulation of LAP2α 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? How do the authors think the membrane integrated LAP2β fits into the story? Any further insight provided would greatly help to address biological mechanism.

In the revised manuscript, we discuss a potential biological function in great detail.

As we have shown previously, LAP2α loss clearly impairs the detection of nucleoplasmic lamins by antibodies in various cell types, including fibroblasts and epidermal and colon progenitor cells (Naetar et al., 2008). Changes in antibody-stained lamin A/C levels in the nuclear interior can also be seen in physiological conditions within cell populations in a cell culture, expressing different levels of LAP2α (see e.g. Figure 1—figure supplement 1E, depicting cells with different levels of LAP2α side by side).

As for a potential biological function and regulation of nucleoplasmic lamins we discuss the following points: While the response of lamin A solubility to mechanical stretch works via lamin A phosphorylation (Buxboim et al., 2014), changes in the detectability of lamin A/C in the nuclear interior during differentiation may be regulated by differentiation-specific downregulation of LAP2α (Markiewicz, Ledran, and Hutchison, 2005; Naetar et al., 2007; Pekovic et al., 2007). Similar effects can be seen in progeria premature aging disease (Vidak et al., 2018; Vidak et al., 2015). We hypothesize that a mobile nucleoplasmic lamin A is important for chromatin regulation and gene expression [see (Bronshtein et al., 2015; Gesson et al., 2016), our unpublished data, and shown in this manuscript in Figure 6], as well as for the regulation of retinoblastoma protein (Dorner et al., 2006) during cell cycle exit and initiation of cell differentiation.

LAP2β was shown to bind lamin B, not lamin A/C (Foisner and Gerace, 1993), while LAP2α binds lamin A/C via a LAP2α-specific domain encoded by exon 4 (Dechat et al., 2000) absent in LAP2β. Thus, we feel that the function of LAP2α regulating lamin A/C in the nuclear interior is unique to the protein and not affected by the other LAP2 isoforms.

7) Lamin assembly mechanism. 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? The authors suggest that Lap2α keeps lamin in a less polymerized state based in part on the findings that Lap2α inhibits lamin A assembly in vitro. However, 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. This suggests 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 should discuss these views in the revised manuscript. Greater use of the ΔK32 mutant was also suggested, specifically as related to Figure 1, where the data on this assembly mutant should be included in the primary figures and with regards to the sensitivity of the N18 antibody – does this detect the ΔK32 mutated lamin A? Could this provide further insight into the impact of LAP2α by extension?

We discuss now in great detail that lamin A assembly may be different in vivo versus in vitro, in view of the data shown in Zwerger et al., 2015, and we also describe alternative models on the assembly state and interactions of nucleoplasmic lamins (see also our response to point 3 above) (Discussion).

In order to highlight the results of the assembly-deficient ΔK32 prelamin A mutant we moved panels showing assembly of this lamin mutant from Figure 1—figure supplement 2 to Figure 1.

Stainings of ΔK32 lamin A mutant by the anti-lamin A/C N18 antibody in LAP2α WT versus KO cells, as mentioned by the reviewer, were already done and shown in one of our previous papers (Pilat et al., 2013). As expected, unlike WT lamin A, the intranuclear staining of the assembly-deficient ΔK32 lamin A by antibody N18 was not impaired in LAP2α KO cells, which would be in support of lamin A assembly being responsible for loss of N18 staining. We add these findings in the Discussion.

8) Chromatin dynamics. Over what time interval was the volume of movement for the telomeres 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/Lap2α DKO (Figure 6 right panel) significant?

Each nucleus is measured for 20.5 minutes. Our extensive experience on measurements in this time range has shown that this is long enough to prevent that measurements are not affected by short-time motion, which may not represent the actual dynamics. [see also (Zada et al., 2019)].

Furthermore, the motion of the nucleus itself (“nuclear position”) is completely controlled during the analysis by performing a drift-correction algorithm to each measured cell. We use a robust algorithm (see for example: (Bronshtein et al., 2015), section methods; and in “Single particle tracking for studying the dynamic properties of genomic regions in live cells“ , I. Bronshtein Berger, E. Kepten and Y. Garini, In: Imaging gene expression Methods and Protocols, edited by Y. Shav-Tal, Springer 2013). Due to the fact that we can track simultaneously (over 20.5 minutes) at least 20-40 telomeres in each cell, and because they are spread in the nucleus volume, the drift-correction algorithm leads to excellent correction of the cell motion. This is done by calculating for each image (at each time point) the “center of gravity” of the nucleus and shifting the coordinates of the nucleus to the same very point. Only then we calculate the dynamics of each telomere, and therefore, the cell motion is not affecting the calculated track of the telomeres and the calculations of the MSD. In addition, we also perform a rotation-correction of each nucleus by using an adequate algorithm that is part of the routine analysis. These two corrections ensure that the MSD that we calculate is accurate and it is not affected by cell drift or rotation.

Regarding the choice of telomeres, we have performed numerous measurements of the dynamics of telomeres in different cell types (MEFs, U2OS and HeLa) while depleting different proteins, and both telomeres and centromeres in WT and lamin A-depleted U20S cells. It is our experience that the dynamics of both types of probes in all these cells behave in a similar manner, i.e. if the telomere MSD reduces, also the centromere MSD reduces and vice versa (Bronshtein et al., 2015). Therefore, we strongly believe that the effect that we measure is not related to the biological properties of the telomeres themselves, and they only serve as ‘beacons’ for observing the general dynamics of chromatin in the nucleus. Possible modifications to the telomeres biology itself that may result from protein binding or other modifications, may change the dynamics of the telomeres on the scale of sub-telomere size, most probably in a 10-20 nm motion range, while the volume of motion that we measure is roughly a sphere with a diameter of ~250-300 nm. Local internal-dynamics of the telomere cannot affect the volume of its motion as a whole, which is what we are measuring.

As requested, for clarifying the data to the readers, we are adding a graph that shows the MSD of telomeres in WT and LAP2α knockout cells to Figure 6 (new panel B). The graph clearly shows that the absence of LAP2α leads to a ‘slower’, more constraint diffusion with lower MSD. These data are correlated with the change in the volume of motion shown in Figure 6A.

We also calculated the corrected p value for comparing Lmna KO and Lmna/Lap2α DKO, which is highly significant (Dunn's multiple comparisons test p < 0.0001 as part of Kruskal-Wallis post tests). Despite the rather small effect size (Cohen’s d=0.3), this suggests that LAP2α has a small, but significant effect on chromatin motion independent of lamin A/C, where its presence further restricts free diffusion of chromatin. This is indicated by the increase in chromatin motion volume in DKO compared to Lmna single KO cells (Figure 6A). These observations were included in the Results and Discussion sections of the manuscript.

References:

Foisner, R., and Gerace, L. (1993). Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation. Cell, 73(7), 1267-1279. doi:10.1016/0092-8674(93)90355-tPekovic, V., Harborth, J., Broers, J. L., Ramaekers, F. C., van Engelen, B., Lammens, M., von Zglinicki, T., Foisner, R., Hutchison, C., and Markiewicz, E. (2007). Nucleoplasmic LAP2alpha-lamin A complexes are required to maintain a proliferative state in human fibroblasts. J Cell Biol, 176(2), 163-172. doi:10.1083/jcb.200606139Vidak, S., Georgiou, K., Fichtinger, P., Naetar, N., Dechat, T., and Foisner, R. (2018). Nucleoplasmic lamins define growth-regulating functions of lamina-associated polypeptide 2alpha in progeria cells. J Cell Sci, 131(3). doi:10.1242/jcs.208462Zada, D., Bronshtein, I., Lerer-Goldshtein, T., Garini, Y., and Appelbaum, L. (2019). Sleep increases chromosome dynamics to enable reduction of accumulating DNA damage in single neurons. Nat Commun, 10(1), 895. doi:10.1038/s41467-019-08806-w

Author details

1. Nana Naetar

   Max Perutz Labs, Center for Medical Biochemistry, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Writing - review and editing

   Contributed equally with

   Konstantina Georgiou

   For correspondence

   nana.naetar@univie.ac.at

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8978-466X

2. Konstantina Georgiou

   Max Perutz Labs, Center for Medical Biochemistry, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria

   Contribution

   Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Nana Naetar

   Competing interests

   No competing interests declared

3. Christian Knapp

   Max Perutz Labs, Center for Medical Biochemistry, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria

   Present address

   ICFO – Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, Castelldefels, Spain

   Contribution

   Software, Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2463-5775

4. Irena Bronshtein

   Physics Department and Nanotechnology Institute, Bar Ilan University, Ramat Gan, Israel

   Contribution

   Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

5. Elisabeth Zier

   Max Perutz Labs, Center for Medical Biochemistry, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

6. Petra Fichtinger

   Max Perutz Labs, Center for Medical Biochemistry, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

7. Thomas Dechat

   Max Perutz Labs, Center for Medical Biochemistry, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria

   Present address

   Ludwig Boltzmann Institute of Osteology at Hanusch Hospital of OEGK and AUVA Trauma Centre Meidling, 1(st) Medical Department Hanusch Hospital, Vienna, Austria

   Contribution

   Conceptualization, Supervision, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3236-7889

8. Yuval Garini

   Physics Department and Nanotechnology Institute, Bar Ilan University, Ramat Gan, Israel

   Present address

   Biomedical Engineering Department, Technion - Israel Institute of Technology, Haifa, Israel

   Contribution

   Conceptualization, Resources, Supervision, Validation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8783-2015

9. Roland Foisner

   Max Perutz Labs, Center for Medical Biochemistry, Medical University of Vienna, Vienna Biocenter Campus (VBC), Vienna, Austria

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Project administration, Writing - review and editing

   For correspondence

   roland.foisner@meduniwien.ac.at

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4734-4647

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