InteractiveFly: GeneBrief

barrier to autointegration factor: Biological Overview | References

BIOLOGICAL OVERVIEW

The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1-domain (LEM-D) proteins that associate with the chromatin and DNA binding protein Barrier-to-autointegration factor (BAF). This study investigated the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only D-emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where D-emerin/Otefin function is essential. Germ cell-specific BAF knockdown causes phenotypes that mirror d-emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that similar to d-emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, these findings provide evidence for a prominent NL partnership between the LEM-D protein D-emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population (Duan, 2020).

The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear membrane. Comprising lamins and hundreds of associated proteins, the NL builds contacts with the genome to regulate transcription, replication and DNA repair. The NL also connects the nucleus with the cytoskeleton, facilitating transduction of regulatory information between cellular compartments. The composition of the NL is cell-type specific, providing a diverse platform for the integration of developmental regulatory signals. Changes in NL structure occur during physiological aging and disease, suggesting that maintenance of NL function is crucial for cellular health and longevity (Duan, 2020).

One prominent family of NL proteins are LEM domain (LEM-D) proteins, named after the founding human members: LAP2, emerin and MAN1. The defining feature of this conserved family is the LEM domain (LEM-D), an ~40 amino acid domain that directly interacts with the metazoan chromatin-binding protein Barrier-to-autointegration factor (BAF, sometimes referred to as BANF1). Purified human BAF directly binds double-stranded DNA, the A-type lamin and histones in vitro, suggesting that BAF also promotes chromatin-NL connections using non-LEM-D-dependent mechanisms. In dividing metazoan cells, regulated formation of complexes between LEM-D proteins, BAF and lamin controls mitotic spindle assembly and positioning, as well as the reformation of the nucleus. In non-dividing metazoan cells, LEM-D proteins and BAF cooperate to tether the genome to the nuclear periphery and form repressed chromatin. These properties highlight central connections between LEM-D proteins and BAF in NL function (Duan, 2020).

Studies in Drosophila melanogaster have begun to define the role of LEM-D proteins and BAF in development. Drosophila has three NL LEM-D proteins that bind BAF, including two emerin orthologues (Emerin/Otefin and Emerin2/Bocksbeutel) and MAN1. Each LEM-D protein is globally expressed during development. Even so, loss of individual NL LEM-D proteins causes different, non-overlapping defects in the several tissues, including the ovaries, testes, wings and the nervous system. These restricted mutant phenotypes reflect functional redundancy among the Drosophila LEM-D proteins, as loss of any two proteins is lethal. Strikingly, phenotypes of the emerin double mutants (otefin-/-; bocksbeutel-/-) phenocopy baf null mutants (Furukawa, 2003). Both baf and the emerin double mutants die before pupation, resulting from decreased mitosis and increased apoptosis of imaginal discs (Barton, 2014; Furukawa, 2003). In contrast, emerin/otefin; MAN1 or emerin2/bocksbeutel; MAN1 die during pupal development, without associated defects in mitosis or apoptosis (Barton, 2014). Together, genetic studies indicate that the Drosophila emerin orthologues and BAF are important partners (Duan, 2020).

This study extend investigations of the Drosophila NL LEM-D and BAF protein partnership. Using a CRISPR generated gfp-baf allele, this study confirmed that BAF is a globally expressed nuclear protein that shows strong enrichment at the NL in diploid cells. Strikingly, this NL enrichment largely depends upon one LEM-D protein, Emerin/Otefin. Prompted by these observations, BAF contributions were studied in the ovary, a tissue where Emerin/Otefin function is essential. In germline stem cells (GSCs), loss of Emerin/Otefin causes a thickening of the NL and reorganization of heterochromatin. These structural nuclear defects are linked to activation of two kinases of the DNA damage response pathway: Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 2 (Chk2). Although oogenesis in emerin/otefin mutants is rescued by loss of these DDR kinases, canonical triggers are not responsible for pathway activation. Instead, ATR and Chk2 activation is linked to defects in NL structure itself (Barton, 2018). Given the roles of BAF in mitotic nuclear envelope formation and repair (Halfmann, 2019; Samwer, 2017; Mehsen, 2018), it was reasoned that checkpoint activation in emerin/otefin mutants might result from altered BAF function. This prediction was tested using germ cell-specific RNA interference (RNAi) to knockdown BAF. This study shows that BAF depletion disrupts NL structure, blocks differentiation and promotes GSC loss, mutant phenotypes that mirror Emerin/Otefin loss. Additionally, mutation of atr or chk2 partially restores germ cell differentiation in the baf mutant background, supporting the possibility that BAF depletion activates the NL checkpoint. Taken together, these findings suggest that Emerin/Otefin plays a dominant role in the enrichment of BAF to the NL and provide evidence that BAF functions with this prominent partner in the maintenance of an adult stem cell population (Duan, 2020).

This study extended in vivo studies of the BAF and LEM-D partnership. Capitalizing on a newly generated gfp-baf allele, this study shows that NL localization of BAF largely depends upon a single LEM-D protein, Emerin/Otefin. Loss of Emerin/Otefin is sufficient to disperse BAF in cells that express the A- and B-type lamins, Emerin2/Bocksbeutel and MAN1 in the NL. These data establish the in vivo existence of a prominent NL partnership between one LEM-D protein and BAF (Duan, 2020).

The basis for the unexpected reliance on Emerin/Otefin is unknown. One possibility is that LEM-Ds have different affinities for BAF. Pairwise alignment of amino acid residues within LEM-Ds shows the highest conservation between Drosophila emerin orthologues (70% similarity; Barton, 2014). Nonetheless, all LEM-Ds are strongly conserved in BAF-binding residues (42% identical, 67% similar). A second possibility is that the interaction of LEM-D proteins with BAF depends upon how a given LEM-D protein assembles into the NL network. Self-association of emerin influences both BAF and lamin binding. Finally, post-translational modifications (PTMs) of LEM-D proteins might impact BAF partnerships. As an example, O-GlcNAcylation modification of emerin affects BAF association, representing a regulated PTM that has the potential to alter NL function in response to nutrient availability. However, such signal-dependent PTMs are likely to be tissue specific, predicting a tissue-restricted, not global, effect on the NL enrichment of BAF. Further studies are needed to resolve the basis for the strong partnership between Emerin/Otefin and BAF (Duan, 2020).

BAF is essential for viability, with dying baf null larvae exhibiting a typical mitotic mutant phenotype that is associated with high levels of apoptosis (Furukawa, 2007). Several observations suggest that loss of NL BAF is not equivalent to complete loss of BAF. First, emerin/otefin null animals are viable, even though there is a global loss of NL BAF. Second, emerin/otefin null animals have lower levels of apoptosis in larval tissues than baf animals, without effects on the development of adult structures. Third, emerin/otefin mutant imaginal disc cells display an unchanged nuclear shape and chromatin architecture (Barton, 2018), whereas these cells are affected in baf mutants (Furukawa, 2003). Based on these data, it is suggested that BAF function at the NL during interphase is not essential. It is predicted that the essential BAF function relates to its contributions in mitosis and depends upon both Drosophila emerin orthologues, as these double mutant animals die with a mitotic mutant phenotype (Duan, 2020).

Effects of mislocalized BAF share features resulting from BAF overexpression in other systems. In emerin/otefin mutant germ cells, BAF dispersal contributes to the aggregation of heterochromatin. Defects in HP1a distribution have also been found in human cells overexpressing BAF or expressing a BAF mutant defective in interacting with NL components. Furthermore, several diseases affecting expression and processing of lamin A alter the distribution of BAF and resemble a BAF overexpression phenotype. Together, these findings support a model in which BAF contributes to the deleterious effects resulting from lamin or LEM-D mutations (Duan, 2020).

BAF is required for maintenance of Drosophila GSCs. Germ cell-specific BAF knockdown caused GSC loss, with remaining GSCs displaying a thickened and irregular NL structure, a phenotype shared with emerin/otefin mutants. These data support a model in which Emerin/Otefin and BAF function together to build NL structure in this cell type. Such a dependence on Emerin/Otefin for NL structure is consistent with limiting levels of the second Drosophila Emerin ortholog, Emerin2/Bocksbeutel (Barton, 2014). It is predicted that, in GSCs, the Emerin/Otefin and BAF might have a shared function in nuclear reformation at the end of mitosis (Duan, 2020).

Activation of the NL checkpoint is linked to NL deformation (Barton, 2018). Strikingly, baf mutant phenotypes are partially suppressed in atr/chk2; nos>bafRNAi animals, with double mutant ovaries showing increased germ cell survival and differentiation. Yet cell death remained in the double mutant backgrounds. Based on these observations, it is predicted that BAF loss in germ cells has multiple consequences. First, NL structure is affected. Second, loss of nuclear BAF might affect transcriptional networks required for GSC maintenance, suggested from studies showing BAF is an epigenetic regulator (Montes de Oca, 2011). Notably, the maintenance of mammalian stem cells also depends on BAF. Knockdown of BAF in either mouse or human embryonic stem cells promoted premature differentiation and reduced survival, phenotypes associated with an altered cell cycle. It remains possible that loss of Drosophila BAF in GSCs perturbs mitosis, which might induce apoptosis. Additional studies are needed to elucidate cell cycle contributions of BAF in GSCs (Duan, 2020).

These studies emphasize the important role of BAF within the NL network. Evidence is presented for consequences of BAF dispersal and loss during development, showing BAF dysfunction causes cell-type specific responses. Further definition of the developmental contributions of BAF will advance understanding of laminopathies, including the Nestor-Guillermo syndrome: a rare hereditary progeroid disorder caused by a missense mutation in BAF/BANF1 (Duan, 2020).

Recruitment of BAF to the nuclear envelope couples the LINC complex to endoreplication

DNA endoreplication has been implicated as a cell strategy to grow in size and in tissue injury. This study demonstrates that barrier to autointegration factor (BAF), represses endoreplication in Drosophila myofibers. This study shows that BAF localization at the nuclear envelope was eliminated either in mutants of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, in which the LEM-domain protein Otefin was similarly excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope required the activity of the BAF kinase VRK1/Ball, and consistently non-phosphorytable BAF-GFP was excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlated with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication was found to overlap BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope resulted with increased E2F1 levels in the nucleoplasm, and subsequent elevated DNA content. It is suggested that LINC-dependent, and phospho-sensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation at the nucleoplasm (Unnikannan, 2020).

Endoreplication emerges as an important strategy of differentiated cells, enabling them to grow in size or rescue tissue integrity following injury, in a wide range of non-dividing cell types. Recent experimental studies have proposed a functional link between mechanical inputs and endoreplication events in various cell types. Moreover, mechanical signals transmitted across the nuclear membrane have been implicated in the regulation of cell cycle, epigenetic events and gene transcription. As part of the mechanism linking cell cycle events with mechanical inputs, the translocation of specific essential factors into the nucleus has been proposed. However, the molecular link between nuclear translocation of such factors, mechanical inputs on the nuclear envelope and endoreplication is still elusive (Unnikannan, 2020).

The linker of nucleoskeleton and cytoskeleton (LINC) complex has been suggested to mediate mechanically induced nuclear entry of essential factors (Driscoll, 2015; Horn, 2014; Osmanagic-Myers, 2015). It physically connects the cytoskeleton and the nucleoskeleton at the interface of the nuclear envelope and has been associated with various human myopathies. The LINC complex is composed of Nesprin protein family members, which associate at their cytoplasmic N-terminal end with distinct cytoskeletal components, and on their nuclear C-terminal end with SUN domain proteins at the perinuclear space. SUN domain proteins bind to various nuclear lamina components, resulting in a physical link between the cytoskeleton and the nucleoskeleton. Recent results indicate that, in Drosophila larval muscles, the LINC complex is essential for arresting endoreplication in the muscle nuclei (myonuclei) and that LINC mutants exhibit additional rounds of DNA replication, resulting in elevated polyploidy (Brayson, 2018; Volk, 2012; Wang, 2018). The molecular nature of this process is currently elusive (Unnikannan, 2020).

In an attempt to reveal the components downstream of the LINC-dependent arrest of DNA endoreplication, a screen was performed for genes whose transcription changes in Drosophila Nesprin/klar mutant muscles. One of the identified genes was barrier-to-autointegration factor (baf), shown to be significantly reduced at the transcription level (Wang, 2018). BAF is a small protein of 89 amino acids that binds dsDNA as well as the nuclear envelope, and in addition forms homodimers. Furthermore, BAF binds to the inner components of the nuclear membrane, including the Lap-2, Emerin, MAN1 (LEM) domain proteins, as well as to lamins A/C and B. Thus, BAF dimers might bridge between dsDNA and the nuclear envelope. Proteomic analysis of BAF partners indicate its potential association with additional proteins, including transcription factors, damage-specific DNA binding proteins and histones. Furthermore, the binding of BAF to its potential partners might be regulated by its phosphorylation state. For example, phosphorylated BAF associates with LEM-domain proteins, whereas de-phosphorylated BAF favors binding to dsDNA. One kinase that has been implicated in BAF phosphorylation is the threonine-serine VRK1 kinase, whose homolog in Drosophila is Ballchen (Ball, also known as NHK-1) (Unnikannan, 2020).

BAF has a crucial role in the condensation and assembly of post-mitotic DNA. Its interaction with both dsDNA and the nuclear lamina enables DNA compaction through cross-bridges between chromosomes and the nuclear envelope, a process essential for the assembly of DNA within a single nucleus following mitosis (Samwer, 2017). Likewise, BAF is recruited to the sites of ruptured nuclear membrane, where it is essential for resealing the ruptured nuclear membrane (Halfmann, 2019). Interestingly, in humans a single amino acid substitution of BAF causes Nestor-Guillermo progeria syndrome (NGPS); however, the molecular basis for the disease awaits further investigation (Unnikannan, 2020).

Previous studies demonstrated that in Drosophila, muscle-specific knockdown of BAF increases the levels of DNA endoreplication, phenocopying the LINC mutant outcome (Wang, 2018). This led to the hypothesis that BAF acts downstream of the LINC complex-dependent mechanotransduction in promoting the arrest of DNA endoreplication in muscle. This study demonstrates that BAF localization at the nuclear envelope is crucial for that process, and that it is downstream of the LINC complex, depends on nucleus-sarcomere connections, and is phosphosensitive. Importantly, elimination of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei and a concomitant increase in E2F1 levels in the nucleoplasm. Taken together, these findings suggest a model in which a LINC-dependent localization of BAF at the nuclear envelope promotes E2F1 tethering to the nuclear envelope to inhibit its accumulation in the nucleoplasm (Unnikannan, 2020).

This study demonstrates the contribution of a novel mechanosensitive component, BAF, in controlling the nuclear accumulation of E2F1, a crucial transcription factor required for the regulation of endoreplication. Whereas previous reports implicated BAF in promoting the condensation and assembly of post-mitotic dsDNA into single nuclei (Samwer, 2017), this study demonstrates that BAF is also essential for the arrest of DNA endoreplication in fully differentiated muscle fibers. Importantly, only BAF that localizes to the nuclear envelope appears to be relevant for this function in post-mitotic differentiated cells. The contribution of BAF to larval muscle functionality is unclear, as baf mutants did not survive up to third instar stage and BAF knockdown in muscles by using RNAi did not eliminate BAF very efficiently (Unnikannan, 2020).

In Drosophila muscle fibers, it was found that BAF was detected in various subcellular sites, including the cytoplasm, nuclear envelope, nucleoplasm and at the nucleolus borders. Yet, only the portion of BAF localized at the nuclear envelope was found to change following elimination of a functional LINC complex. It is well accepted that the LINC complex transmits cytoplasmic mechanical inputs from the cytoskeleton to the nucleoskeleton in various cell types. Moreover, nuclear deformations (from oval into spheroid shape) observed both in larval muscles of LINC complex mutants and in conditions where nuclei detach from the sarcomeres [e.g., Wang (2015) or following Sls knockdown] are indicative of changes in the mechanical inputs applied on the nuclear envelope. Because BAF localization at the nuclear envelope was specifically impaired in both conditions, it is proposed that maintenance of BAF at the nuclear envelope is mechanically sensitive (Unnikannan, 2020).

In control myofibers, BAF exhibited a relatively broad distribution along the outlines of the nuclear envelope, often extending beyond the Lamin C expression domain towards the cytoplasm, overlapping with the nucleus-associated microtubules. This suggested that, in addition to its association with the inner aspects of the nuclear membrane through binding to LEM-domain proteins and Lamin A/C, BAF associates with the outer aspects of the nuclear membrane. Previous experiments indicate that despite its small size BAF does not diffuse passively from the cytoplasm to the nucleus. Furthermore, photobleaching experiments with GFP-BAF indicate that BAF-dependent repair of nuclear ruptures occurs when cytoplasmic BAF, but not nuclear BAF, rapidly associates with the ruptured sites and further recruits LEM-domain proteins to establish membrane sealing (Halfmann, 2019). The authors suggest that their findings are consistent with a dynamic exchange of BAF between cytoplasmic and nuclear pools, where BAF in the cytoplasm primarily responds to mechanical signals. Because the current experiments indicate that BAF phosphorylation is crucial for its maintenance at the nuclear membrane, it is possible that the exchange of BAF localization between the cytoplasm and the nucleus is stabilized by its phosphorylation. The contribution of the LINC complex to BAF association with the nuclear envelope could be either direct (e.g. by binding to components of the LINC complex) or indirect (e.g. through an effect of the LINC complex on the distribution of LEM proteins at the nuclear envelope). The results support the latter model, in which the LINC complex maintains the localization of the LEM protein Otefin at the nuclear envelope to mediate BAF association with the nuclear envelope. Hence, a model is suggested in which the contribution of the LINC complex to BAF localization at the nuclear envelope is through an effect on Otefin localization at the nuclear envelope (Unnikannan, 2020).

Endoreplication has been implicated in a wide variety of differentiated cells in a broad range of species, including human tissues. A link between mechanical tension and endoreplication has been recently suggested. However, the molecular mechanism coupling mechanical tension with the endoreplication process is still elusive. This study found that a key regulator of endoreplication, E2F1, exhibits a specific distribution at the nuclear envelope in fully differentiated myofibers, where it probably resides non-actively. Changes in the mechanical environment of the nuclear envelope correlate with the localization of E2F1 and promote its accumulation within the nucleoplasm, where it is expected to promote DNA synthesis. It will be of interest to find which proteins associate directly with E2F1 at the nuclear envelope. Attempts to co-immunoprecipitate BAF with Msp300 or E2F1 failed to show a specific protein interaction between these proteins. From a physiological point of view, no detectable changes in muscle size or movement were observed in the baf knockdown muscles, and the larvae developed up to adult stage. The baf homozygous mutant did not develop up to the third instar larval stage, so the full physiological contribution of BAF to muscle growth awaits experiments in which a more efficient reduction in BAF levels is induced in muscle tissue (Unnikannan, 2020).

In summary, these results reveal a novel insight into the role of the LINC complex in coupling endoreplication with changes in the nuclear envelope composition in mature muscle fibers. In particular, the mechanosensitive component, BAF, whose localization at the nuclear envelope is tightly regulated by the LINC complex, is shown to negatively control the nuclear accumulation of the cell cycle regulator E2F1 at the level of the nuclear envelope. The localization of Otefin in the nuclear envelope and BAF phosphorylation by Ball kinase are both crucial in this context. This process might be part of a mechanosensitive pathway that regulates polyploidy in a wide variety of differentiated cells (Unnikannan, 2020).

A fraction of barrier-to-autointegration factor (BAF) associates with centromeres and controls mitosis progression

Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. This study shows that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression (Torras-Llort, 2020).

Cell division involves major architectural rearrangements. Metazoa generally undergo open mitosis, which implies that the nuclear envelope (NE) disassembles at prometaphase and reassembles in telophase, after chromosome segregation is completed. A principal player in the regulation of NE dynamics during mitosis is barrier-to-autointegration factor (BAF). BAF is an essential 10 kDa chromatin-binding protein that is highly conserved in metazoan, being involved in multiple pathways including nuclear envelope reassembly (NER), chromatin epigenetics, DNA damage response, and defense against viral DNA infection. Of great importance for its role in the regulation of NE dynamics, BAF interacts with the LEM-domain containing proteins LAP2, EMERIN, and MAN that, together with lamins, form the nuclear lamina. These interactions help anchoring chromatin to the NE in interphase and, in late mitosis, are essential for the recruitment of membranes to the ensemble of decondensing chromosomes. A still poorly understood contribution of BAF to chromosome segregation has also been reported, since loss of BAF leads to strong chromosome segregation defects and high embryonic lethality in both C. elegans and Drosophila (Torras-Llort, 2020).

Phosphorylation plays a key role in regulating BAF localization and function. The mitotic kinase VRK1/NHK1 phosphorylates BAF in mitosis and meiosis. This phosphorylation weakens the binding of BAF to both chromatin and the LEM-domain proteins, and is required for NE disassembly. BAF plays also a crucial role in postmitotic NER. At mitotic exit, BAF is dephosphorylated and reassociates with chromatin and the LEM-domain proteins, concentrating at the 'core region' that surrounds the bulk of decondensing chromosomes, where its mobility and the mobility of the LEM-domain proteins decrease, and nucleates NER. Two protein phosphatases, PP2A and PP4, have been shown to dephosphorylate BAF in different species. In C. elegans and HeLa cells, PP2A is targeted to BAF by the LEM-domain protein Ankle2/LEM4, which is required for BAF dephosphorylation. Ankle2/LEM4 also associates with VRK1/NHK1 and inhibits its activity, which enhances BAF dephosphorylation5. PP2A-mediated BAF dephosphorylation regulates BAF reassociation with chromatin at mitotic exit and is required for NER. PP4 has also been shown to regulate BAF dephosphorylation during mitosis in HEK293 cells (Torras-Llort, 2020).

This study shows that in Drosophila BAF is also a centromere-associated protein that is required for proper centromere assembly and function. Centromeric BAF (cenBAF) localization depends on the PP4 regulatory subunit Falafel (Flfl), which is recruited to centromeres by the constitutive centromeric protein CENP-C. These results suggest that, together with PP4/Flfl and CENP-C, cenBAF forms a centromeric network that controls phosphorylation and association with chromatin of the bulk of BAF, and regulates mitosis progression (Torras-Llort, 2020).

This study has unveiled a novel centromere-based mechanism that controls mitosis progression. Central to this mechanism is the NE component BAF. A fraction of BAF (cenBAF) associates with centromeres. BAF is known to bind across chromatin in interphase, but, in mitosis, VRK1/NHK1 phosphorylates BAF, resulting in its release from chromatin. These results suggest that, at the centromere, PP4 prevents phosphorylation and release of cenBAF in mitosis. cenBAF is a very small proportion of total BAF. In this regard, the vast majority of BAF is phosphorylated and free in mitosis, resulting in high non-chromosomal background that likely precluded the identification of cenBAF in previous IF studies (Torras-Llort, 2020).

cenBAF forms a functional network with PP4 and CENP-C, as all three factors are interdependent for their centromeric localization. Whether they physically interact to form a centromeric complex remains to be determined. In favor of this possibility, CENP-C interacts directly with Flfl in vitro and, moreover, BAF and CENP-C co-immunoprecipitate, suggesting that, either directly or indirectly, CENP-C also interacts with BAF. Along the same lines, constitutive targeting of BAF to centromeres stabilizes centromeric Flfl as well as CENP-C (Torras-Llort, 2020).

These results suggest that cenBAF stabilizes CENP-C at centromeres and, thus, it is required for accurate chromosome segregation. CENP-C connects centromeric chromatin with the outer kinetochore and loss-of-function mutations induce strong chromosome segregation defects, mostly chromosome misalignment in metaphase. Interestingly, metaphase misalignment is the most frequent chromosome segregation defect observed in BAF-depleted cells, supporting that destabilization of CENP-C is their principal cause. The mechanism by which BAF stabilizes CENP-C at centromeres remains unknown. It is possible that cenBAF modifies centromeric chromatin in a way that stabilizes CENP-C, since BAF has been shown to affect histone modifications and higher-order chromatin organization. It is also possible that the stabilization is through the action of PP4, since cenBAF is required for centromeric localization of Flfl. On the other hand, CENP-C destabilization at centromeres likely involves tension exerted by spindle microtubules since, when centromeric localization of cenBAF and PP4 are impaired in CENP-CΔFIM-expressing cells, CENP-C delocalizes to centrosomes and across the spindle in metaphase chromosomes. The results also show that cenBAF is reciprocally stabilized by CENP-C via the recruitment of Flfl. Altogether these observations suggest that the network of interactions between CENP-C, PP4, and cenBAF forms a positive feedback loop that reinforces assembly of centromeric chromatin and, hence, ensures faithful chromosome segregation. BAF depletion also affected centromeric CENP-ACID levels. This effect is likely a consequence of CENP-C destabilization, since CENP-ACID was reduced to a much lesser extent than CENP-C and it is known that CENP-ACID and CENP-C are interdependent for their centromeric localization (Torras-Llort, 2020).

The small fraction of cenBAF regulates the behavior of the large pool of free pBAF in mitosis. Disrupting cenBAF localization induces PP2A-mediated dephosphorylation of free pBAF in mitosis and the accumulation of BAF in a perichromosomal layer that surrounds chromosomes (see cenBAF, PP4, and CENP-C form a centromeric network that prevents PP2A-mediated dephosphorylation and perichromosomal accumulation of BAF in mitosis). Normally, PP2A is inactivated at the entry into mitosis. Thus, the results suggest that in the absence of cenBAF, PP2A remains active in mitosis. How might cenBAF regulate PP2A activity in mitosis remains to be determined. In this regard, PP4 could play a central role, since the results suggest that it regulates PP2A-mediated pBAF dephosphorylation. Whether the centromere-bound fraction of PP2A39 participates in this regulatory mechanism remains to be determined too (Torras-Llort, 2020).

PP2A selectively dephosphorylates mono- (1pBAF) , but not di-phosphorylated (2pBAF) BAF, suggesting that various phosphatases specifically target pBAF. In this regard, in Drosophila, a second unidentified phosphatase has been proposed to dephosphorylate pBAF at the exit from mitosis. PP4 might be involved in 2pBAF dephosphorylation since, though weakly, Flfl depletion increased 2pBAF levels. Further work is required to clarify the actual phospho-sites in 1pBAF and 2pBAF and the potential site-specific activity of the various phosphatases involved in BAF dephosphorylation (Torras-Llort, 2020).

cenBAF disruption compromises progression through mitosis, delaying anaphase onset (AO) and increasing total mitosis duration. Several factors could contribute to these effects. On one hand, defects in centromere and kinetochore assembly are known to delay or arrest mitosis progression, particularly before AO. Furthermore, altering PP2A activity could impact mitosis in many different ways. In this regard, impaired BAF phosphorylation was shown to affect mitosis progression, since VRK1 depletion in mammalian cells, which also prevents BAF phosphorylation and its release from chromatin during mitosis, delays AO and increases mitosis duration too. Delayed AO could reflect a defect in NEBD since BAF phosphorylation is important to weaken anchoring of chromatin to the NE. On the other hand, exiting mitosis, pBAF dephosphorylation is crucial for NER. Thus, it is also possible that, due to the ectopic activation of PP2A in mitosis, cenBAF disruption induces premature pBAF dephosphorylation and NER. The increased proportion of mitotic cells showing assembled NE, and the persistence of Nup-107::mRFP signal through mitosis, support a contribution of cenBAF to NE disassembly/reassembly. Along the same lines, cenBAF disruption induces strong NE morphological defects. Altered nuclear morphology is widely associated with generic mitotic problems. However, the defects observed upon impairing cenBAF localization are rescued by constitutive targeting of BAF to centromeres, indicating that they are linked to cenBAF disruption. Moreover, BAF mutations that affect its ability to polymerize and cross-bridge distant DNA sites (Samwer, 2017), or when BAF phosphorylation is impeded by VRK1 depletion, induce similar nuclear morphology defects. Altogether these results suggest that cenBAF, although localized at centromeres, participates in the global regulation of the structural rearrangements that the NE undergoes during mitosis. Further work is required to reach a better understanding of this contribution (Torras-Llort, 2020).

In summary, these results suggest that, together with PP4 and CENP-C, cenBAF forms a functional centromeric network that is required for accurate chromosome segregation and controls mitosis progression by regulating PP2A-mediated BAF dephosphorylation. It is tempting to speculate that this network helps to coordinate chromosome segregation with the crucial NE rearrangements that mark mitosis progression. Interestingly, other NE components have also been reported to associate with the centromere/kinetochore during mitosis and contribute to spindle assembly, revealing the strong functional links that exist between the NE and the centromere/kinetochore (Torras-Llort, 2020).

Drosophila female germline stem cells undergo mitosis without nuclear breakdown

Stem cell homeostasis requires nuclear lamina (NL) integrity. In Drosophila germ cells, compromised NL integrity activates the ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 2 (Chk2) checkpoint kinases, blocking germ cell differentiation and causing germline stem cell (GSC) loss. Checkpoint activation occurs upon loss of either the NL protein emerin or its partner barrier-to-autointegration factor, two proteins required for nuclear reassembly at the end of mitosis. This study examined how mitosis contributes to NL structural defects linked to checkpoint activation. These analyses led to the unexpected discovery that wild-type female GSCs utilize a non-canonical mode of mitosis, one that retains a permeable but intact nuclear envelope and NL. The interphase NL is remodeled during mitosis for insertion of centrosomes that nucleate the mitotic spindle within the confines of the nucleus. Depletion or loss of NL components causes mitotic defects, including compromised chromosome segregation associated with altered centrosome positioning and structure. Further, in emerin mutant GSCs, centrosomes remain embedded in the interphase NL. Notably, these embedded centrosomes carry large amounts of pericentriolar material and nucleate astral microtubules, revealing a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that defects in centrosome structure are upstream of checkpoint activation, suggesting that these centrosome defects might trigger checkpoint activation and GSC loss. Connections between NL proteins and centrosome function have implications for mechanisms associated with NL dysfunction in other stem cell populations, including NL-associated diseases, such as laminopathies (Duan, 2021).

Nuclear structure is shaped by proteins resident in the nuclear lamina (NL). This extensive network is composed of lamins and hundreds of lamin-associated proteins that line the inner nuclear membrane. The NL confers nuclear rigidity and contributes to chromatin organization important for regulation of transcription, replication, and DNA repair. Additionally, NL proteins transmit regulatory information between cellular compartments through connections that link the nucleoskeleton with the cytoskeleton. Nuclear structure correlates with cell-type-specific changes in NL composition, differences that impact genome organization and function during development (Duan, 2021).

The LAP2-emerin-MAN1 domain (LEM-D) protein family has a prominent role in the NL. These proteins share an ~40-amino-acid domain (LEM-D) that interacts with Barrier-to-autointegration factor (BAF) (sometimes referred to as BANF1), a conserved chromatin protein. In non-dividing cells, interactions between LEM-D proteins and BAF link the genome with the nuclear periphery. In dividing cells, these interactions control mitotic spindle assembly and positioning, as well as nuclear reassembly at the end of mitosis. These properties highlight mechanisms wherein the LEM-D and BAF partnership contributes to nuclear architecture (Duan, 2021).

Physiological aging and many diseases are associated with changes in nuclear structure. Indeed, misshapen and lobulated nuclei are common features of laminopathies, diseases that result from mutations in genes encoding NL proteins. Laminopathies affect some cell types more than others, with primary defects found in skeletal muscle, skin, fat, and bone. Age-associated worsening of laminopathic diseases has been linked to failures in stem cell maintenance suggesting that the NL plays an important role in balancing stem cell proliferation with differentiation. Although contributions of the NL to stem cell function are being investigated, mechanisms that preserve healthy stem cell populations and promote tissue homeostasis remain poorly understood (Duan, 2021).

Studies in Drosophila melanogaster have identified roles for LEM-D proteins and BAF in adult stem cell maintenance. Drosophila encodes three NL LEM-D proteins that bind BAF, including two emerin orthologs (emerin also known as otefin and emerin2 also known as Bocksbeutel) and MAN1. Notably, loss of emerin compromises homeostasis of adult stem cells in the female and male germlines, blocking germ cell differentiation and causing germline stem cell (GSC) loss. These defects are coupled with GSC-restricted deformation of the NL and accumulation of DNA damage, phenotypes that mirror those found in laminopathic cells. Strikingly, gametogenesis of Drosophila emerin mutant germ cells is rescued by mutation of two DNA damage response (DDR) kinases, the responder kinase ataxia telangiectasia and Rad3-related (ATR) or the transducer kinase checkpoint kinase 2 (Chk2). Although emerin mutant GSCs carry DNA damage, genetic and molecular analyses suggest that ATR and Chk2 activation occurs independently of canonical DNA damage triggers and is linked to the NL structural deformation. Germ-cell-specific BAF depletion also causes NL deformation and GSC loss that is partially rescued by chk2 mutation. These findings indicate that emerin and BAF contribute to shared NL functions needed for GSC maintenance (Duan, 2021).

This study extends an investigations of events associated with activation of the NL checkpoint. Prompted by shared requirements for emerin and BAF in nuclear reassembly, this study examined whether defects in mitosis were responsible for NL deformation and checkpoint activation. To this end, the structure of the NL was followed throughout female GSC (fGSC) mitosis. These analyses led to the unexpected discovery that wild-type fGSCs use a non-canonical mode of mitosis, wherein a permeable but intact nuclear envelope (NE) and remodeled NL remain throughout mitosis. This mode of mitosis imposes requirements for NL components, evidenced by observations that depletion or loss of NL components causes defects in centrosome positioning and spindle structure. In emerin mutant fGSCs, centrosomes remain embedded in the interphase NL and retain large amounts of pericentriolar material (PCM) that nucleates astral microtubules. These observations reveal a role for emerin in the regulation of centrosome structure. Epistasis studies demonstrate that PCM retention in emerin mutant GSCs is upstream of Chk2 activation, indicating that the altered structure of the interphase centrosome is linked with NL checkpoint activation. Based on these data, it is proposed that other stem cells might employ distinct modes of mitosis that sensitize these cells to defects in the NL. These findings have implications for mechanisms associated with NL dysfunction in other systems, including laminopathies (Duan, 2021).

The NL has a central role in establishing structures important for the homeostasis of diverse stem cells. Indeed, survival of Drosophila GSCs depends upon the integrity of the NL, wherein NL deformation is linked to activation of the ATR and Chk2 kinases that leads to GSC loss. This studu investigated mechanisms leading to NL deformation in fGSCs, examining the role of mitosis in shaping the NL (Duan, 2021).

Two main modes of mitosis exist, open and closed. In open mitosis, the NE and NL break down, enabling spindle microtubules nucleated from cytoplasmic centrosomes to capture and segregate chromosomes. In closed mitosis, mitotic spindles are nucleated by spindle pole bodies (centrosome equivalents) that are embedded in a retained NE. It is generally assumed that metazoan cells use open mitosis, whereas fungi use closed mitosis. This assumption is linked with metazoan-limited expression of lamin and BAF, two proteins that are required for nuclear reassembly at the end of open mitosis. However, several exceptions to this rule exist, indicating that open and closed mitoses represent extremes of a continuum of mitotic strategies. A classic example of an exceptional mode of mitosis occurs in the Drosophila early embryo. In these early divisions, limited nuclear breakdown occurs, wherein local NE and NL breakdown occurs near centrosomes, with large portions of the NE and NL remaining until metaphase. This NL has a stabilizing function on spindle microtubules, as disruption of the lamin-B delays prometaphase spindle assembly. However, progression to anaphase requires NL and NE dispersal, as increased stabilization of the lamin-B prevents spindle elongation. These mitotic events indicate that the regulation of NE and NL remodeling optimizes progression through mitosis (Duan, 2021).

Mitotic divisions of Drosophila female germ cells also deviate from open mitosis. In contrast to somatic cells in the ovary that show universal NL dispersal, germ cells change the mode of mitosis depending on developmental stage. In larval PCGs, nuclear breakdown begins in prometaphase, forming large, broken patches of NL, whereas in adult fGSCs, the NL remains intact, even into anaphase. As a result, spindle microtubules form within the mitotic fGSC nucleus, emanating from centrosomes embedded in the NL. Notably, features of this non-canonical mitosis are shared with fungi. For example, in Aspergillus nidulans, the never-in-mitosis kinase partially disassembles NPCs, increasing permeability of an otherwise intact mitotic NE to allow mitotic regulators access to the prophase nucleoplasm. Similarly, it was found that NPCs in mitotic fGSCs are partially disassembled, allowing for exchange of nuclear and cytoplasmic components. However, other features differ between fGCS and fungal mitosis. For example, Saccharomyces cerevisiae carry a centriole-less spindle pole body that is embedded in the NE, which allows nucleation of spindle and astral microtubules within the nuclear compartment. In contrast, the centriole-containing centrosomes of fGCSs move into the NE and NL to promote spindle microtubule assembly within the nucleoplasm. fGCS centrosomes are inserted into a cup-like structure composed of lamin-B and emerin, suggesting that localized remodeling of the NE and NL occurs. Based on these comparisons with fungi, it is proposed that fGSCs employ an intermediate form of mitosis, one that is not completely closed because the NE becomes permeable or completely open because the NE and NL remain intact. The current data add additional evidence that metazoans do not solely employ an open mode of mitosis. Indeed, in Drosophila, modes of mitosis are cell type and developmental stage specific (Duan, 2021).

Genetic studies suggest that NL proteins are required for execution of fGSC mitosis. Loss of emerin or depletion of lamin-B alters the structure and positioning of mitotic spindles. Both NL mutants increase the frequency of lagging chromosomes in anaphase, suggesting that the quality of fGSC mitosis is compromised upon NL dysfunction. Defects in the mitotic spindle might result from disruption of mitotic spindle assembly or mitotic matrix formation, as mammalian lamin B is a structural component of the spindle matrix that promotes microtubule organization in mitosis. Alternatively, mitotic spindle defects might result from an altered distribution of nuclear pores, as centrosome separation in mitotic prophase is linked to nuclear pore distribution. Further studies are needed to address these possibilities (Duan, 2021).

The significance of the developmental switch between PGCs and fGSCs mitosis is unclear. Notably, this switch correlates with the transition from symmetric to asymmetric division, suggesting that retention of a mitotic NL might contribute to acquisition of distinct cell fates that occur within a single cell division. Indeed, fGSC homeostasis is linked to the asymmetric inheritance of two nuclear factors that regulate rRNA transcription and maturation, Wicked/U3 small nucleolar RNA (snoRNA)-associated protein 18 and UnderdevelopedTAF1 (TATA-Box Binding Protein Associated Factor 1). The data indicate that asymmetric trafficking of these proteins occurs within an intact mitotic NL, which might help establish the distinct distribution of these proteins and possibly others. For example, microtubules direct the asymmetric distribution of pSmad to one centrosome for its degradation in cultured human embryonic stem cells. Although niche signaling has a dominant role in fGSC maintenance, it is suggested that retention of a mitotic NL might amplify mechanisms used in asymmetric division (Duan, 2021).

Although emerin and lamin-B are required for fGSC mitosis, only loss of emerin leads to fGSC death. Indeed, oogenesis in nos > lam RNAi females occurs without evidence of Chk2 activation (Duan, 2021).

Although the low levels of lamin-B that remain in nos > lam RNAi fGSCs might be sufficient to guide mitosis, it is also possible that emerin and lamin-B make distinct contributions. Observations that the structure of the interphase centrosome differs in the two mutant backgrounds provide support for the latter possibility. In emerin mutant fGSCs, centrosomes remain embedded in the NL, and these centrosomes retain increased amounts of PCM, defects not observed in nos>RNAi mutants. Embedded centrosomes might contribute to the extensive structural deformation of the NL found in these emerin mutant fGSCs, because the expanded PCM retains γ-tubulin, the major microtubule nucleating component of the PCM.82 As a result, emerin, but not nos > lam RNAi, mutants nucleate astral microtubules in interphase fGSCs. Epistasis studies demonstrate that both PCM expansion and microtubule nucleation remain in chk2, emerin double mutants, implying that these features are independent or upstream of checkpoint activation. It is predicted that differences in interphase centrosome structure are connected to NL checkpoint activation (Duan, 2021).

Mechanisms responsible for expanded PCM in emerin mutants are unknown. Centrosome maturation and disassembly involve regulated activities of kinases that promote PCM expansion and phosphatases that reverse phosphorylation of PCM proteins. Structural defects of the interphase emerin mutant centrosome might originate from incomplete or partial PCM disassembly or from premature recruitment of PCM. Effects on centrosome structure might be direct, as emerin is a component of mitotic centrosomes in Drosophila embryos. This association appears to be conserved, as human emerin is also found in mitotic centrosomes. Further, phosphorylation of Drosophila emerin by Aurora-A kinase is required for mitotic exit in SL2 cells, indicating that emerin has a regulatory function at the centrosome. Alternatively, effects of loss of emerin might be indirect, resulting from gene expression changes that alter levels of mitotic regulators. Regardless of mechanism, the current data suggest that emerin has a role in PCM regulation. Further investigations are needed to test contributions of emerin to the centrosome cycle in fGSC mitosis (Duan, 2021).

ATR and Chk2 kinases localize to mitotic centrosomes. Studies in human cells indicate that ATR associates with γ-tubulin and influences the kinetics of microtubule formation at centrosomes. Localization of ATR to the centrosome provides a link between mitosis and the DDR. However, DDR proteins localize to centrosomes even in the absence of DNA damage, raising the possibility that ATR is a general sensor of structure and function at centrosomes. Building from these observations, it is predicted that the structurally defective centrosome in emerin mutant fGSCs might be responsible for transmitting signals to ATR and Chk2 kinases, ultimately leading to fGSC loss (Duan, 2021).

Mutations in NL LEM-D proteins cause diseases linked to compromised stem cell homeostasis. This study links centrosome dysfunction with failures in stem cell homeostasis due to mutation of the Drosophila NL protein Emerin. These studies align with observations in human fibroblasts that emerin anchors interphase centrosomes to the nucleus through direct interactions with microtubules and that expression of mutant forms of emerin in HeLa cells causes aberrant nuclear shape and mislocalization of tubulin and centrosomes. Taken together, these observations reinforce connections between emerin and centrosomes. As mechanisms of stem cell homeostasis are shared between cell types and organisms, it is possible that other stem cell populations used non-canonical modes of mitosis to ensure robustness of the asymmetric division, which might sensitize division of these cells to defects in the NL composition (Duan, 2021).

The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes

Chromosome condensation during cell division is one of the most dramatic events in the cell cycle. Condensin and topoisomerase II are the most studied factors in chromosome condensation. However, their inactivation leads to only mild defects and little is known about the roles of other factors. This study took advantage of Drosophila oocytes to elucidate the roles of potential condensation factors by performing RNA interference (RNAi). Consistent with previous studies, depletion of condensin I subunits or topoisomerase II in oocytes only mildly affected chromosome condensation. In contrast, severe undercondensation of chromosomes was found after depletion of the Mi-2-containing NuRD nucleosome remodelling complex or the protein kinase NHK-1 (also known as Ballchen in Drosophila). The further phenotypic analysis suggests that Mi-2 and NHK-1 are involved in different pathways of chromosome condensation. The main role of NHK-1 in chromosome condensation is to phosphorylate Barrier-to-autointegration factor (BAF) and suppress its activity in linking chromosomes to nuclear envelope proteins. It was further shown that NHK-1 is important for chromosome condensation during mitosis as well as in oocytes (Nikalayevich, 2015).

This report is the first to use Drosophila oocytes to study chromosome condensation. It is argued that the Drosophila oocyte combined with RNAi is an excellent system for research of chromosome condensation, which complements commonly used mitotic systems. Firstly, Drosophila oocytes grow enormously in volume between completion of pre-meiotic mitosis and recombination and chromosome condensation. shRNA expression can be induced after the protein executes its role in the previous mitosis and/or recombination but prior to oocyte growth. Even if the target protein is stable, it becomes sufficiently diluted before chromosome condensation in oocytes. This is in contrast to mitotic cycles where cells only double in size between divisions. Secondly, Drosophila oocytes arrest in metaphase of the first meiotic division. This allows chromosome defects to be studied in the first division after the target protein is depleted, rather than as a mixture of defects accumulated through multiple divisions caused by a gradual decrease of the protein. Finally, as oocytes are large, the condensation state of chromosomes can be clearly observed without mechanical treatment such as squashing or spreading. Therefore, RNAi in Drosophila oocytes could be a powerful system to study chromosome condensation, although negative results should be interpreted with caution as they might be caused by insufficient depletion, genetic redundancy or cell-type-specific function (Nikalayevich, 2015).

Indeed, in this study, a small-scale survey of chromosomal proteins, new chromosome condensation factors were identified in addition to well-known ones, demonstrating the effectiveness of Drosophila oocytes as a research system. Well-known factors, including condensin I subunits, topoisomerase II and Aurora B, showed milder chromosome condensation defects. Knockdown of topoisomerase II or condensin I showed similar condensation defects, and appeared to affect mainly centromeric and/or pericentromeric regions. The previous reports in mitosis are consistent with this result, suggesting that these two factors are not the main condensation factors in mitosis or in meiosis (Nikalayevich, 2015).

A previous study of Mi-2 in Drosophila suggested that it promotes decondensation of chromosomes because overexpression of wild-type Mi-2 results in chromosome decondensation in polytene or mitotic cells and overexpression of dominant-negative Mi-2 results in overcondensation. In the current study, Mi-2 RNAi in oocytes showed chromosome decondensation, whereas in a preliminary study in neuroblasts Mi-2 RNAi did not show chromosome decondensation. The difference from the previous study might be due to the method of disrupting the Mi-2 function or cell types used for the studies. It is argued that the phenotype caused by RNAi in oocytes is a better reflection of the in vivo function. RNAi of other NuRD subunits indicated that the NuRD complex is important for chromosome condensation (Nikalayevich, 2015).

How does the NuRD complex promote chromosome condensation? It is possible that nucleosome remodelling is directly required during chromosome condensation. For example, proper positioning of nucleosomes might be important for full chromosome condensation. Indeed, other nucleosome remodelling complexes have been suggested to be involved in chromosome condensation in fission yeast. Alternatively, histone deacetylase acivity of the NuRD complex might be important for chromosome condensation, as histone modifications are a major way to regulate chromosome structure. The possibility cannot be excluded that NuRD acts through transcription of other chromosome condensation factors, as it is known to regulate gene transcription. Further studies using more sophisticated mutations would help to distinguish these possibilities (Nikalayevich, 2015).

This study found that knockdown of NHK-1 resulted in severe chromosome condensation defects in nearly all oocytes. Previously, involvement of NHK-1 or its orthologues in metaphase chromosome condensation has not been reported, although overexpression of the human orthologue disrupts chromatin organisation in interphase. None of the three female sterile nhk-1 mutants showed chromosome condensation defects in metaphase I in oocytes. This might be because the minimal NHK-1 activity required for producing viable adults is sufficient to allow chromosome condensation in oocytes. Female-germline-specific RNAi is likely to have achieved greater depletion of NHK-1 in oocytes. This study showed that phosphorylation of BAF, thus inactivating its linking of DNA to LEM-domain-containing inner nuclear membrane proteins, is the major role of NHK-1 in chromosome condensation in oocytes. However, NHK-1 might regulate multiple pathways during condensation, for example, it has been shown that it is required for histone 2A phosphorylation and condensin recruitment in prophase I oocytes (Nikalayevich, 2015).

A crucial question is whether the chromosome condensation defect is a direct consequence of NHK-1 loss or a secondary consequence of a karyosome defect in prophase I oocytes. Evidence indicates that the compact karyosome in the prophase I nucleus and chromosome condensation in metaphase I are at least partly independent. In female-sterile hypomophic nhk-1 mutants, chromatin organisation in prophase I oocytes is defective, but metaphase I chromosomes are properly condensed in mature oocytes (Cullen, 2005; Ivanovska, 2005). By contrast, in Mi-2 RNAi oocytes, the karyosome is normal in prophase I, but chromosomes become undercondensed after nuclear envelope breakdown in some metaphase I oocytes. Furthermore, as chromosome condensation in mitosis is also defective in nhk-1 mutants, the role for NHK-1 in chromosome condensation must be at least partly independent from meiosis-specific chromatin organisation. Therefore, release of LEM-containing nuclear envelope proteins from chromosomes might be a prerequisite for proper chromosome condensation (Nikalayevich, 2015).

In conclusion, this targeted survey using RNAi in Drosophila oocytes has already identified new factors required for chromosome condensation. Further analysis provided new insights into the molecular mechanism of condensation including the release of nuclear envelope proteins from chromosomes and nucleosome remodelling and/or histone deacetylation as essential steps for condensation. In future, a larger scale screen of putative chromosomal proteins might prove to be fruitful (Nikalayevich, 2015).

Unique and shared functions of nuclear lamina LEM domain proteins in Drosophila

The nuclear lamina is an extensive protein network that contributes to nuclear structure and function. LEM domain (LEM-D) proteins are components of the nuclear lamina, identified by a shared ~45 amino acid motif that binds Barrier to Autointegration Factor (BAF), a chromatin interacting protein. Drosophila melanogaster has three nuclear lamina LEM-D proteins, named Otefin (Ote), Bocksbeutel (Bocks) and dMAN1. Although these LEM-D proteins are globally expressed, loss of either Ote or dMAN1 causes tissue-specific defects in adult flies that differ from each other. The reason for such distinct tissue-restricted defects is unknown. This study shows that null alleles of bocks cause no overt adult phenotypes. Phenotypes associated with lem-d double mutants were studied. Although the absence of individual LEM-D proteins does not affect viability, loss of any two proteins causes lethality. Mutant phenotypes displayed by lem-d double mutants differ from baf mutants, indicating that BAF function is retained in animals with a single nuclear lamina LEM-D protein. Interestingly, lem-d double mutants displayed distinct developmental and cellular mutant phenotypes, suggesting that Drosophila LEM-D proteins have developmental functions that are differentially shared with other LEM-D family members. This conclusion is supported by studies showing that ectopically produced LEM-D proteins have distinct capacities to rescue the tissue-specific phenotypes found in single lem-d mutants. These findings predict that cell-specific mutant phenotypes caused by loss of LEM-D proteins reflect both the constellation of LEM-D proteins within the nuclear lamina and the capacity of functional compensation of the remaining LEM-D proteins (Barton, 2014).

PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila

As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. This study conducted a maternal-effect screen in Drosophila melanogaster to identify genes that function with PP2A-B55/Tws in the cell cycle. Eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Mechanistic studies using Drosophila cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes Drosophila as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis (Mehsen, 2018).

The molecular mechanisms mediating an orderly mitotic exit and return into interphase are much less understood than the mechanisms of mitotic entry. Moreover, while phosphatases are known to play crucial roles in promoting the mitosis to interphase transition, their specific contributions to the various events of this process remain largely unknown. This study has used the Drosophila system to search for and dissect the molecular events controlled by the PP2A-B55/Tws phosphatase in the cell cycle. Second-site noncomplementation screens have been used in various model organisms to identify functionally linked genes. This work builds on the power of second-site noncomplementation maternal-effect screens in Drosophila to identify close collaboration between genes in cell cycle regulation (Mehsen, 2018).

The genetic screen uncovered a strong link between PP2A-Tws and NER at the end of M phase. Simultaneously reducing the levels of Tws and Lamin in eggs using heterozygous mutations in mothers causes major defects in NER after meiosis II or after mitosis for embryos that initiated syncytial nuclear divisions. This result is striking considering that Lamin is not an essential protein in several cell types. Hypomorphic lamin mutants develop to adulthood, despite showing nuclear migration defects in photoreceptors and being female sterile. In lamin null mutants, neuroblasts continue to proliferate in the absence of detectable Lamin. In mice, the orthologous B-type lamins are dispensable for cell viability and proliferation, at least in keratinocytes; however, B-type lamins are essential in neurons. In general, B-type lamins may play a crucial role in structuring nuclei and withstanding force in cells where nuclear migration/positioning is essential. Such cell types include Drosophila eggs, where pronuclei must converge before fusing, and syncytial embryos, where nuclei migrate toward the cortex (Mehsen, 2018).

Using cells in culture, this study found that PP2A-Tws promotes the recruitment of several NE components after mitosis, namely BAF, Lamin, and Nup107. In Drosophila oogenesis, BAF phosphorylation by NHK-1 promotes the detachment of chromatin from the germinal vesicle during karyosome formation (Lancaster, 2007). The current work found that BAF requires NHK-1 phosphorylation sites to dissociate from chromatin during NEB, as in C. elegans (Gorjanacz, 2007; Asencio, 2012). The current genetic, biochemical and imaging results suggest that phosphorylation of BAF by NHK-1 is reversed by PP2A-Tws to promote its recruitment on chromatin at the onset of NER. This is consistent with results in C. elegans that showed a role for PP2A in this process, although the relevant phosphorylation sites in BAF were not investigated and the PP2A adaptor subunit involved was unclear (Asencio, 2012). Recent work shows that BAF plays a crucial role in holding chromosomes together just after anaphase to promote the assembly of the NE around a single nucleus (Samwer, 2017). The current findings suggest that PP2A-Tws dephosphorylates BAF to promote this function. The results also suggest that regulation of BAF phosphorylation by NHK-1 and PP2A-Tws regulates its ability to form complexes with Lamin. In vertebrates, BAF is known to interact with lamins via LEM domain proteins at the NE (Schellhaus, 2016). Human BAF phosphorylation by VRK1/NHK-1 decreases its ability to interact with a LEM domain (Nichols, 2006). LEM-domain proteins have also been shown to be phosphorylated to negatively regulate their ability to interact with BAF in X. laevis extracts. Thus, PP2A-B55 could dephosphorylate LEM proteins to further promote their association with BAF during NER, and this possibility should be investigated (Mehsen, 2018).

By inducing the recruitment of BAF on reassembling nuclei, PP2A-Tws likely promotes the recruitment of multiple downstream NE components. Nevertheless, PP2A-Tws likely has other targets in NER, possibly including Lamin and Nup107. Both proteins contain multiple CDK phosphorylation motifs, and PP2A-B55 enzymes have been shown to dephosphorylate many such sites efficiently. Moreover, it was observed that Lamin and Nup107 both associate with Tws. This study found that mutation of all CDK consensus sites in Lamin prevents lamina disassembly in mitosis and, although the attempted phospho-mimetic mutation of all sites did not disrupt lamina assembly in live cells, it increases Lamin solubility in cell lysates. The CDK sites on Lamin are grouped in two clusters flanking the coiled region, and some of these sites have already been shown to negatively regulate homotypic interactions of Lamin. The effect of mutating CDK sites in Nup107 was not examined. However, Nup107 is rapidly dephosphorylated at multiple sites during mitotic exit in human cells and dephosphorylation of at least one CDK site in Nup107 was shown to depend on PP2A-B55. However, although PP2A-B55 is capable of dephosphorylating several CDK sites, many of these sites are probably regulated mainly by another phosphatase in vivo. Moreover, numerous examples of PP2A-B55-dependent, non-CDK sites were recently identified. This is further exemplified in this study by the dephosphorylation of BAF by PP2A-Tws at a NHK-1 site, which cannot be a CDK site, as it lacks the proline residue in position +1. Nevertheless, with its positively charged amino acid residues in positions +2 to +4, this site resembles the recently defined PP2A-B55 consensus motif and a consensus motif for sites lacking a Pro residue at position +1 but that are rapidly dephosphorylated during mitotic exit (Mehsen, 2018).

Overall, PP2A-B55 appears to target multiple proteins, dephosphorylating them at various sites that depend on multiple kinases, to promote NER cooperatively. A recent phosphoproteomic study found that several proteins of the NE are particularly prone to rapid dephosphorylation during mitotic exit, in a process that likely involves other phosphatases. Much work remains to be done to fully dissect the mechanisms at play. The fact that NER is only delayed and not completely prevented when PP2A-Tws is silenced in cell culture could be due to an incomplete inactivation of PP2A-Tws inherent to the RNAi approach. Alternatively, other phosphatases may partially compensate for the loss of PP2A-Tws activity. Protein phosphatase 4 (PP4) may function in this way as it has been shown to dephosphorylate BAF in human cells. In addition, protein phosphatase 1 enzymes likely contribute to NER in Drosophila, as they promote this process through multiple mechanisms in vertebrates, including the dephosphorylation of Lamin B (Mehsen, 2018).

The screen results point at other functions of PP2A-Tws in the completion of M phase that remain to be explored, although some of the genetic interactions identified could reflect roles of PP2A-Tws unrelated to mitotic regulation. Preliminary, unpublished results suggest that the genetic interaction between tws and CycB3 reflects their collaboration in the completion of meiosis. Interestingly, this study uncovered genetic interactions between tws and genes that encode nucleocytoplasmic transport factors. Mutations in the gene for Cse1/CAS, which transports importin α back to the cytoplasm to promote its function in nuclear import, enhances tws-dependent embryonic lethality. Conversely, mutations in embargoed (emb), which encodes the nuclear export factor Crm1, rescues tws-dependent embryonic lethality. These results suggest that active nuclear import plays an important role in NER or other aspects of the establishment of interphase nuclei after mitosis and/or meiosis, presumably by promoting the nuclear localization of crucial enzymes or structural factors. Defining these factors and the regulation of their nucleocytoplasmic transport during mitotic exit should be the topic of future investigations (Mehsen, 2018).

This work has used a genetic strategy to search for the roles of PP2A-Tws in the cell cycle in vivo. PP2A-Tws was found to promote NER, and studies have begun to dissect the mechanisms at play. This study opens the door to the use of Drosophila to gain a better mechanistic understanding of NER at the molecular level. Moreover, it will be a powerful system to further dissect the functions of PP2A-Tws and other phosphatases in the coordination of mitotic exit (Mehsen, 2018).

DNA cross-bridging shapes a single nucleus from a set of mitotic chromosomes

Eukaryotic cells store their chromosomes in a single nucleus. This is important to maintain genomic integrity, as chromosomes packaged into separate nuclei (micronuclei) are prone to massive DNA damage. During mitosis, higher eukaryotes disassemble their nucleus and release individualized chromosomes for segregation. How numerous chromosomes subsequently reform a single nucleus has remained unclear. Using image-based screening of human cells, this study identified barrier-to-autointegration factor (BAF) as a key factor guiding membranes to form a single nucleus. Unexpectedly, nuclear assembly does not require BAF's association with inner nuclear membrane proteins but instead relies on BAF's ability to bridge distant DNA sites. Live-cell imaging and in vitro reconstitution showed that BAF enriches around the mitotic chromosome ensemble to induce a densely cross-bridged chromatin layer that is mechanically stiff and limits membranes to the surface. This study reveals that BAF-mediated changes in chromosome mechanics underlie nuclear assembly with broad implications for proper genome function (Samwer, 2017).

Nearly all eukaryotic cells store their genome in a single nuclear compartment. The genome itself, however, is divided into numerous chromosomes. If individual chromosomes form a separate nucleus (micronucleus), they are prone to DNA damage or even complete chromosome pulverization by chromothripsis. Micronuclei are a common feature of cancer cells and thought to be a major driver in the evolution of cancer genomes. The packaging of all chromosomes into a single nucleus is therefore critical for the maintenance of genome integrity and health (Samwer, 2017).

Cells of higher eukaryotes disassemble their nucleus during mitosis to form individualized chromosomes that move independently on the mitotic spindle. If individual chromosomes attach incorrectly to the mitotic spindle, they can lag behind the mass of segregating anaphase chromosomes and then often package into a separate micronucleus during mitotic exit. Thus, individual chromosomes can, in principle, function as a template for nuclear reformation. Yet, in normally segregating cells, each set of anaphase chromosomes consistently packages into a single nucleus (Samwer, 2017).

During interphase of the cell cycle, various transmembrane proteins link the nuclear envelope (NE) to chromatin either by direct binding to DNA or by binding to adaptor proteins like barrier-to-autointegration factor (BAF). Upon mitotic entry, these linkages are disrupted by protein phosphorylation. Consequently, transmembrane proteins of the NE disperse in the endoplasmic reticulum (ER), while other NE proteins dissolve into the cytoplasm. During mitotic exit, dephosphorylation of these proteins promotes rebinding of ER-derived membranes to chromatin. How nuclear membranes are guided along the surface of a chromosome set to form a single nucleus, rather than enwrapping individual chromosomes remains unknown (Samwer, 2017).

Membrane-chromatin interactions might be limited by the spindle-mediated compaction of chromosomes. The chromokinesin Kid localizes along microtubules within the anaphase chromosome ensemble and Kid knock-out mice display micronucleation during early development. However, the Kid knock-out did not perturb nuclear assembly in cells of adult mice, suggesting the existence of alternative mechanisms that restrict nuclear membranes to the surface of the chromosome ensemble (Samwer, 2017).

The formation of a single nuclear surface might also be explained by a limited amount of membranes that can associate with chromatin. While this is a conceivable hypothesis, experimental support has yet to be corroborated (Samwer, 2017).

To gain insights into the morphogenesis of the nucleus, this study manipulated microtubules and chromosome geometries in human cells. This revealed that neither the mitotic spindle nor limiting amounts of membranes explain the formation of a single nuclear surface. By image-based screening, BAF was identified as a key factor driving formation of a single nucleus in a spindle-independent manner. These investigations in cells and in vitro suggest that BAF shapes a single nucleus through the formation of a dense chromatin network that limits membranes to the surface of the chromosome ensemble (Samwer, 2017).

Repair of nuclear ruptures requires barrier-to-autointegration factor

Cell nuclei rupture following exposure to mechanical force and/or upon weakening of nuclear integrity, but nuclear ruptures are repairable. Barrier-to-autointegration factor (BAF), a small DNA-binding protein, rapidly localizes to nuclear ruptures; however, its role at these rupture sites is unknown. This study shows that it is predominantly a nonphosphorylated cytoplasmic population of BAF that binds nuclear DNA to rapidly and transiently localize to the sites of nuclear rupture, resulting in BAF accumulation in the nucleus. BAF subsequently recruits transmembrane LEM-domain proteins, causing their accumulation at rupture sites. Loss of BAF impairs recruitment of LEM-domain proteins and nuclear envelope membranes to nuclear rupture sites and prevents nuclear envelope barrier function restoration. Simultaneous depletion of multiple LEM-domain proteins similarly inhibits rupture repair. LEMD2 is required for recruitment of the ESCRT-III membrane repair machinery to ruptures; however, neither LEMD2 nor ESCRT-III is required to repair ruptures. These results reveal a new role for BAF in the response to and repair of nuclear ruptures (Halfmann, 2019).

Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis

Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. This study shows that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAF's mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L (Drosophila homolog: Ankle2) interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans (Asencio, 2012).

Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly

Barrier-to-autointegration factor (BAF) is an essential, highly conserved, metazoan protein. BAF interacts with LEM (LAP2, emerin, MAN1) domain-carrying proteins of the inner nuclear membrane. This study analyzed the in vivo function of BAF in Caenorhabditis elegans embryos using both RNA interference and a temperature-sensitive baf-1 gene mutation and found that BAF is directly involved in nuclear envelope (NE) formation. NE defects were observed independent of and before the chromatin organization phenotype previously reported in BAF-depleted worms and flies. Vaccinia-related kinase (VRK) was identified as a regulator of BAF phosphorylation and localization. VRK localizes both to the NE and chromatin in a cell-cycle-dependent manner. Depletion of VRK results in several mitotic defects, including impaired NE formation and BAF delocalization. It is proposed that phosphorylation of BAF by VRK plays an essential regulatory role in the association of BAF with chromatin and nuclear membrane proteins during NE formation (Gorjanacz, 2007).

NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus

Accurate chromosome segregation in meiosis requires dynamic changes in chromatin organization. In Drosophila melanogaster, upon completion of recombination, meiotic chromosomes form a single, compact cluster called the karyosome in an enlarged oocyte nucleus. This clustering is also found in humans; however, the mechanisms underlying karyosome formation are not understood. This study reports that phosphorylation of barrier to autointegration factor (BAF) by the conserved kinase nucleosomal histone kinase-1 (NHK-1; Drosophila Vrk1) has a critical function in karyosome formation. The noncatalytic domain of NHK-1 is crucial for its kinase activity toward BAF, a protein that acts as a linker between chromatin and the nuclear envelope. A reduction of NHK-1 or expression of nonphosphorylatable BAF results in ectopic association of chromosomes with the nuclear envelope in oocytes. It is proposed that BAF phosphorylation by NHK-1 disrupts anchorage of chromosomes to the nuclear envelope, allowing karyosome formation in oocytes. These data provide the first mechanistic insight into how the karyosome forms (Lancaster, 2007).

The vaccinia-related kinases phosphorylate the N' terminus of BAF, regulating its interaction with DNA and its retention in the nucleus

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. This study demonstrates that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N' terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. This study demonstrates that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, these data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture (Nichols, 2006).

Search PubMed for articles about Drosophila Baf

Asencio, C., Davidson, I. F., Santarella-Mellwig, R., Ly-Hartig, T. B., Mall, M., Wallenfang, M. R., Mattaj, I. W. and Gorjanacz, M. (2012). Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis. Cell 150(1): 122-135. PubMed ID: 22770216

Barton, L. J., Wilmington, S. R., Martin, M. J., Skopec, H. M., Lovander, K. E., Pinto, B. S. and Geyer, P. K. (2014). Unique and shared functions of nuclear lamina LEM domain proteins in Drosophila. Genetics 197(2): 653-665. PubMed ID: 24700158

Barton, L. J., Duan, T., Ke, W., Luttinger, A., Lovander, K. E., Soshnev, A. A. and Geyer, P. K. (2018). Nuclear lamina dysfunction triggers a germline stem cell checkpoint. Nat Commun 9(1): 3960. PubMed ID: 30262885

Brayson, D., Ho, C. Y. and Shanahan, C. M. (2018). Muscle tensions merge to cause a DNA replication crisis. J Cell Biol 217(6): 1891-1893. PubMed ID: 29769233

Cullen, C. F., Brittle, A. L., Ito, T. and Ohkura, H. (2005). The conserved kinase NHK-1 is essential for mitotic progression and unifying acentrosomal meiotic spindles in Drosophila melanogaster. J Cell Biol 171(4): 593-602. PubMed ID: 16301329

Driscoll, T. P., Cosgrove, B. D., Heo, S. J., Shurden, Z. E. and Mauck, R. L. (2015). Cytoskeletal to nuclear strain transfer regulates YAP signaling in mesenchymal stem cells. Biophys J 108(12): 2783-2793. PubMed ID: 26083918

Duan, T., Kitzman, S. C. and Geyer, P. K. (2020). Survival of Drosophila germline stem cells requires the chromatin binding protein Barrier-to-autointegration factor. Development. PubMed ID: 32345742

Duan, T., Cupp, R. and Geyer, P. K. (2021). Drosophila female germline stem cells undergo mitosis without nuclear breakdown. Curr Biol. PubMed ID: 33548191

Furukawa, K., Sugiyama, S., Osouda, S., Goto, H., Inagaki, M., Horigome, T., Omata, S., McConnell, M., Fisher, P. A. and Nishida, Y. (2003). Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila. J Cell Sci 116(Pt 18): 3811-3823. PubMed ID: 12902403

Furukawa, K., Aida, T., Nonaka, Y., Osoda, S., Juarez, C., Horigome, T. and Sugiyama, S. (2007). BAF as a caspase-dependent mediator of nuclear apoptosis in Drosophila. J Struct Biol 160(2): 125-134. PubMed ID: 17904382

Gorjanacz, M., Klerkx, E. P., Galy, V., Santarella, R., Lopez-Iglesias, C., Askjaer, P. and Mattaj, I. W. (2007). Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly. EMBO J 26(1): 132-143. PubMed ID: 17170708

Halfmann, C. T., Sears, R. M., Katiyar, A., Busselman, B. W., Aman, L. K., Zhang, Q., O'Bryan, C. S., Angelini, T. E., Lele, T. P. and Roux, K. J. (2019). Repair of nuclear ruptures requires barrier-to-autointegration factor. J Cell Biol 218(7): 2136-2149. PubMed ID: 31147383

Horn, H. F. (2014). LINC complex proteins in development and disease. Curr Top Dev Biol 109: 287-321. PubMed ID: 24947240

Ivanovska, I., Khandan, T., Ito, T. and Orr-Weaver, T. L. (2005). A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes. Genes Dev 19(21): 2571-2582. PubMed ID: 16230526

Lancaster, O. M., Cullen, C. F. and Ohkura, H. (2007). NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus. J Cell Biol 179(5): 817-824. PubMed ID: 18039935

Mehsen, H., Boudreau, V., Garrido, D., Bourouh, M., Larouche, M., Maddox, P. S., Swan, A. and Archambault, V. (2018). PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila. J Cell Biol 217(12): 4106-4123. PubMed ID: 30309980

Montes de Oca, R., Andreassen, P. R. and Wilson, K. L. (2011). Barrier-to-Autointegration Factor influences specific histone modifications. Nucleus 2(6): 580-590. PubMed ID: 22127260

Nichols, R. J., Wiebe, M. S. and Traktman, P. (2006). The vaccinia-related kinases phosphorylate the N' terminus of BAF, regulating its interaction with DNA and its retention in the nucleus. Mol Biol Cell 17(5): 2451-2464. PubMed ID: 16495336

Nikalayevich, E. and Ohkura, H. (2015). The NuRD nucleosome remodelling complex and NHK-1 kinase are required for chromosome condensation in oocytes. J Cell Sci 128(3): 566-575. PubMed ID: 25501812

Osmanagic-Myers, S., Dechat, T. and Foisner, R. (2015). Lamins at the crossroads of mechanosignaling. Genes Dev 29(3): 225-237. PubMed ID: 25644599

Samwer, M., Schneider, M. W. G., Hoefler, R., Schmalhorst, P. S., Jude, J. G., Zuber, J. and Gerlich, D. W. (2017). DNA cross-bridging shapes a single nucleus from a set of mitotic chromosomes. Cell 170(5): 956-972 e923. PubMed ID: 28841419

Schellhaus, A. K., De Magistris, P. and Antonin, W. (2016). Nuclear reformation at the end of mitosis. J Mol Biol 428(10 Pt A): 1962-1985. PubMed ID: 26423234

Torras-Llort, M., Medina-Giro, S., Escudero-Ferruz, P., Lipinszki, Z., Moreno-Moreno, O., Karman, Z., Przewloka, M. R. and Azorin, F. (2020). A fraction of barrier-to-autointegration factor (BAF) associates with centromeres and controls mitosis progression. Commun Biol 3(1): 454. PubMed ID: 32814801

Unnikannan, C. P., Reuveny, A., Grunberg, D. and Volk, T. (2020). Recruitment of BAF to the nuclear envelope couples the LINC complex to endoreplication. Development. PubMed ID: 33168584

Volk, T. (2013). Positioning nuclei within the cytoplasm of striated muscle fiber: cooperation between microtubules and KASH proteins. Nucleus 4(1): 18-22. PubMed ID: 23211643

Wang, S., Stoops, E., Cp, U., Markus, B., Reuveny, A., Ordan, E. and Volk, T. (2018). Mechanotransduction via the LINC complex regulates DNA replication in myonuclei. J Cell Biol 217(6): 2005-2018. PubMed ID: 29650775

date revised: 5 November 2023

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