Nucleoskeleton proteins for nuclear dynamics

Abstract

The eukaryotic nucleus shows organized structures of chromosomes, transcriptional components and their associated proteins. It has been believed that such a dense nuclear environment prevents the formation of a cytoskeleton-like network of protein filaments. However, accumulating evidence suggests that the cell nucleus also possesses structural filamentous components to support nuclear organization and compartments, which are referred to as nucleoskeleton proteins. Nucleoskeleton proteins including lamins and actin influence nuclear dynamics including transcriptional regulation, chromatin organization and DNA damage responses. Furthermore, these nucleoskeleton proteins play a pivotal role in cellular differentiation and animal development. In this commentary, we discuss how nucleoskeleton-based regulatory mechanisms orchestrate nuclear dynamics.

In the 1970s, nuclear proteins that remained after the removal of chromatin components with high-salt buffer extraction were identified (1) and the identification of the residual protein fractions led to the idea of nuclear matrix, also referred to as the nucleoskeleton (2). Later, nuclear intermediate filament proteins, named lamins, were discovered as a nucleoskeleton component (3, 4), followed by the identification of titin, actin, myosin, etc. (5). Nucleoskeleton proteins play a major role in maintaining the mechanical property of not only the nucleus but also the whole cell (5). Furthermore, the nucleoskeleton is mechanically coupled to the cytoskeleton through the linkers of the nucleoskeleton to the cytoskeleton (LINC) complex (6) and this interaction is important for mechanotransduction. Apart from these functions, considerable attention is currently being focused on whether dynamic regulations of nucleoskeleton proteins, such as the polymerization of actin, are involved in nuclear events (Fig. 1).

Actin protein exists in the monomeric (G-actin) or polymeric filamentous form (F-actin), and functions in many important biological processes in the cytoplasm including cell shape regulation and contraction by dynamically exchanging its polymerized state. Recent studies consolidate the idea that dynamics of actin polymerization in the nucleus also profoundly affect cellular functions. The Grosse lab developed a probe for visualizing nuclear actin dynamics in a living cell, referred to as nuclear actin chromobody-GFP (nAC-GFP) (7). Using this probe, they have shown that nuclear actin undergoes a dynamic assembly process when cells are divided after mitosis (8). Importantly, this transient formation of nuclear F-actin ensures nuclear expansion and chromatin decompaction (8). Furthermore, the signalling pathway that governs nuclear F-actin formation has been revealed (9). Thus, like in the case of the cytoskeleton, actin organizes the nucleus by altering its polymerized states. In this issue, Fuderer et al. further extend their previous findings that the elevation of nuclear calcium levels induces transient actin filament assembly (9) by developing the monitoring system of rapid nuclear calcium transients, characterizing the calcium-triggered dynamic assembly/disassembly processes of nuclear actin (10).

Another important feature of actin is that it has been identified as a component of a variety of chromatin remodelling factors (CRFs) and transcription machineries (11, 12). Pioneering works by Percipalle and othersidentified that nuclear actin interacts with all three RNA polymerase (RNAP) complexes and nascent transcripts (13–18). Recent studies demonstrated that nuclear actin is critical for Pol II clustering for transcriptional activation upon serum stimulation (19). Chromatin remodelling complexes also contain actin and actin-related proteins (ARPs) (11, 20). Among the chromatin remodelling complexes that contain actin, the remodelling activity of the Brahma-associated factor (BAF) complex might be affected by nuclear actin (21). The Percipalle group found that nuclear beta-actin is important for the chromatin-binding activity of the BAF complex to its target sites for gene expression regulation by taking advantage of beta-actin knockout cells (22, 23). They summarize our current understanding on nuclear actin’s functions in chromatin remodelling and regulation of gene expression in this special issue (24). In addition, they propose a novel concept of ‘mitoskeleton’, in which actin plays a role in the regulation of mitochondrial genes.

Nuclear actin and its dynamics thus influence chromatin structures and gene expression. The regulation of gene expression is spatiotemporally controlled during animal development. It is therefore plausible that the regulation of nuclear actin dynamics could be a part of developmental programmes that entail reprogramming of germ cells after fertilization and differentiation of embryos. Indeed, nuclear actin is involved in a number of cellular differentiation processes (25–27) and its in vivo function to ensure transcription has been shown during Drosophila oogenesis (28). Furthermore, when gene expression programmes are drastically reprogrammed after the nuclear transfer of somatic nuclei to oocytes, nuclear actin and actin-binding proteins play important roles (29, 30). The Miyamoto group has shown that interconnected nuclear actin filaments are formed in pronuclei of mouse fertilized embryos at the 1-cell stage (31). The disturbance of nuclear actin dynamics in pronuclei impaired mouse embryonic development to term (31), thus demonstrating the importance of nuclear actin dynamics in in vivo embryonic development. In this issue, this notion is further extended by examining endogenous nuclear F-actin in mouse fertilized and cloned embryos, and the group discovered that abnormal nuclear actin polymerization is observed in cloned embryos, suggesting abnormal reprogramming of nucleoskeleton structures (32).

As mentioned, nuclear actin dynamics are key for establishing and maintaining cellular functions. The assembly and disassembly of nuclear actin are regulated by actin-binding proteins. Formins polymerize actin inside the mammalian nucleus (33). ARPs are also known as regulators for actin polymerization in the cytoplasm, and are found in the nucleus as well (34). The Arp2/3 complex, known as the inducer of cytoplasmic F-actin, induces nuclear actin polymerization in response to DNA damage (35). Importantly, the Harata group has shown that Arp4, a nuclear Arp, serves as a suppressor for nuclear actin polymerization and knockdown of Arp4 leads to enhanced nuclear actin polymerization (36).

Actin filaments are utilized by motor protein myosins. The first myosin to be discovered in the nucleus is Nuclear myosin I, which is colocalized with RNAP II (37) and the small ribosomal subunit (38). Subsequently, many members of myosins have been found in the nucleus, and their involvement in transcriptional regulation has been demonstrated (39). The Toseland group revealed that Myosin VI binds to DNA and is also associated with the RNAP II complex as a tethering factor and/or auxiliary motor driving transcription (40). In addition to the transcriptional roles of nuclear myosins through their interactions with RNAPs, they are also engaged in chromosomal movements. Nuclear myosin I, together with nuclear actin, is implicated in the long-range interphase chromatin movements (41). Such myosin’s function as a nuclear motor is required for the spatial changes in chromosome territories following DNA damage (42). In addition, nuclear myosin in concert with nuclear F-actin relocalizes DNA damage sites for repair (43). The Toseland lab summarizes recent findings on DNA damage response and discusses mechanisms of how nuclear motor myosins regulate DNA repair processes in this issue (44).

One of the most studied nucleoskeleton proteins is the nuclear lamin. Lamins are classified as A- or B-type. Nuclear lamins are implicated in a number of nuclear processes including DNA replication and repair, chromatin organization and transcription (45). Furthermore, lamin A is responsible for the mechanical properties of nuclei (46). The interaction of lamins with other nucleoskeleton proteins has been shown (47) and, to note, nuclear actin directly binds to lamin (48). Several studies support the idea that lamins are involved in the regulation of nuclear actin dynamics (7, 49, 50). The Harata group has recently revealed that a lamin A mutant called progerin (51) disturbs nuclear F-actin formation and that the decrease of nuclear F-actin is involved in the aetiology of Hutchinson–Gilford progeria syndrome (52). As well as lamins, inner nuclear membrane proteins play roles in the association of chromatin with nucleoskeleton. In this issue, Oda et al. (53) report that, whereas lamin B receptor functions to tether chromatin to the nuclear envelope, this chromatin tethering system is circumvented in the Xenopus blastula, in which nuclear F-actin is responsible for tethering chromatin to the nuclear envelope (54).

To study the dynamic feature of nucleoskeleton proteins, an experimental tool that enables to spatiotemporally monitor and control activities of nuclear proteins would be required. To analyse and manipulate actin functions in cells, various small actin-binding molecules are utilized; however, the small molecules affect both cytoplasmic and nuclear actin. In this issue, the Harata group shows bicyclic peptides selectively binding to G-actin as a novel tool (55). The bicyclic peptide fused to a nuclear localization signal (NLS) is accumulated in the nucleus and represses the formation of nuclear F-actin and its functions, suggesting the possibility of the NLS-bicyclic peptide for analysing and manipulating nuclear F-actin.

Dynamic appearance/disappearance is a feature of nuclear F-actin. For example, nuclear actin polymerization can be induced within a minute (9), which needs to be captured by live-cell imaging. In addition, nuclear actin dynamics are altered during embryonic development and cellular differentiation at a certain place and time. Thus, in order to manipulate dynamics of nucleoskeleton proteins, an optogenetic approach that allows to control protein activity at precise places and times would be ideal. The Di Ventura lab has developed an optogenetic tool for light-inducible protein import to the nucleus (56). They combined NLS with the second light-oxygen-voltage (LOV2) domain of Avena sativa phototropin 1 that sterically conceals the NLS from the nuclear import machinery in dark. Upon blue light illumination, the conformational change of the LOV2 domain is induced and the NLS is recognized by importins for nuclear import. They have also applied the LOV2 domain for light-inducible nuclear export (57). Thus, nuclear localization of proteins can be spatiotemporally controlled. This system has been used to regulate nuclear localization of cofilin, an F-actin disassembly factor, to alter nuclear actin polymerization (8, 31). LOV2 domain-based optogenetic tools are also effective to spatiotemporally control actin polymerization by modulating activities of actin-binding proteins (33, 58). In this special issue, Forlani and Di Ventura (59) comprehensively summarize optogenetic approaches to study nuclear events.

To sum up, recent findings uncover the previously unknown functions of nucleoskeleton proteins. Considerable attention has been paid as to how dynamic changes of nucleoskeleton proteins actively regulate genomic functions. An important clue to tackle this question is that dynamics of nucleoskeleton proteins can affect nuclear architecture, which almost inevitably causes altered gene expression, as reviewed by the Biggiogera group in this issue (60). The relevance of nucleoskeleton-oriented regulation of gene expression to development, cellular differentiation, ageing and/or diseases needs to be further investigated in future studies.

Acknowledgements

We thank Ms. N. Backes Kamimura and Mr. J. Horvat for proof reading.

Funding

K.M. is supported by Human Frontier Science Programme (RGP0021/2016), by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP17H05045, JP19H05271, JP19H05751 and JP20K21376, by The Naito Foundation, by Takeda Science Foundation to K.M., and by a Kindai University Research Grant (19-II-1). M.H. is supported by the JSPS KAKENHI grant numbers JP19K22347, JP18H02164 and JP18H03946, and also by the JSPS Core-to-Core Programme (Advanced Research Networks) entitled ‘Establishment of international agricultural immunology research-core for a quantum improvement in food safety’.

Author Contributions

K.M. and M.H. wrote the manuscript.

Conflict of Interest

None declared.

References

Abbreviations

 
• ARP

  actin-related protein

 
• BAF complex

  Brahma-associated factor complex

 
• CRF

  chromatin remodeling factor

 
• F-actin

  filamentous actin

 
• G-actin

  globular actin

 
• LINC complex

  linkers of the nucleoskeleton to the cytoskeleton complex

 
• LOV2 domain

  second light-oxygen-voltage (LOV2) domain

 
• nAC-GFP

  nuclear actin chromobody-green fluorescent protein

 
• NLS

  nuclear localization signal

 
• RNAP

  RNA polymerase.