Cytoplasmic actin forms striking arrays of filaments, which can be readily visualized by conventional light microscopy methods in most cell types. The mechanical, trans­port and scaffolding roles of the actin cyto­skeleton are well known, and cytoplasmic actin polymerization plays a part in processes such as cell motility, adhesion, cytokinesis and morphogenesis. At first glance, the situation seems completely different in the cell nucleus, where conventional actin fila­ments had not been detected and therefore remained enigmatic. Phalloidin, the most commonly used drug for visualizing cellular actin filaments, does not usually stain the nucleus, but this could be explained by the low abundance of actin in this compartment compared with the cytoplasm. By contrast, antibodies raised against special forms of actin that are mainly monomeric stain the nucleus very strongly1, which suggests that nuclear actin is mainly monomeric or adopts specific conformations.

In this Progress article, we first briefly summarize evidence indicating that a pool of nuclear actin is monomeric, before discussing recent data showing that poly­merized actin structures are present in the nucleus of eukaryotic cells. Many actin filament­binding proteins are being linked to gene expression, and new probes have permitted the first actual visualizations of polymeric actin in the somatic cell nucleus.

Nuclear roles of monomeric actinDespite the fact that actin has been linked to crucial gene expression processes for decades2,3, the mechanistic details of nuclear actin functions have remained largely unclear. Actin has a role in promoting transcription by all three eukaryotic RNA polymerases, it is a component of many chromatin remodelling complexes and it also participates in pre­mRNA processing and export (reviewed in REFS 4–6). In addi­tion, actin regulates the activity of specific transcription factors7. Thus, actin seems to influence the whole gene transcription pathway, from gene activation to export of the mature mRNA. Although very little biochemical data exist for the interaction of nuclear actin with specific proteins, some experiments seem to support the conclu­sion that nuclear actin is mainly monomeric. Actin interacts with a subset of hetero genous nuclear ribonucleoproteins (hnRNPs) to promote the recruitment of histone acetyl­transferases (HATs) to active genes. The actin in these complexes also binds to deoxyribo­nuclease I (DNAseI)8–10, and this interaction is a hallmark for the detection of monomeric actin. Another transcription­associated actin monome r­binding protein is positive elon­gation factor b (pTEFb), which helps RNA poly merase II (Pol II) to escape pausing11. In the inositol­requiring 80 (INO80) chro­matin remodelling complex, the actin­related

proteins ARP4 and ARP8 negatively influ­ence actin polymerization in vitro, and they help to keep actin in its monomeric form within the complex12. In addition, the barbed end of actin, from which polymerization occurs, is not accessible for polymerization within the INO80 chromatin remodeller13. These data, as well as additional evidence, have evoked models suggesting that nuclear actin mainly operates as a monomer4, pos­sibly acting as an inter action platform for the recruitment of different gene expression complexes to facilitate transcription. Thus, although scaffolding roles for monomeric actin have been proposed in many studies over the years, more detailed mechanistic insight as well as in vivo analysis are required to better understand the precise nuclear functions of these interactions.

Towards polymeric nuclear actinAlthough evidence for the functional requirement of both monomeric and poly­meric nuclear actin in mammalian cells is available, the existence and role of polymer­ized actin in the nucleus was largely elusive and controversial. Early but indirect clues that polymeric nuclear actin existed came from the fact that many nuclear actin­dependent processes are disrupted by drugs that interfere with actin polymerization or the motor activity of actin­dependent myosins. These processes include transcrip­tion14,15 and the intra­nuclear movement of chromosomal loci16–18 or promyelocytic leukaemia (PML) bodies (punctate structures that contain PML protein)19. Many myosin motor proteins have been linked to various nuclear tasks, such as Pol I transcription and chromatin modifications15,20. In addition, the derepression of inflammatory response genes downstream of Toll­like receptors (TLRs) involves the actin filament­binding protein coronin 2A, which acts as a com­ponent of the nuclear receptor co­receptor complex (NCoR). Binding of coronin 2A to oligomeric actin seems to mediate TLR­induced NCoR turnover21. The presence of proteins that interact with actin filaments in transcription complexes, in combination with the cellular effects of drugs that inhibit actin polymerization, suggest a requirement for polymerized nuclear actin.

To be or not to be assembled: progressing into nuclear actin filamentsRobert Grosse and Maria K. Vartiainen

Abstract | The paradigm states that cytoplasmic actin operates as filaments and nuclear actin is mainly monomeric, acting as a scaffold in transcription complexes. However, why should a powerful function of actin, namely polymerization, not be used in the nucleus? Recent progress in the field forces us to rethink this issue, as many actin filament assembly proteins have been linked to nuclear functions and new experimental approaches have provided the first direct visualizations of polymerized nuclear actin.

PROGRESS

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Further support for this comes from the nuclear presence of actin­filament assembly proteins and their links to basic transcriptional processes. For example, the actin nucleation complex ARP2/3 and its activators Wiskott–Aldrich syndro me pro­tein (WASP) and neural WASP (NWASP) are found in the nucleus, and biochemica l assays for actin assembly and Pol II activit y using nuclear extracts support the notion that they may be involved in Pol II­mediated transcription22,23. In addi­tion, the F­actin­severing protein cofilin has been linked to the elongation phase of Pol II function, during which it might facilitate the association of actin with the elongat­ing polymerase24. Locally controlled actin polymerization and depolymerization may therefore be required for transcription.

Finally, exciting evidence for nuclear F­actin structures came from the observa­tion that a sponge­like actin meshwork is required to stabilize the mechanical integrity of the giant Xenopus laevis oocyte nucleus, which maintains very high actin concentrations in contrast to somatic cell nuclei25. Although the structural details of this actin network have been debated26, a recent study revealed the nuclear presence of many actin filament­binding proteins, such as supervillin and filamin A, in the X. laevis oocyte27. This strongly suggests that the meshwork is indeed formed from canonical actin filaments. Interestingly, reactivation of the pluripotency gene oct4, after the transfer of a C2C12 cell nucleus into a X. laevis oocyte at the germinal vesi­cle stage, seems to be mediated by nuclear actin polymerization that is regulated by formin­binding protein­like 1 (also known as TOCA1), which supports actin assembly by NWASP28.

Together, these studies planted a seed and argued in favour for the potential exist­ence of polymeric actin structures also in mammalian somatic cell nuclei.

Visualizing nuclear actinApproaches to monitor actin assembly in living cells usually involve the expression of isolated actin­binding domains (ABDs) from various species, which, when fused to fluorescent proteins such as GFP, allow actin polymers to be visualized. For example, a 25 kDa ABD from a crosslinking protein of Dictyostelium discoideum was used to visualize contractile actin networks in the nucleoplasmic region after nuclear envelope breakdown in starfish oocytes29. In addi­tion, common F­actin­binding peptides isolated from the dystrophin homologue utrophin have been extensively character­ized and used to study cytoplasmic and oocyte F­actin structures28,30–32. However, although actin is the most abundant protein in the cytoplasm of somatic cells, its nuclear concentration is low; hence, the labelling of cytoplasmic actin filaments obscures poten­tial signals from the nuclear compartment. Two recent studies have overcome this prob­lem by using ABDs fused to nuclear localiza­tion sequences (NLS) (BOX 1). In one case, an utrophin truncation mutant (Utr230) was generated to reduce the inherent actin bundling activity of this protein. The expres­sion of nuclear targeted Utr230–enhanced GFP–NLS in mammalian cells revealed the presence of punctate nuclear actin struc­tures of submicron length. Interestingly, signals detected with this probe emanated from the interchromatin space33, suggesting that these actin filament structures are not directly involved in chromatin interactions or functions.

In another study, the F­actin marker that is currently used most frequently by researchers — Lifeact — was fused to a NLS to restrict the detection of actin filaments to the nucleus34. When expressed in human cells at low levels, Lifeact–GFP–NLS displayed a homogenous and diffuse nuclear signal. However, within 20 seconds after serum stimulation, transient but distinct actin filament formation was observed, and this required the activity of nuclear formins (which have essential roles in remodelling the actin and microtubule cytoskeletons35). This suggests that nuclear actin assembly is signal­operated and can be rapidly reorganized. Thus, actin polymeriza­tion in the nucleus seems to be dynamic and tightly regulated. Using phalloidin labelling, this study also demonstrates, for the first time, the existence of a native actin filament struc­ture in fibroblast nuclei that forms in response to serum34 (FIG. 1).

Important questions now are how do these two studies fit together and what are the possible functions of these filaments? It seems likely that nuclear filaments are short under steady state conditions33, but that they form higher­ordered structures upon serum stimulation or nuclear formin activa­tion34. These organized structures could in principle serve for short­term intranuclear transport processes. Another possibility is that other signals or stimuli may promote intranuclear actin filament structures over longer time periods or in subnuclear regions. Clearly, these issues remain to be investigated. In addition, the existence of nuclear F­actin punctae, as detected using Utr230–enhanced GFP–NLS, may require further confirmation using phalloidin staining in untransfected cells or the co­expression of both the utrophin and Lifeact probes, if possible. It is also con­ceivable that the actin structures visualized by these two probes represent two different pools of nuclear actin with different functions, generated by different actin nucleation factors (FIG. 2). Moreover, it must also be remem­bered that so far these probes have only been used in light microscopy experiments and have thus been hampered by the resolution limit of this technique. It is likely that super­resolutio n microscopy or the adaptation of the new probes for other imaging techniques, such as electron microscopy, will be key for studying the organization and functions of nuclear actin filaments in various cells and tissues in the coming years.

Regulation of nuclear actinWith the emergence of polymerized nuclear actin, the next obvious question is how are actin dynamics regulated in the nucleus?

Box 1 | Probes for visualizing monomeric and polymeric nuclear actin

Monomeric actinRPEL1 motif. MAL (megakaryocytic acute leukaemia), a transcriptional activator of serum response factor (SRF), contains an RPEL domain composed of three RPEL repeats, which bind actin monomers45. When fused to a nuclear localization signal (NLS), RPEL1 concentrates to nuclear speckles33.

Polymeric actinLifeact. This 17‑amino acid long peptide is derived from the budding yeast protein Abp140, which binds actin monomers and filaments46. When targeted to the nucleus, Lifeact–NLS recognizes linear nuclear actin filaments that are induced by serum stimulation and the activation of the diaphanous‑related formin mDia but shows a diffuse localization in unstimulated cells34.

Utr230. Tandem calponin homology domains of the actin‑binding protein utrophin are frequently used to study actin dynamics in the cytoplasm31, but the targeting of this construct to the nucleus may result in ectopic nuclear actin polymerization or filament stabilization. To circumvent this problem, a truncated version, Utr230, was generated that highlights the appearance of discrete nuclear actin punctae of submicron length. These actin punctae did not colocalize with RNA polymerase I (Pol I), Pol II or Pol III and are excluded from chromatin‑rich regions33.

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Phalloidin Lamin A/C DAPI Lamin A/C Phalloidin

10 µm

Nuclei contain many (if not most) types of actin­binding protein that are found in the cytoplasm. Thus, being equipped with this toolkit, actin treadmilling (whereby one end of a filament grows while the other end dis assembles into monomers) could, in principle, also take place in the nuclear compartment. The key nuclear actin regula­tors include actin­filament nucleating factors (the ARP2/3 complex, formins and junction­mediating and regulatory protein (JMY)), the regulators of actin monomer pools (cofilin, profilin and thymosin β4), as well as several actin capping proteins (such as tro­pomodulin and gelsolin­like capping protein (CAPG))36–38. However, the experimental evi­dence that these proteins indeed control actin dynamics in the nucleus is still questionable, mainly due to the difficulty in assessing their nuclear­specific functions. Moreover, the relative contributions of different actin regulators may vary between cell types and experimental settings. For example, serum­stimulated actin assembly rates in nuclear extracts were decreased using siRNAs against the diaphanous­related formins mDia1 and mDia2, whereas inhibition of the ARP2/3 complex or the formin FHOD1 (FH1 and FH2 domain­containing protein 1) had no effect on actin assembly34. Also, a recent study identifying actin­filament binding proteins from X. laevis oocyte extracts, using a biochemical phalloidin affinity matrix, did not find nuclear ARP2/3, but instead inverted formin 2 (REF. 27). How nuclear formin activity is regulated, and by which signals, remains elusive. As the ARP2/3 complex has been linked to Pol II function23, it is possible that it regulates nuclear actin poly merization only locally at the transcribed genes or in other special circumstances. Future work

will be necessary to reveal whether the same upstream signalling components that control the activities of actin regulators in the cyto­plasm also operate in the nucleus. The new actin filament probes33,34 will likely have a crucial role in these studies, by allowing, for example, RNAi screening experiments for regulators of specific nuclear actin pools.

Cytoplasmic actin dynamics are likely to have an effect on nuclear actin function. This is due to the fact that actin constantly and rapidly shuttles in and out of the nucleus, and therefore nuclear and cytoplasmic actin pools are in constant communication. As actin is transported through the nuclear pore complexes as monomers, the level of actin monomers seems to limit the transport rate in both directions. Therefore, the actin polymer­ization status or other actin­binding events in either the nucleus or cytoplasm can affect the shuttling speed of monomers and hence modulate the levels and polymerization prop­erties of nuclear actin39. For example, DNA replication stress, which induces enhanced nuclear localization of actin and its regula­tors RAC and IQGAP1 (IQ motif­containing GTPase­activating protein 1), results in faster shuttling of actin due to its decreased cellular retention40. The new nuclear actin probes will also aid in studies that examine changes in nuclear actin levels and its regulation. For example, nuclear staining by the Utr230 probe has already highlighted how nuclear actin level s are altered by the siRNA­mediated depletion of the nuclear transport factors of actin, exportin 6 and importin 9 (REF. 33).

The nuclear lamina may specifically regu­late nuclear actin dynamics. In addition to the extensive connections of the lamina with cytoplasmic actin networks through LINC (linker of nucleoskeleton and cytoskeleton)

complexes41, A­type and B­type lamins42 as well as emerin have been reported to directly interact with nuclear actin. The interaction of emerin with nuclear actin is especially intriguing, as emerin interacts with the pointed ends of actin filaments and can pro­mote actin polymerization in vitro43. Recent results indicated that lamin A/C and emerin operate together to control actin dynamic s by regulating the MAL (megakaryocytic acute leukaemia; also known as MKL1 or MRTFA)–serum response factor (SRF) circuit, in which MAL is an actin­regulated co­activator of the SRF transcription factor that regulates the expression of cytoskeletal genes (FIG. 2). MAL binds actin monomers through its RPEL domain, and actin bind­ing regulates the cellular localization of MAL by preventing its nuclear import and enhancing its export. Additionally, only actin­free MAL can activate SRF­mediated transcription7. MAL therefore acts as an actin monomer sensor, which at low actin monomer concentrations accumulates in the nucleus to activate SRF. The nuclear translocation of MAL is impaired in cells deficient in lamin A/C or in cells expressing a laminopathy­causing LMNA­mutant. This defect was due to altered actin dynamics and could be rescued by the ectopic expression of emerin, which was mislocalized in the mutant cells. Importantly, the actin­binding activity of emerin was required for rescue, further confirming that the defect observed in lamin A/C­deficient cells was the result of altered nuclear actin dynamics44. This sug­gests that structural changes in the nuclear lamina impair the activity of tissue­specific transcription factors (such as SRF via the MAL–SRF pathway) and implies a role for emerin as a key regulator of nuclear actin dynamics. In the future, it will be interesting to study whether nuclear envelope proteins contribute to or alter the formation of either Utr230­ or Lifeact­stainable nuclear actin polymers.

Future directionsDuring the past few years, considerable progress has been made in the nuclear actin field, with possibly one of the most important developments being the visualization of poly­meric nuclear actin. However, many questions still remain. In the cytoplasm, actin filaments form many types of higher­order structur e, from bundles to branched networks, which all function in different cell biological processes. It is entirely reasonable that the recently detected nuclear actin polymers33,34 also represen t different actin pools that are regulate d by different mechanisms (FIG. 2).

Figure 1 | Seeing is believing: visualization of polymeric nuclear actin. Confocal fluorescence microscopy images showing serum-stimulated NIH3T3 cells stained for lamin A/C (red), DNA (DAPI; blue) and actin filaments (phalloidin; green). Nuclear actin filaments are visible. The image on the left shows phalloidin-staining only (in black and white for the best contrast). The image in the middle is a merge of lamin A/C staining and DAPI staining. The image on the right is a merge of lamin A/C staining and phalloidin staining. Images courtesy of C. Baarlink, University of Marburg, Germany.

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c F-actin network (treadmill-competent pool)

Chromatin or RNA–Pol II-associated pool

d F-actin punctae (treadmill-competent pool)

mDia

mDia

Emerin

MAL

MAL

EXP6

IPO

9

G-actina

?

?

ARP2/3complex

Nucleus

Cytoplasm

?

G-actin

NWASP

MA

L

SRE

SRF

b

The nuclear presence of numerous actin regu­lators suggests that it is indeed plausible to build many different types of polymeric actin structure in the nucleus. Their formation could then be regulated through different sig­nalling pathways and could vary between dif­ferent cell types and tissues; these parameters would also apply to their nuclear functions. The functional significance of the different polymeric actin assemblies in the nucleus still remains enigmatic, but it is tempting to speculate that the different polymer struc­tures fulfil distinct tasks. For example, the short actin polymers detected with the Utr230 probe could contribute to the viscoelasticity

of the nucleoplasm33. Formin­nucleated fila­ments, being longer in length, could have a role in regulating intranuclear transport, chromatin state and gene expression, beyond their established role in regulating SRF activ­ity34. What type of actin polymers emerin44 contributes to in the nucleus still needs to be clarified. Equally unclear is the structure and function of the actin polymers that may be nucleated by the nuclear ARP2/3 complex and its activators5,23.

Another interesting issue is the role of actin in regulating general transcription as a component of, for example, the Pol II and chromatin remodelling complexes, and its

impact on specific transcription factors, such as SRF. In the case of SRF, actin monomers clearly have a negative effect on SRF­target gene expression7,whereas, in general, actin has a positive role in transcription39. However, although actin seems to function as a mono­mer in chromatin remodelling complexes13, the ‘functional’ form of actin in the Pol II complex is still unclear, although some studies have suggested a requirement for polymer­ized actin in transcription14,15. Detailed bio­chemical analyses of the interactions between nuclear actin and the transcription machin­ery, as well as the identification of direct bind­ing events, are required to resolve these issues.

Figure 2 | Schematic of the possible regulation of nuclear actin dynamics and nuclear actin filament assembly. Nuclear actin can exist in several different forms. a | Actin monomers shuttle in and out of the nucleus, and this process is controlled by importin 9 (IPO9) and exportin 6 (EXP6). b | Actin monomers can also bind to chromatin remodelling complexes and transcription complexes, for example, through interactions with actin-related proteins (ARPs) and positive elongation factor b (pTEFb) (not shown), which may keep actin monomeric in these complexes. ARP2/3 and neural Wiskott–Aldrich syndrome protein (NWASP) are also associated with RNA polymerase II (Pol II) in the nucleus. The dashed lines indicate that there is little or very slow exchange between free nuclear G-actin and actin bound to chromatin, which remains stably associated. c | Monomeric actin can also assemble into long actin filaments, which form an F-actin network, in the nucleus. This process is stimulated by serum, which by unknown mechanisms activates mDia formins, such as mDia2, that are actively shuttling in and out

of the nuclear compartment. The resulting decrease in actin monomers causes the serum response factor (SRF) co-activator MAL to be retained in the nucleus, as MAL requires monomeric actin binding for its export. This results in SRF-mediated transcriptional activation. d | Actin can also form nuclear F-actin punctae, but how they assemble from monomeric actin and their relationship with the other nuclear F-actin structures is still unclear (as indicated by the question mark). It is nevertheless unlikely that these F-actin punctae would have a role in the chromatin-related activities of actin. Another protein that may contribute to nuclear actin assembly is the inner nuclear membrane protein emerin, which also contributes to MAL-SRF regu-lation. However, the exact nuclear actin pool that is regulated by emerin remains to be determined, as indicated by the question mark. Certain nuclear actin pools may be competent for nucleocytoplasmic shuttling and/ or may be involved in nuclear actin treadmilling, whereas others may be stably associated with chromatin or RNA–Pol II transcription complexes.

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Robert Grosse is at the Biochemical-Pharmacological Center (BPC), Institute of Pharmacology, University of

Marburg, 35043 Germany.

Maria K. Vartiainen is at the Institute of Biotechnology, Program in Cell and Molecular Biology,

University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.

e-mails:robert.grosse@staff.uni-marburg.de; maria.vartiainen@helsinki.fi

doi:10.1038/nrm3681 Published online 3 October 2013

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AcknowledgementsThe authors thank members of their laboratories for discus-sions and C. Baarlink for the image in figure 1. The work in the laboratory of M.K.V is funded by the European Research Council (ERC) Starting grant, Academy of Finland and Sigrid Juselius foundation. Work in the laboratory of R.G. is partly funded by the Deutsche Forschnungsgemeinschaft (GR 2111/2 and SFB 593).

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