The presence of actin in nuclei: a critical appraisal.

To assess the significance of actin associations with nuclei, we have examined Amoeba proteus nuclei for the presence of labeled actin under a variety of circumstances without (in most instances) isolating nuclei or breaking up cytoplasms prior to the extraction of proteins.We first established that: the 42,000 dalton proteins (presumed to be actin) present in cytoplasm and non-isolated nuclei are identical electrophoretically; the putative actin of amebas has the same size and almost the same isoelectric point as rat muscle actin; and the peptide “fingerprints” of putative ameba actin and rat actin are very similar after tryptic digestion. We therefore concluded that the 42,000 dalton protein of ameba is actin.We determined that: the concentrations of actin in the cytoplasm and nucleus of amebas are the same; actin is readily lost from nuclei that are released from lysed cells; shortly after a ³⁵S-labeled nucleus is transplanted into unlabeled cytoplasm, or an unlabeled nucleus is transplanted into ³⁵S-labeled cytoplasm, the concentration of ³⁵S-actin in nucleus and cytoplasm is the same; and when cells containing ³⁵S-actin are subjected to long chase periods on unlabeled food, the concentrations of ³⁵S-actin in nucleus and cytoplasm fall in parallel. These observations taken together suggest that actin is not tightly associated with nuclei. Rather, actin may associate with nuclei for the trivial reason that the nuclear envelope is no barrier to free movement of that protein between the two compartments.We conclude that the mere presence of actin in nuclei is insufficient grounds for assuming that it has any role in nuclear functions, such as, for example, chromosome condensation.     

Actin in the nucleus: what form and what for?

Actin is an abundant protein in most nonmuscle cells. It has often been observed in isolated nuclei, yet cytoplasmic contamination was of course initially regarded as the most plausible origin. Numerous studies on nuclear actin appeared in the 1970s and 1980s, but the picture remained rather muddy. The viewpoint at that time was that actin-shown to move freely between cytoplasm and nucleus-was a mere "thermodynamic wanderer," transiently occupying the nucleus. More recently, evidence has been mounting that actin's presence in the nucleus is not simply governed by the laws of diffusion. The same holds true for the finding of various actin-related proteins in the nucleus, and the case for nuclear myosin, specifically myosin I, is now quite convincing. Moreover, the first intimations of functional roles of nuclear actin are now emerging. Here we examine the overall subject from cell biological and chemical perspectives. The major issue is no longer the presence of actin in the nucleus but rather its supramolecular organization, intranuclear locations, and, of course, functions. These issues interface with recent findings that reveal a surprisingly diverse repertoire of actin conformations and oligomer and polymer forms beyond monomeric G-actin and polymeric F-actin. We present ideas for advancing the nuclear actin field and call for a renewed attack on this major problem in cell biology.     

Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm

For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked 'lower dimer' (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 [Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

Heterochromatin protein (HP)1γ is not only in the nucleus but also in the cytoplasm interacting with actin in both cell compartments

Confocal and electron microscopy images, and WB analysis of cellular fractions revealed that HP1γ is in the nucleus but also in the cytoplasm of C2C12 myoblasts, myotubes, skeletal and cardiac muscles, N2a, HeLa and HEK293T cells. Signal specificity was tested with different antibodies and by HP1γ knockdown. Leptomycin B treatment of myoblasts increased nuclear HP1γ, suggesting that its nuclear export is Crm-1-dependent. HP1γ exhibited a filamentous pattern of staining partially co-localizing with actin in the cytoplasm of myotubes and myofibrils. Immunoelectron microscopic analysis showed high-density immunogold particles that correspond to HP1γ localized to the Z-disk and A-band of the sarcomere of skeletal muscle. HP1γ partially co-localized with actin in C2C12 myotubes and murine myofibrils. Importantly, actin co-immunoprecipitated with HP1γ in the nuclear and cytosolic fractions of myoblasts. Actin co-immunoprecipitated with HP1γ in myoblasts incubated in the absence or presence of the actin depolymerizing agent cytochalasin D, suggesting that HP1γ may interact with G-and F-actin. In the cytoplasm, HP1γ was associated to the perinuclear actin cap that controls nuclear shape and position. In the nucleus, re-ChIP assays showed that HP1γ-actin associates to the promoter and transcribed regions of the house keeping gene GAPDH, suggesting that HP1γ may function as a scaffold protein for the recruitment of actin to control gene expression. When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes. In summary, HP1γ is present in the nucleus and the cytoplasm interacting with actin, a protein complex that may exert different functions depending on its subcellular localization.     

Nuclear actin: ancient clue to evolution in eukaryotes?

Until recently it was widely accepted that the dynamic cytoskeletal matrix is exclusive to the cytoplasm of eukaryotes, evolving before the emergence of the cell nucleus to enable phagocytosis, cell motility and the sophisticated functioning of the endomembrane system within the cytosol. The discovery of the existence of a prokaryotic cytoskeleton has changed this picture significantly. As a result, the idea has taken shape that the appearance of actin occurred in the very first cell; therefore, the emergence of microfilaments precedes that of the eukaryotic cytoskeleton. The discovery of nuclear actin opened new perspective on the field, suggesting that the nuclear activities of actin reflect the functions of primordial actin-like proteins. In this paper, we review the recent literature to explore the evolutionary origin of nuclear actin. We conclude that both ancient and eukaryotic features of the actin world can be detected in the nucleus today, which supports the idea that the cytoskeleton attained significant eukaryotic innovations before the tandem evolution of the cytoskeleton and nucleus occurred.     

Nuclear export of actin: a novel mechanism regulating the subcellular localization of a major cytoskeletal protein.

Actin is a highly conserved, ubiquitous cytoskeletal protein, which is essential for multiple cellular functions. Despite its small size (Mr = 42 000), unpolymerized forms of actin, as well as polymerized forms, exist primarily in the cytoplasm, excluded from the nucleus. Although spatial control of actin is crucially important, the molecular mechanisms ensuring the cytoplasmic localization of unpolymerized actin have not been revealed so far. In this paper we report that actin contains two leucine-rich type nuclear export signal (NES) sequences in the middle part of the molecule, which are both shown to be functional. Monomeric actin, when injected into the nucleus, was rapidly exported in a manner which was sensitive to leptomycin B (LMB), a specific inhibitor of NES-dependent nuclear export. LMB treatment of cells prevented nuclear exclusion of endogenous actin, inducing its nuclear accumulation. Furthermore, actin mutants with disrupted NESs accumulated in the nucleus. Expression of these NES-disrupted actin mutants, but not of wild-type actin, induced a decrease in the proliferative potential of the cell. These results reveal a novel molecular mechanism controlling the subcellular distribution of actin.     

Exportin 6: a novel nuclear export receptor that is specific for profilin.actin complexes

Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin beta-superfamily. Here we identify exportin 6 (Exp6) as a novel family member from higher eukaryotes and show that it mediates nuclear export of profilin.actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only approximately 20% identical amino acid residues, their function in profilin.actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. In contrast to a previous report, we found no indications of a major and direct role for CRM1 in actin export from mammalian or insect nuclei.     

Characterization of nuclear localization and nuclear export signals of yeast actin-binding protein Pan1

Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.     

Nucleus-associated actin in Amoeba proteus

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.     

Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.     

Critical role for a stage-specific actin in male exflagellation of the malaria parasite

Male gametogenesis occurs directly after uptake of malaria parasites by the mosquito vector and leads to the release of eight nucleated flagellar gametes. Here, we report that one of the two parasite actin isoforms, named actin II, is essential for this process. Disruption of actin II in Plasmodium berghei resulted in viable asexual blood stages, but male gametogenesis was specifically inhibited. Upon activation, male gametocyte DNA was replicated normally and axonemes assembled, but egress from the host cell was inhibited, and axoneme motility abolished. The major actin isoform, actin I, displayed dual localization to the cytoplasm and the nucleus in male gametocytes. After activation actin I was found to be restricted to the cytoplasm. In actII(-) mutant parasites, this re-localization was abolished and actin I remained in both cellular compartments. These findings reveal vital and pleiotropic functions for the actin II isoform in male gametogenesis of the malaria parasite.     

Nuclear actin: to polymerize or not to polymerize

The form and function of actin in the nucleus have been enigmatic for over 30 years. Recently actin has been assigned numerous functional roles in the nucleus, but its form remains a mystery. The intricate relationship between actin form and function in the cytoplasm implies that understanding the structural properties of nuclear actin is elementary to fully understanding its function. In this issue, McDonald et al. (p. 541) use fluorescence recovery after photobleaching (FRAP) to tackle the question of whether nuclear actin exists as monomers or polymers.