Upload
jose-r-cruz
View
215
Download
0
Embed Size (px)
Citation preview
Cell Biology International 32 (2008) 584e587www.elsevier.com/locate/cellbi
Nuclear actin in plants
Jose R. Cruz, Consuelo de la Torre, Susana Moreno Dıaz de la Espina*
Centro Investigaciones Biologicas, CSIC Ramiro de Maeztu 9, Madrid 28040, Spain
Abstract
Actins constitute a wide family of proteins that are major components of the cytoskeleton. Animal cells have nuclear G-actin forms thatassemble into several nuclear macromolecular complexes and are substrates for myosin I b (NMI). The nuclear actin related proteins(ARPs) are part of the chromatin-remodelling complex, while nuclear acting binding proteins (nABPs) comprise either nuclear forms of cyto-plasmic ABPs (as NMI) or specific nABPs. No evidence of the presence of nuclear actin exists in plants, which lack orthologues of the mainanimal structural nABPs. Here we prove the presence of actin forms with different solubility, and their associated protein NMI in the plant nu-cleus, as components of the transcription complexes and the nucleoskeleton. For this, WB and confocal immunofluorescence with antibodiesagainst human actin and NMI were used.� 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
Keywords: Actin; Myosin I; Nucleus; Nucleoskeleton; Transcription foci; Allium cepa
1. Introduction
Actin constitutes a ubiquitous highly conserved multigenicfamily of proteins that assemble dynamically in a cytoplasmicnetwork of microfilaments. Animal cells have nuclear non-filamentous b-actin forms that assemble into nuclear macro-molecular complexes and are substrates for nuclear myosin I(NMI), a member of the super family of the actin motors(Bettinger et al., 2004). The actin super family also includesactin related proteins (ARPs), some displaying homology toconventional actins and sharing the actin fold. Some nuclearARPs (nARPs) are a part of chromatin-remodelling com-plexes as BAF53. Nuclear acting binding proteins (nABPs)control the dynamics of actin in the nucleus and compriseeither nuclear forms the cytoplasmic ABPs as NMI, profilin,cofilin and p 4.1, or specific nABPs like emerin, lamin A,exportin 6, etc. (Blessing et al., 2004). Nuclear actin can adoptdifferent unconventional conformations (Schoenenbergeret al., 2005; Jockusch et al., 2006; McDonald et al., 2006)
* Corresponding author. Tel.: þ34 918 373 112; fax: þ34 915 360 432.
E-mail address: [email protected] (S. Moreno Dıaz de la Espina).
1065-6995/$ - see front matter � 2007 International Federation for Cell Biology.
doi:10.1016/j.cellbi.2007.11.004
that might contribute to its multiple nuclear functions(Bettinger et al., 2004). Thus it is involved in transcription(Grummt, 2006), chromatin remodelling (Sjolinder et al.,2005), transport and signal transduction (Forest et al., 2005),nuclear structure (Kiseleva et al., 2004; De Lanerolle et al.,2005), etc. However, there is no evidence for the presenceor functionality of nuclear actin in plant cells that lack theorthologues of lamins and lamin-associated proteins (LAPs)and p 4.1 that constitute the main structural nABPs in animalcells (Shumaker et al., 2003).
In plants, actins constitute a wide family of proteins(Kandasamy et al., 2004). They contain well-characterizednARPs involved in chromatin remodelling as AtARPs 4, 6and 7 (Kandasamy et al., 2004; Meagher et al., 2005). Never-theless the proteomic analysis failed to detect actin in theArabidopsis nucleoskeleton (NSK) (Calikowski et al., 2003)or nucleolus, although its associated protein NMI was present(Brown et al., 2005).
In this preliminary work, we investigated the presence,intranuclear distributions and functionality of nuclear actinand NMI in nuclei of meristematic root cells of Allium cepa,by western blot (WB) and confocal immunofluorescence usingantibodies against different regions of human actin (AC15 and8227) and NMI (M3567).
Published by Elsevier Ltd. All rights reserved.
585J.R. Cruz et al. / Cell Biology International 32 (2008) 584e587
2. Results and discussion
2.1. Onion meristematic nuclei have actin forms withdifferent solubility
The anti-human actin Mab and Pabs recognized an intensemonomeric actin band and, in some cases, a second band atabout 52 kDa in HeLa and endothelial human cells by WB.In onion meristematic root cells they showed a weaker reactiv-ity. Up to three bands with variable intensities were revealed inisolated onion nuclei: a main band at 44 kDa and two addi-tional ones with higher apparent molecular masses (about 52and 80 kDa; Fig. 1). Based on their mobilities and reactivity,and taking into account that the isolation of nuclei was donein a medium containing 50 mM Mg acetate that would pro-mote actin dimerization, the upper bands may correspond tonon-denatured low dimer (LD) actin and complexes of mono-meric actin with small ABPs as profilin that display similarmobilities (Millonig et al., 1988; Aguda et al., 2006) and existin a dynamic equilibrium in the nucleus (McDonald et al.,2006). Putative proteolytic fragments of the protein wereobserved in some cases, although a cross-reaction with nuclearARPs could not be discarded either. Sequential extraction ofnuclei with non-ionic detergent, 0.25 mM (NH4)2SO4 and2 M NaCl buffers, revealed the existence of actin forms withdifferent solubility in the nucleus (Fig. 1: S1, S2). The nuclearactin bands at 44 and 53 kDa were also detected in the NSKfraction (Fig. 1). The anti-NMI revealed a single band inboth nuclei and NSKs from onion cells (Fig. 1).
2.2. Actin and NMI assemble in multimeric complexes attranscription foci
Confocal microscopy immunofluorescence with the sameantibodies revealed a consistent distribution of actin andNMI in discrete transcription foci of isolated nuclei fromonion meristems (Fig. 2a, b), confirmed by run on transcrip-tion assays in situ, after a Br-UTP pulse (Fig. 2c), consistentlywith data in HeLa cells (Grummt, 2006; Schoenenbergeret al., 2005). rRNA transcription foci, clustered in the
Fig. 1. Representative WBs revealing the presence of b-actin and NMI in protein f
istematic root cells with antibodies against human b-actin (Pab 8227 and Mab AC15
(S1), and 0.25 mM (NH4)2SO4 and 50 mM NaCl (S2). Human endothelial (hE) and
without the corresponding primary antibodies.
nucleolus, were enriched in actin, in contrast with results ofthe proteomic analysis (Brown et al., 2005). This demon-strates the sensitivity of this in situ approach (Fig. 2a, b).Experiments with RNA synthesis inhibitors showed thatNMI is highly sensitive to the block of RNA polymerases,while the effects on actin staining were delayed (not shown).The numbers of actin-enriched nucleolar foci alwaysexceeded those involved in active transcription in the run onassays (Fig. 2c). This suggests that actin association to thetranscription complex is independent of active transcription.A small fraction of the nuclear set of CBs appeared to containactin (not shown). The in situ results ruled out the possibilitythat the nuclear actin forms detected by WB correspond tocytoplasmic contamination. Instead they support that nuclearactin is a component of transcription and snRNP processingmultimeric nuclear complexes and suggest its involvementin these processes, as reported in animal cells (Grummt,2006).
2.3. Actin is a component of the plant nucleoskeleton
The immunofluorescence analysis of the onion NSK (Yuand Moreno Dıaz de la Espina, 1999) validated the results ofWBs, confirming the association of b-actin with this macromo-lecular complex (Figs. 1 and 2d, e). This contrasted with the re-sults of the proteomic analysis in Arabidopsis thaliana thatfailed to detect the protein in the NKS (Calikowski et al.,2003). The actin staining of the NSK was much weaker thanthat of the transcription foci and never displayed linear arraysof actin filaments similar to the cytoplasmic ones. Actin distrib-uted diffusely in the residual nucleolus, probably associated toits underlying fibrillar network, and also in the nucleoskeletalfilaments (Yu and Moreno Dıaz de la Espina, 1999). The laterstaining probably corresponds to the diffused nucleoplasmicstaining observed in nuclei (Fig. 2a) and could distribute ina similar way to pore-linked filaments (PLFs) in Xenopus laevisoocyte nuclei (Kiseleva et al., 2004). Actin-enriched transcrip-tion sites were not observed in the NSK (Fig. 2d). Occasionally,they showed some bright spots that could correspond to resid-ual nuclear bodies enriched in the protein, as demonstrated by
ractions from isolated nuclei (N) and nucleoskeletons (NSK) from onion mer-
) and NMI (M3567). Nuclear protein fractions extracted by 0.5% Triton X-100
HeLa cells were used as positive controls. (�) Negative controls: incubations
Fig. 2. In situ distribution of b-actin and NMI in onion nuclei (N) and nucleoskeletons (NSK) as shown by confocal immunofluorescence with the same antibodies
used for the WB analysis. In the nuclei, both proteins were enriched in transcription foci (a, b), that were visualized by run on transcription assays after a Br-UTP
pulse, revealed by anti-Br immunolabelling (c). A weak actin staining was also associated to nucleoplasmic filaments (a). In the NSK, actin staining appeared
homogeneously associated to the residual nucleolus and to nucleoskeleton filaments (d). These are better observed at higher magnification (d0) and in DIC images
(e, e0).
586 J.R. Cruz et al. / Cell Biology International 32 (2008) 584e587
the overlay of immunofluorescence and DIC images of nucle-oskeletal fractions (Fig. 2d, e). They probably correspond toCajal bodies, the main subnuclear domains for snRNP process-ing that are usual components of the onion NSK (Acevedoet al., 2002).
3. Conclusions
Actin forms with different solubility as well as the actinmotor protein NMI are components of the plant nucleus.They are probably involved in transcription, as componentsof the transcription complexes, and also play a structuralrole in the NSK. As orthologs of the main structural nABPs(lamins, emerin, LAPS, p 4.1) are not present in the plant ge-nomes, but both systems have a similar nuclear organization,plants probably would have specific functional homologs ofthese proteins, as occurs with the NIFs (nuclear intermediatefilament proteins), considered to be the plant functional homo-logs of lamins (Blumenthal et al., 2004). Future investigations
will clear up the molecular mechanisms involved in nuclearactin functionality in plants.
Acknowledgements
We thank Mrs. M. Carnota for expert technical assistance.Work supported by the Spanish DGI (projects: BFU 2004-03071, BFU 2006-00379 and BFU 2007-60142/BFI).
References
Acevedo R, Samaniego R, Moreno Dıaz de la Espina S. Coiled bodies in
nuclei from plant cells evolving from dormancy to proliferation. Chromo-
soma 2002;110:559e69.
Aguda AH, Xue B, Irobi E, Preat Th, Robinson RC. The structural basis of
actin interaction with multiple WH2/b-thymosin motif-containing
proteins. Structure 2006;14:469e76.
Bettinger BT, Gilbert DM, Amberg DC. Actin up in the nucleus. Nat Rev Mol
Cell Biol 2004;5:410e5.
Blessing CA, Ugrinova GT, Goodson HV. Actin and ARPs: action in the
nucleus. Trends Cell Biol 2004;14:435e42.
587J.R. Cruz et al. / Cell Biology International 32 (2008) 584e587
Blumenthal SSD, Clark GB, Roux SJ. Biochemical and immunological char-
acterization of pea nuclear intermediate filament proteins. Planta
2004;218:965e75.
Brown JWS, Shaw PJ, Shaw P, Marshall DF. Arabidopsis nucleolar protein
database (AtNoPDB). Nucleic Acids Res 2005;33:633e6.
Calikowski TT, Meulia T, Meier I. A proteomic study of the Arabidopsis
nuclear matrix. J Cell Biochem 2003;90:361e78.
De Lanerolle P, Johnson T, Hoffman WA. Actin and myosin I in the nucleus:
what next? Nat Struct Mol Biol 2005;12:742e6.
Forest Th, Barnard S, Baines JD. Active intranuclear movement of herpes
virus capsids. Nat Cell Biol 2005;7:429e31.
Grummt I. Actin and myosin as transcription factors. Curr Opin Genet Dev
2006;16:191e6.
Jockusch BM, Schoenenberger C-A, Stetefeld J, Aebi U. Tracking down the
different forms of nuclear actin. Trends Cell Biol 2006;16:391e6.
Kandasamy M, Deal RB, Mckinney EC, Meagher RB. Plant actin-related
proteins. Trends Plant Sci 2004;9:196e202.
Kiseleva E, Drummond SP, Goldeberg MW, Rutherford SA, Allen TD,
Wilson KL. Actin- and protein-4.1-containing filaments link nuclear
pore complexes to subnuclear organelles in Xenopus oocyte nuclei.
J Cell Sci 2004;117:2481e90.
McDonald D, Carrero G, Andrin C, De Vries G, Hendzel MJ. Nucleoplas-
mic b-actin exists in a dynamic equilibrium between low-mobility
polymeric species and rapidly diffusing populations. J Cell Biol 2006;
172:541e52.
Meagher RB, Deal RB, Kandasamy MK, McKinney EC. Nuclear actin-related
proteins as epigenetic regulators of development. Plant Physiol 2005;139:
1576e85.
Millonig R, Salvo H, Aebi U. Probing actin polymerization by intermolecular
cross-linking. J Cell Biol 1988;106:785e96.
Schoenenberger C-A, Buchmeier S, Boerries M, Sutterlin R, Aebi U,
Jockusch BM. Conformation-specific antibodies reveal distinct actin struc-
tures in the nucleus and the cytoplasm. J Struct Biol 2005;157:157e68.
Shumaker DK, Kuczmarski ER, Goldman RD. The nucleoskeleton: lamins and
actin are major players in essential nuclear functions. Curr Opin Cell Biol
2003;15:358e66.
Sjolinder M, Bjork P, Soderberg E, Sabri N, Farrants AKO, Visa N. The
growing pre-mRNA recruits actin and chromatin-modifying factors to
transcriptionally active genes. Genes Dev 2005;19:1871e84.
Yu W, Moreno Dıaz de la Espina S. The plant nucleoskeleton: ultrastructural
organization and identification of NuMA homologues in the nuclear matrix
and mitotic spindle of plant cells. Exp Cell Res 1999;246:516e26.