BACKGROUND: Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. CONCLUSIONS/SIGNIFICANCE: We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the ASCR v v i Prague Czech Republic

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Nowak G, Pestic-Dragovich L, Hozak P, Philimonenko A, Simerly C, et al. Evidence for the presence of myosin I in the nucleus. J Biol Chem. 1997;272:17176–17181. PubMed

Pestic-Dragovich L, Stojiljkovic L, Philimonenko AA, Nowak G, Ke Y, et al. A myosin I isoform in the nucleus. Science. 2000;290:337–341. PubMed

Kahle M, Pridalova J, Spacek M, Dzijak R, Hozak P. Nuclear myosin is ubiquitously expressed and evolutionary conserved in vertebrates. Histochem Cell Biol. 2007;127:139–148. PubMed

Hofmann WA, Richards TA, de Lanerolle P. Ancient animal ancestry for nuclear myosin. J Cell Sci. 2009;122:636–643. PubMed PMC

Fomproix N, Percipalle P. An actin-myosin complex on actively transcribing genes. Exp Cell Res. 2004;294:140–148. PubMed

Hofmann WA, Stojiljkovic L, Fuchsova B, Vargas GM, Mavrommatis E, et al. Actin is part of pre-initiation complexes and is necessary for transcription by RNA polymerase II. Nat Cell Biol. 2004;6:1094–1101. PubMed

Philimonenko VV, Zhao J, Iben S, Dingova H, Kysela K, et al. Nuclear actin and myosin I are required for RNA polymerase I transcription. Nat Cell Biol. 2004;6:1165–1172. PubMed

Philimonenko VV, Janacek J, Harata M, Hozak P. Transcription-dependent rearrangements of actin and nuclear myosin I in the nucleolus. Histochem Cell Biol. 2010;134:243–249. PubMed

Grummt I. Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. Genes Dev. 2003;17:1691–1702. PubMed

Grummt I. Actin and myosin as transcription factors. Curr Opin Genet Dev. 2006;16:191–196. PubMed

Ye J, Zhao J, Hoffmann-Rohrer U, Grummt I. Nuclear myosin I acts in concert with polymeric actin to drive RNA polymerase I transcription. Genes Dev. 2008;22:322–330. PubMed PMC

Percipalle P, Farrants AK. Chromatin remodelling and transcription: be-WICHed by nuclear myosin 1. Curr Opin Cell Biol. 2006;18:267–274. PubMed

Obrdlik A, Louvet E, Kukalev A, Naschekin D, Kiseleva E, et al. Nuclear myosin 1 is in complex with mature rRNA transcripts and associates with the nuclear pore basket. FASEB J. 2010;24:146–157. PubMed

Cisterna B, Necchi D, Prosperi E, Biggiogera M. Small ribosomal subunits associate with nuclear myosin and actin in transit to the nuclear pores. FASEB J. 2006;20:1901–1903. PubMed

Cisterna B, Malatesta M, Dieker J, Muller S, Prosperi E, et al. An active mechanism flanks and modulates the export of the small ribosomal subunits. Histochem Cell Biol. 2009;131:743–753. PubMed

Mehta IS, Amira M, Harvey AJ, Bridger JM. Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts. Genome Biol. 2010;11:R5. PubMed PMC

Hu X, Li X, Valverde K, Fu X, Noguchi C, et al. LSD1-mediated epigenetic modification is required for TAL1 function and hematopoiesis. Proc Natl Acad Sci U S A. 2009;106:10141–10146. PubMed PMC

Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, et al. Long-range directional movement of an interphase chromosome site. Curr Biol. 2006;16:825–831. PubMed

Kysela K, Philimonenko AA, Philimonenko VV, Janacek J, Kahle M, et al. Nuclear distribution of actin and myosin I depends on transcriptional activity of the cell. Histochem Cell Biol. 2005;124:347–358. PubMed

Hokanson DE, Laakso JM, Lin T, Sept D, Ostap EM. Myo1c binds phosphoinositides through a putative pleckstrin homology domain. Mol Biol Cell. 2006;17:4856–4865. PubMed PMC

Laakso JM, Lewis JH, Shuman H, Ostap EM. Myosin I can act as a molecular force sensor. Science. 2008;321:133–136. PubMed PMC

Frangioni JV, Neel BG. Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal. J Cell Sci. 1993;105(Pt 2):481–488. PubMed

Mohr D, Frey S, Fischer T, Guttler T, Gorlich D. Characterisation of the passive permeability barrier of nuclear pore complexes. EMBO J. 2009;28:2541–2553. PubMed PMC

Cokol M, Nair R, Rost B. Finding nuclear localization signals. EMBO Rep. 2000;1:411–415. PubMed PMC

Dumont RA, Zhao YD, Holt JR, Bahler M, Gillespie PG. Myosin-I isozymes in neonatal rodent auditory and vestibular epithelia. Journal of the Association for Research in Otolaryngology: JARO. 2002;3:375–389. PubMed PMC

Chen J, Wagner MC. Altered membrane-cytoskeleton linkage and membrane blebbing in energy-depleted renal proximal tubular cells. American journal of physiology Renal physiology. 2001;280:F619–627. PubMed

Pemberton LF, Paschal BM. Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic. 2005;6:187–198. PubMed

Kutay U, Bischoff FR, Kostka S, Kraft R, Gorlich D. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell. 1997;90:1061–1071. PubMed

Gillespie PG, Cyr JL. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides. BMC Biochem. 2002;3:31. PubMed PMC

Lindsay AJ, McCaffrey MW. Myosin Vb localises to nucleoli and associates with the RNA polymerase I transcription complex. Cell Motil Cytoskeleton. 2009;66:1057–1072. PubMed

Suetsugu S, Takenawa T. Translocation of N-WASP by nuclear localization and export signals into the nucleus modulates expression of HSP90. J Biol Chem. 2003;278:42515–42523. PubMed

Hirono M, Denis CS, Richardson GP, Gillespie PG. Hair cells require phosphatidylinositol 4,5-bisphosphate for mechanical transduction and adaptation. Neuron. 2004;44:309–320. PubMed

Harley VR, Lovell-Badge R, Goodfellow PN, Hextall PJ. The HMG box of SRY is a calmodulin binding domain. FEBS letters. 1996;391:24–28. PubMed

Sim H, Rimmer K, Kelly S, Ludbrook LM, Clayton AH, et al. Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal. Molecular endocrinology. 2005;19:1884–1892. PubMed

Argentaro A, Sim H, Kelly S, Preiss S, Clayton A, et al. A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal. The Journal of biological chemistry. 2003;278:33839–33847. PubMed

Antonsson A, Hughes K, Edin S, Grundstrom T. Regulation of c-Rel nuclear localization by binding of Ca2+/calmodulin. Molecular and cellular biology. 2003;23:1418–1427. PubMed PMC

Houdusse A, Gaucher JF, Krementsova E, Mui S, Trybus KM, et al. Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:19326–19331. PubMed PMC

Manceva S, Lin T, Pham H, Lewis JH, Goldman YE, et al. Calcium regulation of calmodulin binding to and dissociation from the myo1c regulatory domain. Biochemistry. 2007;46:11718–11726. PubMed PMC

Kahl CR, Means AR. Regulation of cell cycle progression by calcium/calmodulin-dependent pathways. Endocrine reviews. 2003;24:719–736. PubMed

Yip MF, Ramm G, Larance M, Hoehn KL, Wagner MC, et al. CaMKII-mediated phosphorylation of the myosin motor Myo1c is required for insulin-stimulated GLUT4 translocation in adipocytes. Cell metabolism. 2008;8:384–398. PubMed

Dumont RA, Zhao YD, Holt JR, Bahler M, Gillespie PG. Myosin-I isozymes in neonatal rodent auditory and vestibular epithelia. J Assoc Res Otolaryngol. 2002;3:375–389. PubMed PMC

Galvez T, Duthey B, Kniazeff J, Blahos J, Rovelli G, et al. Allosteric interactions between GB1 and GB2 subunits are required for optimal GABA(B) receptor function. EMBO J. 2001;20:2152–2159. PubMed PMC

Kutay U, Lipowsky G, Izaurralde E, Bischoff FR, Schwarzmaier P, et al. Identification of a tRNA-specific nuclear export receptor. Mol Cell. 1998;1:359–369. PubMed

Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:8424–8428. PubMed PMC

Leneuve P, Colnot S, Hamard G, Francis F, Niwa-Kawakita M, et al. Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles. Nucleic Acids Res. 2003;31:e21. PubMed PMC

Nagata T, Redman RS, Lakshman R. Isolation of intact nuclei of high purity from mouse liver. Anal Biochem. 2010;398:178–184. PubMed

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