Tissue specific expression of Myosin IC Isoforms
© Sielski et al.; licensee BioMed Central Ltd. 2014
Received: 23 December 2013
Accepted: 3 March 2014
Published: 11 March 2014
Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus and is implicated in a variety of processes in both compartments. We recently identified a novel isoform of myosin IC and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C) that differ only in the addition of short isoform-specific N-terminal peptides. The expression pattern of the isoforms and the mechanisms of expression regulation remain unknown.
To determine the expression patterns of myosin IC isoforms, we performed a comprehensive expression analysis of the two myosin IC isoforms (isoform A and B) that contain isoform-specific sequences. By immunoblotting with isoform-specific antibodies and by qRT-PCR with isoform-specific primer we demonstrate that myosin IC isoforms A and B have distinct expression patterns in mouse tissues. Specifically, we show that myosin IC isoform A is expressed in a tissue specific pattern, while myosin IC isoform B is ubiquitously expressed at comparable levels in mouse tissues.
The differences in the expression profile of the myosin IC isoforms indicate a tissue-specific MYOIC gene regulation and further suggest that the myosin IC isoforms, despite their high sequence homology, might have tissue-specific and isoform-specific functions.
KeywordsMyosin IC Isoforms Gene expression Tissue specificity Protein expression
Myosin IC (formerly known as myosin I-β; ) is a single headed class I myosin that localizes to the nucleus and the cytoplasm. In the cytoplasm, myosin IC has been implicated, among other processes, in lipid raft arrangements , transport of vesicles containing membrane proteins such as the glucose transporter , and in ion channel regulation in hair cells of the inner ear [4–6]. In the nucleus, myosin IC is involved in various aspects of transcription [7–10], in chromatin remodeling [11–13], and in dynamic organization of chromosomal structures .
Interestingly, despite the high sequence homology, initial studies on isoform localization and function indicate that the myosin IC isoforms localize to different cellular compartments and are functionally distinct [17, 18]. However, the underlying factors that facilitate the functional difference between the isoforms are not fully understood. In addition to the potential functional differences between the isoforms and their distinct intracellular localizations, our previous analysis of expression of the newly identified myosin IC isoform A in tissue culture cells also indicated a potential difference in expression patterns between the isoforms .
Previous studies analyzing expression of total myosin IC with antibodies directed against an epitope in the C-terminal domain that is common to all myosins as well as studies analyzing protein and mRNA expression of myosin IC isoform B (NMI) in a variety of organisms and tissues demonstrated a ubiquitous and conserved expression of myosin IC [20–22]. However, our comparison of myosin IC isoforms A and B expression in HeLa, COS-7, and NIH 3T3 cells showed that while all three cell types express myosin IC isoform B at comparable levels, isoform A was strongly expressed only in COS-7 cells but could barely be detected in NIH 3T3 and HeLa cells  which suggests a difference in the expression pattern of the myosin IC isoforms. Therefore, we extended our studies and present here a comprehensive analysis of the expression pattern of myosin IC isoform A and B in mouse organs and tissues.
Results and discussion
As shown in Figure 1, only two of the three myosin IC isoforms that are expressed by the MYOIC gene, namely isoforms A and B, contain nucleotide and amino acid sequences that are isoform-specific and thus can be detected individually . To determine protein expression of the two isoforms, we performed immunoblot analysis of a panel of 33 different organs and tissues that were collected from 2-4 month old male and female C57Bl/6 mice. Protein extracts were analyzed using antibodies that recognize the individual isoforms. Figure 1 shows a schematic of the 5’ region of MYOIC, the resulting N-terminal amino acid sequences of the myosin IC isoforms, and the amino acid sequences that are recognized by the isoform-specific antibodies. The antibody that recognizes specifically myosin IC isoform A, is a monoclonal antibody that was generated using a peptide sequence that is encoded by the isoform A-specific exon -2 as immunogen . The antibody that recognizes myosin IC isoform B (NMI) is a polyclonal antibody that was generated using the isoform B-specific 16 amino acid long N-terminal peptide as immunogen .
In contrast to the tissue-specific expression of isoform A, myosin IC isoform B protein is easily detectable in all analyzed tissues and shows only moderate variations in expression levels, with the lowest expression observed in skeletal muscle and heart and the highest expression observed in liver, thymus, and in the various adipose depot tissues (Figure 2A and C). However, it should be noted that the maximum difference in relative expression of isoform B between the analyzed tissues is only 2.3 fold while the difference in relative expression of isoform A between the various tissues is up to 22 fold (compare Figure 2B and C).
In contrast, isoform B mRNA is expressed in all analyzed tissues with only slight variations in expression levels (Figure 3C).
In summary, we have identified and characterized a tissue-specific expression pattern of a recently identified, novel myosin IC isoform and we demonstrate that two of the myosin IC isoforms exhibit substantial differences in the expression profiles. Our data show that in contrast to the ubiquitously expressed myosin IC isoform B, the newly discovered myosin IC isoform A exhibits tissue-specific expression patterns on both, protein and mRNA levels. Previous studies analyzing the cellular distribution of the myosin IC isoforms in COS-7 cells, i.e. in a cell line that expresses all three myosin IC isoforms at high levels , revealed that each of the three isoforms localizes to specific cellular regions and interacts with different binding partners [17, 18] which strongly suggests isoform-specific cellular functions. Our identification of differential expression profiles further strengthens the notion that the myosin IC isoforms are functionally distinct. Future work is aimed at understanding the regulatory mechanisms that lead to tissue-specific expression of myosin IC isoform A and at identifying potential tissue-specific functions of this isoform.
The MYOIC gene expresses three different isoforms two of which exhibit significant differences in expression patterns. While myosin IC isoform B is ubiquitously expressed, myosin IC isoform A exhibits a tissue-specific expressed pattern that suggests tissue-specific functions of this myosin IC isoform.
Figure 1 shows the isoform-specific sequences that were used to generate myosin IC isoform specific antibodies. Antibodies that recognize various isoforms of myosin IC are: 1. the anti-NMI antibody is a rabbit polyclonal antibody that was raised against the 16 amino acid long N-terminal peptide of NMI, here called isoform B [9, 23] (Sigma-Aldrich, St Louis, MO); 2. the myosin IC-isoform A antibody is a mouse monoclonal antibody that was raised against the myosin IC isoform A specific N-terminal peptide and recognizes exclusively myosin IC isoform A . Monoclonal antibodies to β-actin were obtained from Sigma (Sigma-Aldrich, St Louis, MO). Peroxidase-conjugated secondary anti–mouse or anti–rabbit antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Animals and tissue collection
Tissue was isolated from male and female C57BL/6 mice 2–4 month old. All animal work was approved by the institutional animal care and use committee (University at Buffalo; protocol # PGY58128N). For each tissue type, tissues were extracted and analyzed from at least 3 male and 3 female mice.
For preparation of protein extracts, tissues were homogenized by sonication at 4°C for 5–20 seconds (depending on tissue) in extraction buffer [1x PBS, 1% NP40; 5 mM EDTA; 2% SDS, 1% Na-deoxycholate, with protease inhibitors (Sigma-Aldrich, St Louis, MO)] followed by a 30 min incubation on a rocking platform at 4°C. The samples were then centrifuged at maximum speed in a tabletop centrifuge for 10 min at room temperature. The supernatant was used to determine protein concentration using a Bradford protein assay according to manufacturer’s instructions (Bio-Rad, Hercules, CA). Equal amounts of protein extracts (40 μg) were separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred onto nitrocellulose membrane. After transfer, the membrane was cut followed by incubation of the lower half with antibodies specific to actin while the upper half was incubated with antibodies specific to myosin IC isoform A. Immunoreactive bands were detected by enhanced chemiluminescence. After detection of isoform A, the blots were stripped and re-probed with the antibody specific to myosin IC isoform B. Densitometric analysis was performed on the selected bands based on their relative intensities using ImageJ software.
Quantitative real-time PCR (qRT-PCR)
Primer used in qRT-PCR analysis
Forward primer (5’-3’)
Reverse primer (5’-3’)
MYOIC (isoform A)
MYOIC (isoform B)
reverse transcription polymerase chain reaction
quantitative real time polymerase chain reaction
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
phosphate buffered saline
- 5’ UTR:
5’ untranslated region.
We gratefully acknowledge Tera Domaradzki for help with tissue isolation. This study was funded in part by the Department of Defense CDMRP PCRP (W81XWH-12-1-0234 to W.A.H.).
- Gillespie PG, Albanesi JP, Bahler M, Bement WM, Berg JS, Burgess DR, Burnside B, Cheney RE, Corey DP, Coudrier E, de Lanerolle P, Hammer JA, Hasson J, Holt JR, Hudspeth AJ, Ikebe M, Kendricks-Jones J, Korn ED, R Li, Mercer JA, Milligan RA, Mooseker MS, Ostap EM, Petit C, Pollard TD, Sellers JR, Soldati T, Titus MA: Myosin-I nomenclature. J Cell Biol. 2001, 155 (5): 703-704. 10.1083/jcb.200110032.PubMed CentralView ArticlePubMedGoogle Scholar
- Brandstaetter H, Kendrick-Jones J, Buss F: Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion. J Cell Sci. 2012, 125 (Pt 8): 1991-2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Bose A, Guilherme A, Robida SI, Nicoloro SM, Zhou QL, Jiang ZY, Pomerleau DP, Czech MP: Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c. Nature. 2002, 420 (6917): 821-824. 10.1038/nature01246.View ArticlePubMedGoogle Scholar
- Cyr JL, Dumont RA, Gillespie PG: Myosin-1c interacts with hair-cell receptors through its calmodulin-binding IQ domains. J Neurosci. 2002, 22 (7): 2487-2495.PubMedGoogle Scholar
- Bond LM, Brandstaetter H, Kendrick-Jones J, Buss F: Functional roles for myosin 1c in cellular signaling pathways. Cell Signal. 2013, 25 (1): 229-235. 10.1016/j.cellsig.2012.09.026.PubMed CentralView ArticlePubMedGoogle Scholar
- Barylko B, Jung G, Albanesi JP: Structure, function, and regulation of myosin 1C. Acta Biochim Pol. 2005, 52 (2): 373-380.PubMedGoogle Scholar
- Hofmann WA, Johnson T, Klapczynski M, Fan JL, de Lanerolle P: From transcription to transport: emerging roles for nuclear myosin I. Biochem Cell Biol. 2006, 84 (4): 418-426. 10.1139/o06-069.View ArticlePubMedGoogle Scholar
- Hofmann WA, Vargas GM, Ramchandran R, Stojiljkovic L, Goodrich JA, de Lanerolle P: Nuclear myosin I is necessary for the formation of the first phosphodiester bond during transcription initiation by RNA polymerase II. J Cell Biochem. 2006, 99 (4): 1001-1009. 10.1002/jcb.21035.View ArticlePubMedGoogle Scholar
- Pestic-Dragovich L, Stojiljkovic L, Philimonenko AA, Nowak G, Ke Y, Settlage RE, Shabanowitz J, Hunt DF, Hozak P, de Lanerolle P: A myosin I isoform in the nucleus. Science. 2000, 290 (5490): 337-341. 10.1126/science.290.5490.337.View ArticlePubMedGoogle Scholar
- Philimonenko VV, Zhao J, Iben S, Dingova H, Kysela K, Kahle M, Zentgraf H, Hofmann WA, de Lanerolle P, Hozak P, Grummt I: Nuclear actin and myosin I are required for RNA polymerase I transcription. Nat Cell Biol. 2004, 6 (12): 1165-1172. 10.1038/ncb1190.View ArticlePubMedGoogle Scholar
- Percipalle P, Farrants AK: Chromatin remodelling and transcription: be-WICHed by nuclear myosin 1. Curr Opin Cell Biol. 2006, 18 (3): 267-274. 10.1016/j.ceb.2006.03.001.View ArticlePubMedGoogle Scholar
- Percipalle P, Fomproix N, Cavellan E, Voit R, Reimer G, Kruger T, Thyberg J, Scheer U, Grummt I, Farrants AK: The chromatin remodelling complex WSTF-SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription. EMBO Rep. 2006, 7 (5): 525-530.PubMed CentralPubMedGoogle Scholar
- Sarshad A, Sadeghifar F, Louvet E, Mori R, Bohm S, Al-Muzzaini B, Vintermist A, Fomproix N, Ostlund AK, Percipalle P: Nuclear myosin 1c facilitates the chromatin modifications required to activate rRNA gene transcription and cell cycle progression. PLoS Genet. 2013, 9 (3): e1003397-10.1371/journal.pgen.1003397.PubMed CentralView ArticlePubMedGoogle Scholar
- Chuang CH, Carpenter AE, Fuchsova B, Johnson T, de Lanerolle P, Belmont AS: Long-range directional movement of an interphase chromosome site. Curr Biol. 2006, 16 (8): 825-831. 10.1016/j.cub.2006.03.059.View ArticlePubMedGoogle Scholar
- Nowak G, Pestic-Dragovich L, Hozak P, Philimonenko A, Simerly C, Schatten G, de Lanerolle P: Evidence for the presence of myosin I in the nucleus. J Biol Chem. 1997, 272 (27): 17176-17181. 10.1074/jbc.272.27.17176.View ArticlePubMedGoogle Scholar
- Dzijak R, Yildirim S, Kahle M, Novak P, Hnilicova J, Venit T, Hozak P: Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner. PLoS ONE. 2012, 7 (1): e30529-10.1371/journal.pone.0030529.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwab RS, Ihnatovych I, Yunus SZ, Domaradzki T, Hofmann WA: Identification of signals that facilitate isoform specific nucleolar localization of myosin IC. Exp Cell Res. 2013, 319 (8): 1111-1123. 10.1016/j.yexcr.2013.02.008.View ArticlePubMedGoogle Scholar
- Ihnatovych I, Migocka-Patrzalek M, Dukh M, Hofmann WA: Identification and characterization of a novel myosin Ic isoform that localizes to the nucleus. Cytoskeleton (Hoboken). 2012, 69 (8): 555-565. 10.1002/cm.21040.View ArticleGoogle Scholar
- Barylko B, Wagner MC, Reizes O, Albanesi JP: Purification and characterization of a mammalian myosin I. Proc Natl Acad Sci U S A. 1992, 89 (2): 490-494. 10.1073/pnas.89.2.490.PubMed CentralView ArticlePubMedGoogle Scholar
- Hofmann WA, Richards TA, de Lanerolle P: Ancient animal ancestry for nuclear myosin. J Cell Sci. 2009, 122 (Pt 5): 636-643.PubMed CentralView ArticlePubMedGoogle Scholar
- 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 (2): 139-148. 10.1007/s00418-006-0231-0.View ArticlePubMedGoogle Scholar
- Wagner MC, Barylko B, Albanesi JP: Tissue distribution and subcellular localization of mammalian myosin I. J Cell Biol. 1992, 119 (1): 163-170. 10.1083/jcb.119.1.163.View ArticlePubMedGoogle Scholar
- Fomproix N, Percipalle P: An actin-myosin complex on actively transcribing genes. Exp Cell Res. 2004, 294 (1): 140-148. 10.1016/j.yexcr.2003.10.028.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.