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Identification of biotin carboxyl carrier protein in Tetrahymena and its application in in vitro motility systems of outer arm dynein
Journal of Microbiological Methods 105 (2014) 150–154
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Identification of biotin carboxyl carrier protein in Tetrahymena and its application in in vitro motility systems of outer arm dynein Masaki Edamatsu ⁎ Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 26 July 2014 Accepted 26 July 2014 Available online 6 August 2014
a b s t r a c t Axonemal dynein plays a central role in ciliary beating. Recently, a functional expression system of axonemal dynein was established in the ciliated protozoan Tetrahymena. This study identifies biotin carboxyl carrier protein (BCCP) in Tetrahymena and demonstrates its application in in vitro motility systems of outer arm dynein. © 2014 Elsevier B.V. All rights reserved.
Keywords: Tetrahymena Ciliated protozoan Axonemal dynein Biotin carboxyl carrier protein (BCCP) Biomolecular motor Ciliary movement
Ciliary movement is driven by axonemal dyneins and the impaired motility of the dyneins causes primary ciliary dyskinesia (PCD) (Badano et al., 2006; Marshall, 2008; Satir and Christensen, 2008). Axonemal dyneins are large AAA+ (ATPases associated with diverse cellular activities)-type motors (500–1500 kDa) and are divided into inner arm and outer arm dyneins (Gibbons, 1995; Vale, 2000; Kamiya, 2002). In Tetrahymena, there are a total of 23 axonemal dynein heavy chain genes (Wilkes et al., 2008). Axonemal dyneins are activated in a coordinated manner during ciliary beating, and this coordinated activation is a specific property of axonemal dyneins. These motors could be utilized as unique motile elements for nanoscale devices in the field of nanobiotechnology (Bachand et al., 2014; van den Heuvel and Dekker, 2007; Hess, 2011). An expression system for the genetic engineering of axonemal dynein has recently been developed in Tetrahymena (Edamatsu, 2014). Although this expression system is useful for functional and structural studies of axonemal dyneins and ciliary movement, a versatile system for tagging axonemal dyneins is needed to further advance molecular studies of these proteins. In this study, a versatile BCCP tag was identified in Tetrahymena and applied to in vitro motility systems of axonemal dynein. BCCP is one part of a biotin-dependent enzyme and is found in most prokaryotes and eukaryotes (Chen et al., 2012). BCCP consists of approximately 70 amino acids and is biotinylated by biotin protein ligase ⁎ Corresponding author at: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-0041, Japan. Tel.: +81 3 5454 6158. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.mimet.2014.07.036 0167-7012/© 2014 Elsevier B.V. All rights reserved.
(BPL) in culture. The high affinity and specificity of BCCP for avidin offers an advantage in the binding of biotinylated motors to avidinconjugated or coated materials. It has been reported that intrinsic BCCP is efficiently biotinylated in host cells (Healy et al., 2010; Polyak et al., 2001); therefore, a search for the BCCP sequence was performed in the Tetrahymena Genome Database (TGD) Wiki (http://ciliate.org/index.php/home/welcome). The BCCP family is classified into 4 subfamilies, and 15 representative species from each subfamily [listed in (Chen et al., 2012)] were used for the homology search. One uncharacterized protein (gene ID: TTHERM_00502240) homologous to carbamoyl phosphate synthase (CPSase) was identified in the database. The domain structure of this protein is shown in Fig. 1A. The C-terminus of the protein is a BCCPlike domain containing the biotin-attachment consensus sequence (Healy et al., 2010) (Fig. 1B and D). As described below, this domain was biotinylated in Tetrahymena cells and was thus identified as Tetrahymena BCCP (TtBCCP). TtBCCP was close to subfamily C members in the phylogenetic analysis (Fig. 1E). Fig. 1C shows a homology model of TtBCCP based on the structure of Escherichia coli BCCP. TtBCCP was found to lack the “thumb loop”, which contributes to substrate specificity in the BPL enzyme (Healy et al., 2010) (Fig. 1C and D). Next, a TtBCCP-fused outer arm dynein was generated. The Tetrahymena outer arm dynein forms a three-headed structure comprising alpha (DYH3), beta (DYH4) and gamma (DYH5) heavy chains (Wilkes et al., 2008). The expression cassette targeting the N-terminus of DYH3 is shown in (Fig. 2A and B). The PCR primers used in this study are shown in Supplemental Table S1. Transformations were performed according to (Edamatsu, 2014), and homologous recombination was confirmed by PCR analysis (Fig. 2C and D). Cilia were labeled with
M. Edamatsu / Journal of Microbiological Methods 105 (2014) 150–154
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Fig. 1. Identification and characterization of TtBCCP. (A) Domain structure of the CPSase-like protein identified in Tetrahymena. (B) Nucleotide and deduced amino acid sequences of TtBCCP. (C) Homology modeling of TtBCCP using SWISS-MODEL tools (Guex and Peitsch, 1997; Schwede et al., 2003; Arnold et al., 2006). TtBCCP (green) and Escherichia coli BCCP (gray, PDB code 1 BDO) were superimposed using PyMOL software (http://www.pymol.org). The biotinylated lysine in E. coli BCCP is also shown. (D) Alignment of BCCP domains. The aligned sequences are E. coli BCCP (EcBCCP) [P0ABE0], Arabidopsis thaliana BCCP (AtBCCP) [Q9LLC1], Schizosaccharomyces pombe BCCP (SpBCCP) [P78820], Homo sapiens BCCP (HsBCCP) [Q13085], and Tetrahymena thermophila (TtBCCP) [TTHERM_00502240]. The arrow represents the biotin attachment site. The underline represents the thumb loop. (E) Phylogenic tree of BCCPs constructed using MEGA 6 software (http://www.megasoftware.net).
Cy3-streptavidin (Cy3-SA) in permeabilized cells (Fig. 2E). The Tetrahymena oral apparatus was also labeled with Cy3-SA because it contains a large number of cilia. In control experiments, fluorescence in the cilia and in the oral apparatus was quenched by preincubation of Cy3-SA with 5 mM biotin (Fig. 2E).
Biotinylation of TtBCCP-dynein was performed in proteose peptone medium (containing 74 nM biotin, as estimated from the manufacturer's manual), and the addition of 0–100 μM biotin to the medium did not significantly enhance biotinylation of the recombinant dynein (Fig. 2F). TtBCCP-dynein was purified from ciliary extracts
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M. Edamatsu / Journal of Microbiological Methods 105 (2014) 150–154
A
B
D
C
E
a
F
b
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Fig. 2. Construction and purification of the TtBCCP-outer arm dynein in Tetrahymena. (A) Domain structure of DYH3. (B) The expression cassette for TtBCCP-DYH3. F1 (1.8-kb untranslated region of DYH3), neo (neomycin resistance gene), BTU1P (beta-tubulin 1 promoter), His6-FLAG-TtBCCP, and F2 (0.9-kb coding region of DYH3) were sequentially cloned into the pGBKT7 vector. (C) Schematic representation of homologous recombination in the expression cassette. The arrows represent the PCR primers used in (D). (D) PCR analysis. Lanes 1 and 2: PCR using cFw1 and cRv1 primers; lanes 3 and 4: PCR using cFw2 and cRv2 primers. Lanes 1 and 3: wild-type; lanes 2 and 4: transformants. (E) Localization of TtBCCP-DYH3. Permeabilized cells labeled with Cy3-SA (a) or Cy3-SA preincubated with 5 mM biotin (b). Scale bar represents 5 μm. (F) Biotinylation of TtBCCP-DYH3. Axonemal proteins of transformants grown in culture medium supplemented with 0–100 μM biotin were examined by avidin-blot analysis. CBB staining (upper panel) and HRP-streptavidin staining (lower panel) are shown. (G) Purification and avidin-blot analysis of TtBCCP-outer arm dynein. Lanes 1 and 2: CBB staining; lanes 3 and 4: Cy3–SA staining. Lanes 1 and 3: native outer arm dynein; lanes 2 and 4: TtBCCP-outer arm dynein.
according to (Edamatsu, 2014). The purified protein was specifically labeled with Cy3-SA and confirmed to be biotinylated dynein (Fig. 2G). Next, TtBCCP was applied to in vitro motility assays of outer arm dynein. First, recombinant dynein was fixed on glass slides via streptavidin–biotin binding, and dynein motility was examined (gliding assay, Fig. 3A). Biotinylated BSA, streptavidin, casein, TtBCCP-dynein, and microtubules were introduced sequentially into the flow chamber. The gliding velocity was 4.4 ± 1.3 μm/s (Fig. 3B and C), which was comparable to the gliding velocity of GFP-fused outer arm dynein (Edamatsu, 2014). In control experiments, few microtubules were found in the flow chamber after preincubation with 5 mM biotin (Fig. 3C).
TtBCCP-dynein was also applicable to another type of motility assay. In a TIRF (total internal reflection fluorescence) assay, TtBCCP-dynein was labeled with Qdot605-streptavidin (Life Technologies; Tokyo, Japan) (SA-Qdot), and the behavior of Qdot-labeled dynein on microtubules was examined (Fig. 3D). Anti-FLAG antibody (for fixation of microtubules), microtubules, casein, TtBCCP-dynein, and SA-Qdot were introduced sequentially into the flow chamber. This procedure avoided aggregation of the SA-Qdot–dynein complex. After the addition of ATP, moving spots were observed along the microtubules (Fig. 3E). In previous studies, GFP-fused outer arm dynein moved processively along microtubules (Edamatsu, 2014), and similar results were obtained in this study. Qdot shows much higher fluorescence than GFP,
M. Edamatsu / Journal of Microbiological Methods 105 (2014) 150–154
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A
C
B
a
b
c
D
E d
Fig. 3. In vitro motility assays of TtBCCP-outer arm dynein. (A) Schematic representation of TtBCCP-based gliding assay. (B) Gliding of microtubules. The frames show 0 s (a), 0.5 s (b) and 1.0 s (c) after recording was initiated. The arrows represent a gliding microtubule. The scale bar represents 3 μm. (C) Histogram of gliding velocity. Average velocity ± standard deviation is shown. The inset shows the control experiment, in which samples were preincubated with 5 mM biotin. The scale bar represents 5 μm. (D) Schematic representation of the TtBCCP-based TIRF assay. (E) Movement of Qdot-labeled dynein. Qdot-labeled dynein spots are colored green, and the microtubule is colored red. The frames represent 0 s (a), 15 s (b), and 30 s (c) after recording was initiated. The scale bar represents 2 μm. The arrows represent movement of Qdot-labeled dynein spots. (d) Kymograph of the Qdot-labeled dynein. The arrows represent the moving spots along a microtubule. The scale bars represent 4 μm.
allowing the motile properties of Qdot-labeled dynein to be analyzed using FIONA (fluorescence imaging with one-nanometer accuracy) (Yildiz et al., 2003). The small TtBCCP tag can be introduced into the tail or other regions of an axonemal dynein and can bind a wide variety of ligands with high affinity and specificity. FIONA analysis using highly fluorescent molecules allows the examination of the behaviors of diverse axonemal dyneins on microtubules at nanometer resolution. Cryo-electron tomography using avidin-conjugated ligands enables the identification of diverse Tetrahymena dyneins in the axoneme, as well as the classification of specific domains in certain isoforms. In addition, various TtBCCPfused axonemal dyneins could be used in novel nanodevices in the field of nano-biotechnology due to their unique properties. Currently, Tetrahymena is the only expression system for motile axonemal dyneins, and the TtBCCP tag system will thus provide opportunities for basic and applied molecular studies of axonemal dyneins.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2014.07.036. References Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a webbased environment for protein structure homology modelling. Bioinformatics 22, 195–201. Bachand, G.D., Bouxsein, N.F., VanDelinder, V., Bachand, M., 2014. Biomolecular motors in nanoscale materials, devices, and systems. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 6, 163–177. Badano, J.L.,Mitsuma, N.,Beales, P.L.,Katsanis, N., 2006. The ciliopathies: an emerging class of human genetic disorders. Annu. Rev. Genomics Hum. Genet. 7, 125–148. Chen, Y., Elizondo-Noriega, A., Cantu, D.C.,Reilly, P.J., 2012. Structural classification of biotin carboxyl carrier proteins. Biotechnol. Lett. 34, 1869–1875. Edamatsu, M., 2014. The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena. Biochem. Biophys. Res. Commun. 447, 596–601. Gibbons, I.R., 1995. Dynein family of motor proteins: present status and future questions. Cell Motil. Cytoskeleton 32, 136–144.
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Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723. Healy, S.,McDonald, M.K.,Wu, X.,Yue, W.W.,Kochan, G.,Oppermann, U.,Gravel, R.A., 2010. Structural impact of human and Escherichia coli biotin carboxyl carrier proteins on biotin attachment. Biochemistry 49, 4687–4694. Hess, H., 2011. Engineering applications of biomolecular motors. Annu. Rev. Biomed. Eng. 13, 429–450. Kamiya, R., 2002. Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants. Int. Rev. Cytol. 219, 115–155. Marshall, W.F., 2008. The cell biological basis of ciliary disease. J. Cell Biol. 180, 17–21. Polyak, S.W., Chapman-Smith, A., Mulhern, T.D., Cronan Jr., J.E., Wallace, J.C., 2001. Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains. J. Biol. Chem. 276, 3037–3045. Satir, P., Christensen, S.T., 2008. Structure and function of mammalian cilia. Histochem. Cell Biol. 129, 687–693.
Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., 2003. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res. 31, 3381–3385. Vale, R.D., 2000. AAA proteins. Lords of the ring. J. Cell Biol. 150, F13–F19. van den Heuvel, M.G., Dekker, C., 2007. Motor proteins at work for nanotechnology. Science 317, 333–336. Wilkes, D.E., Watson, H.E., Mitchell, D.R., Asai, D.J., 2008. Twenty-five dyneins in Tetrahymena: a re-examination of the multidynein hypothesis. Cell Motil. Cytoskeleton 65, 342–351. Yildiz, A., Forkey, J.N., McKinney, S.A., Ha, T., Goldman, Y.E., Selvin, P.R., 2003. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Identification of biotin carboxyl carrier protein in Tetrahymena and its application in in vitro motility systems of outer arm dynein Masaki Edamatsu ⁎ Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Tokyo, Japan
a r t i c l e
i n f o
Article history: Received 26 July 2014 Accepted 26 July 2014 Available online 6 August 2014
a b s t r a c t Axonemal dynein plays a central role in ciliary beating. Recently, a functional expression system of axonemal dynein was established in the ciliated protozoan Tetrahymena. This study identifies biotin carboxyl carrier protein (BCCP) in Tetrahymena and demonstrates its application in in vitro motility systems of outer arm dynein. © 2014 Elsevier B.V. All rights reserved.
Keywords: Tetrahymena Ciliated protozoan Axonemal dynein Biotin carboxyl carrier protein (BCCP) Biomolecular motor Ciliary movement
Ciliary movement is driven by axonemal dyneins and the impaired motility of the dyneins causes primary ciliary dyskinesia (PCD) (Badano et al., 2006; Marshall, 2008; Satir and Christensen, 2008). Axonemal dyneins are large AAA+ (ATPases associated with diverse cellular activities)-type motors (500–1500 kDa) and are divided into inner arm and outer arm dyneins (Gibbons, 1995; Vale, 2000; Kamiya, 2002). In Tetrahymena, there are a total of 23 axonemal dynein heavy chain genes (Wilkes et al., 2008). Axonemal dyneins are activated in a coordinated manner during ciliary beating, and this coordinated activation is a specific property of axonemal dyneins. These motors could be utilized as unique motile elements for nanoscale devices in the field of nanobiotechnology (Bachand et al., 2014; van den Heuvel and Dekker, 2007; Hess, 2011). An expression system for the genetic engineering of axonemal dynein has recently been developed in Tetrahymena (Edamatsu, 2014). Although this expression system is useful for functional and structural studies of axonemal dyneins and ciliary movement, a versatile system for tagging axonemal dyneins is needed to further advance molecular studies of these proteins. In this study, a versatile BCCP tag was identified in Tetrahymena and applied to in vitro motility systems of axonemal dynein. BCCP is one part of a biotin-dependent enzyme and is found in most prokaryotes and eukaryotes (Chen et al., 2012). BCCP consists of approximately 70 amino acids and is biotinylated by biotin protein ligase ⁎ Corresponding author at: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-0041, Japan. Tel.: +81 3 5454 6158. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.mimet.2014.07.036 0167-7012/© 2014 Elsevier B.V. All rights reserved.
(BPL) in culture. The high affinity and specificity of BCCP for avidin offers an advantage in the binding of biotinylated motors to avidinconjugated or coated materials. It has been reported that intrinsic BCCP is efficiently biotinylated in host cells (Healy et al., 2010; Polyak et al., 2001); therefore, a search for the BCCP sequence was performed in the Tetrahymena Genome Database (TGD) Wiki (http://ciliate.org/index.php/home/welcome). The BCCP family is classified into 4 subfamilies, and 15 representative species from each subfamily [listed in (Chen et al., 2012)] were used for the homology search. One uncharacterized protein (gene ID: TTHERM_00502240) homologous to carbamoyl phosphate synthase (CPSase) was identified in the database. The domain structure of this protein is shown in Fig. 1A. The C-terminus of the protein is a BCCPlike domain containing the biotin-attachment consensus sequence (Healy et al., 2010) (Fig. 1B and D). As described below, this domain was biotinylated in Tetrahymena cells and was thus identified as Tetrahymena BCCP (TtBCCP). TtBCCP was close to subfamily C members in the phylogenetic analysis (Fig. 1E). Fig. 1C shows a homology model of TtBCCP based on the structure of Escherichia coli BCCP. TtBCCP was found to lack the “thumb loop”, which contributes to substrate specificity in the BPL enzyme (Healy et al., 2010) (Fig. 1C and D). Next, a TtBCCP-fused outer arm dynein was generated. The Tetrahymena outer arm dynein forms a three-headed structure comprising alpha (DYH3), beta (DYH4) and gamma (DYH5) heavy chains (Wilkes et al., 2008). The expression cassette targeting the N-terminus of DYH3 is shown in (Fig. 2A and B). The PCR primers used in this study are shown in Supplemental Table S1. Transformations were performed according to (Edamatsu, 2014), and homologous recombination was confirmed by PCR analysis (Fig. 2C and D). Cilia were labeled with
M. Edamatsu / Journal of Microbiological Methods 105 (2014) 150–154
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A
B
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Fig. 1. Identification and characterization of TtBCCP. (A) Domain structure of the CPSase-like protein identified in Tetrahymena. (B) Nucleotide and deduced amino acid sequences of TtBCCP. (C) Homology modeling of TtBCCP using SWISS-MODEL tools (Guex and Peitsch, 1997; Schwede et al., 2003; Arnold et al., 2006). TtBCCP (green) and Escherichia coli BCCP (gray, PDB code 1 BDO) were superimposed using PyMOL software (http://www.pymol.org). The biotinylated lysine in E. coli BCCP is also shown. (D) Alignment of BCCP domains. The aligned sequences are E. coli BCCP (EcBCCP) [P0ABE0], Arabidopsis thaliana BCCP (AtBCCP) [Q9LLC1], Schizosaccharomyces pombe BCCP (SpBCCP) [P78820], Homo sapiens BCCP (HsBCCP) [Q13085], and Tetrahymena thermophila (TtBCCP) [TTHERM_00502240]. The arrow represents the biotin attachment site. The underline represents the thumb loop. (E) Phylogenic tree of BCCPs constructed using MEGA 6 software (http://www.megasoftware.net).
Cy3-streptavidin (Cy3-SA) in permeabilized cells (Fig. 2E). The Tetrahymena oral apparatus was also labeled with Cy3-SA because it contains a large number of cilia. In control experiments, fluorescence in the cilia and in the oral apparatus was quenched by preincubation of Cy3-SA with 5 mM biotin (Fig. 2E).
Biotinylation of TtBCCP-dynein was performed in proteose peptone medium (containing 74 nM biotin, as estimated from the manufacturer's manual), and the addition of 0–100 μM biotin to the medium did not significantly enhance biotinylation of the recombinant dynein (Fig. 2F). TtBCCP-dynein was purified from ciliary extracts
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M. Edamatsu / Journal of Microbiological Methods 105 (2014) 150–154
A
B
D
C
E
a
F
b
G
Fig. 2. Construction and purification of the TtBCCP-outer arm dynein in Tetrahymena. (A) Domain structure of DYH3. (B) The expression cassette for TtBCCP-DYH3. F1 (1.8-kb untranslated region of DYH3), neo (neomycin resistance gene), BTU1P (beta-tubulin 1 promoter), His6-FLAG-TtBCCP, and F2 (0.9-kb coding region of DYH3) were sequentially cloned into the pGBKT7 vector. (C) Schematic representation of homologous recombination in the expression cassette. The arrows represent the PCR primers used in (D). (D) PCR analysis. Lanes 1 and 2: PCR using cFw1 and cRv1 primers; lanes 3 and 4: PCR using cFw2 and cRv2 primers. Lanes 1 and 3: wild-type; lanes 2 and 4: transformants. (E) Localization of TtBCCP-DYH3. Permeabilized cells labeled with Cy3-SA (a) or Cy3-SA preincubated with 5 mM biotin (b). Scale bar represents 5 μm. (F) Biotinylation of TtBCCP-DYH3. Axonemal proteins of transformants grown in culture medium supplemented with 0–100 μM biotin were examined by avidin-blot analysis. CBB staining (upper panel) and HRP-streptavidin staining (lower panel) are shown. (G) Purification and avidin-blot analysis of TtBCCP-outer arm dynein. Lanes 1 and 2: CBB staining; lanes 3 and 4: Cy3–SA staining. Lanes 1 and 3: native outer arm dynein; lanes 2 and 4: TtBCCP-outer arm dynein.
according to (Edamatsu, 2014). The purified protein was specifically labeled with Cy3-SA and confirmed to be biotinylated dynein (Fig. 2G). Next, TtBCCP was applied to in vitro motility assays of outer arm dynein. First, recombinant dynein was fixed on glass slides via streptavidin–biotin binding, and dynein motility was examined (gliding assay, Fig. 3A). Biotinylated BSA, streptavidin, casein, TtBCCP-dynein, and microtubules were introduced sequentially into the flow chamber. The gliding velocity was 4.4 ± 1.3 μm/s (Fig. 3B and C), which was comparable to the gliding velocity of GFP-fused outer arm dynein (Edamatsu, 2014). In control experiments, few microtubules were found in the flow chamber after preincubation with 5 mM biotin (Fig. 3C).
TtBCCP-dynein was also applicable to another type of motility assay. In a TIRF (total internal reflection fluorescence) assay, TtBCCP-dynein was labeled with Qdot605-streptavidin (Life Technologies; Tokyo, Japan) (SA-Qdot), and the behavior of Qdot-labeled dynein on microtubules was examined (Fig. 3D). Anti-FLAG antibody (for fixation of microtubules), microtubules, casein, TtBCCP-dynein, and SA-Qdot were introduced sequentially into the flow chamber. This procedure avoided aggregation of the SA-Qdot–dynein complex. After the addition of ATP, moving spots were observed along the microtubules (Fig. 3E). In previous studies, GFP-fused outer arm dynein moved processively along microtubules (Edamatsu, 2014), and similar results were obtained in this study. Qdot shows much higher fluorescence than GFP,
M. Edamatsu / Journal of Microbiological Methods 105 (2014) 150–154
153
A
C
B
a
b
c
D
E d
Fig. 3. In vitro motility assays of TtBCCP-outer arm dynein. (A) Schematic representation of TtBCCP-based gliding assay. (B) Gliding of microtubules. The frames show 0 s (a), 0.5 s (b) and 1.0 s (c) after recording was initiated. The arrows represent a gliding microtubule. The scale bar represents 3 μm. (C) Histogram of gliding velocity. Average velocity ± standard deviation is shown. The inset shows the control experiment, in which samples were preincubated with 5 mM biotin. The scale bar represents 5 μm. (D) Schematic representation of the TtBCCP-based TIRF assay. (E) Movement of Qdot-labeled dynein. Qdot-labeled dynein spots are colored green, and the microtubule is colored red. The frames represent 0 s (a), 15 s (b), and 30 s (c) after recording was initiated. The scale bar represents 2 μm. The arrows represent movement of Qdot-labeled dynein spots. (d) Kymograph of the Qdot-labeled dynein. The arrows represent the moving spots along a microtubule. The scale bars represent 4 μm.
allowing the motile properties of Qdot-labeled dynein to be analyzed using FIONA (fluorescence imaging with one-nanometer accuracy) (Yildiz et al., 2003). The small TtBCCP tag can be introduced into the tail or other regions of an axonemal dynein and can bind a wide variety of ligands with high affinity and specificity. FIONA analysis using highly fluorescent molecules allows the examination of the behaviors of diverse axonemal dyneins on microtubules at nanometer resolution. Cryo-electron tomography using avidin-conjugated ligands enables the identification of diverse Tetrahymena dyneins in the axoneme, as well as the classification of specific domains in certain isoforms. In addition, various TtBCCPfused axonemal dyneins could be used in novel nanodevices in the field of nano-biotechnology due to their unique properties. Currently, Tetrahymena is the only expression system for motile axonemal dyneins, and the TtBCCP tag system will thus provide opportunities for basic and applied molecular studies of axonemal dyneins.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2014.07.036. References Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a webbased environment for protein structure homology modelling. Bioinformatics 22, 195–201. Bachand, G.D., Bouxsein, N.F., VanDelinder, V., Bachand, M., 2014. Biomolecular motors in nanoscale materials, devices, and systems. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 6, 163–177. Badano, J.L.,Mitsuma, N.,Beales, P.L.,Katsanis, N., 2006. The ciliopathies: an emerging class of human genetic disorders. Annu. Rev. Genomics Hum. Genet. 7, 125–148. Chen, Y., Elizondo-Noriega, A., Cantu, D.C.,Reilly, P.J., 2012. Structural classification of biotin carboxyl carrier proteins. Biotechnol. Lett. 34, 1869–1875. Edamatsu, M., 2014. The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena. Biochem. Biophys. Res. Commun. 447, 596–601. Gibbons, I.R., 1995. Dynein family of motor proteins: present status and future questions. Cell Motil. Cytoskeleton 32, 136–144.
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Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723. Healy, S.,McDonald, M.K.,Wu, X.,Yue, W.W.,Kochan, G.,Oppermann, U.,Gravel, R.A., 2010. Structural impact of human and Escherichia coli biotin carboxyl carrier proteins on biotin attachment. Biochemistry 49, 4687–4694. Hess, H., 2011. Engineering applications of biomolecular motors. Annu. Rev. Biomed. Eng. 13, 429–450. Kamiya, R., 2002. Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants. Int. Rev. Cytol. 219, 115–155. Marshall, W.F., 2008. The cell biological basis of ciliary disease. J. Cell Biol. 180, 17–21. Polyak, S.W., Chapman-Smith, A., Mulhern, T.D., Cronan Jr., J.E., Wallace, J.C., 2001. Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains. J. Biol. Chem. 276, 3037–3045. Satir, P., Christensen, S.T., 2008. Structure and function of mammalian cilia. Histochem. Cell Biol. 129, 687–693.
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