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Modulation of the Stathmin-like Microtubule Destabilizing Activity of RB3, a Neuron-specific Member of the SCG10 Family, by Its N-terminal Domain

2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês

10.1074/jbc.m313693200

ISSN

1083-351X

Autores

Chitose Nakao, Tomohiko J. Itoh, Hirokazu Hotani, Nozomu Mori,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

RB3 is a neuron-specific homologue of the SCG10/stathmin family proteins, possessing a unique N-terminal membrane-associated domain and the stathmin-like domain at the C terminus, which promotes microtubule (MT) catastrophe and/or tubulin sequestering. We examined herein the contribution of the N-terminal subdomain of RB3 to the regulation of MT dynamics. To begin with, we determined the effects of full-length (RB3-f) and short truncated (RB3-s) forms of RB3 on the polymerization of MT in vitro. RB3-s had a deletion of amino acids 1–75 from the N terminus, leaving the so-called stathmin-like domain, consisting of residues 76–217. Although both RB3-f and RB3-s exhibited MT-depolymerizing activity, RB3-f was less effective. The binding affinity for tubulin was also lower in RB3-f. Direct observation of the dynamics of individual MTs using dark field microscopy revealed that RB3-s slowed MT elongation velocity, increased catastrophes, and reduced rescues. This effect is almost identical to that by stathmin/oncoprotein 18. On the other hand, the MT elongation rate increased at lower concentrations of RB3-f. In addition, RB3-f, indicated higher rescue frequency than control as well as the catastrophe in a dose-dependent manner. The functionality of RB3-f indicated that full-length RB3 has not only stathmin-like MT destabilizing activity but also MT-associated protein-like MT stabilizing activity. Possibly, the balance of these activities is altered in a concentration-dependent manner in vitro. This interesting regulatory role of the unique N-terminal domain of RB3 in MT dynamics would contribute to the physiological regulation of neuronal morphogenesis. RB3 is a neuron-specific homologue of the SCG10/stathmin family proteins, possessing a unique N-terminal membrane-associated domain and the stathmin-like domain at the C terminus, which promotes microtubule (MT) catastrophe and/or tubulin sequestering. We examined herein the contribution of the N-terminal subdomain of RB3 to the regulation of MT dynamics. To begin with, we determined the effects of full-length (RB3-f) and short truncated (RB3-s) forms of RB3 on the polymerization of MT in vitro. RB3-s had a deletion of amino acids 1–75 from the N terminus, leaving the so-called stathmin-like domain, consisting of residues 76–217. Although both RB3-f and RB3-s exhibited MT-depolymerizing activity, RB3-f was less effective. The binding affinity for tubulin was also lower in RB3-f. Direct observation of the dynamics of individual MTs using dark field microscopy revealed that RB3-s slowed MT elongation velocity, increased catastrophes, and reduced rescues. This effect is almost identical to that by stathmin/oncoprotein 18. On the other hand, the MT elongation rate increased at lower concentrations of RB3-f. In addition, RB3-f, indicated higher rescue frequency than control as well as the catastrophe in a dose-dependent manner. The functionality of RB3-f indicated that full-length RB3 has not only stathmin-like MT destabilizing activity but also MT-associated protein-like MT stabilizing activity. Possibly, the balance of these activities is altered in a concentration-dependent manner in vitro. This interesting regulatory role of the unique N-terminal domain of RB3 in MT dynamics would contribute to the physiological regulation of neuronal morphogenesis. Microtubules (MTs), 1The abbreviations used are: MT, microtubule; RB3-f, full-length RB3; RB3-s, N-terminal short truncated form of RB3; MAP, MT-associated protein; NGF, nerve growth factor; PIPES, 1,4-piperazinedi-ethanesulfonic acid; HAT, histidine affinity tag. polymers of tubulin heterodimers, are an important cytoskeleton in eukaryotes. Their dynamics are regulated, depending on various cellular activities, and a variety of regulatory proteins have been identified so far. Stathmin/oncoprotein 18 family proteins are one of the protein families that make MTs labile (1Mori N. Morii H. J. Neurosci. Res. 2002; 70: 264-273Crossref PubMed Scopus (107) Google Scholar, 2Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (366) Google Scholar, 3Walczak C.E. Curr. Opin. Cell Biol. 2000; 12: 52-56Crossref PubMed Scopus (151) Google Scholar). It has been demonstrated that one stathmin molecule forms a ternary complex with two tubulin heterodimers, termed the "T2S complex," via hydrophobic interaction (4Gigant B. Curmi P.A. Martin-Barbey C. Charbaut E. Lachkar S. Lebeau L. Siavoshian S. Sobel A. Knossow M. Cell. 2000; 102: 809-816Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 5Honnappa S. Cutting B. Jahnke W. Seelig J. Steinmetz M.O. J. Biol. Chem. 2003; 278: 38926-38934Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The formation of the T2S complex can alter conditions leading to the depolymerization of MTs (4Gigant B. Curmi P.A. Martin-Barbey C. Charbaut E. Lachkar S. Lebeau L. Siavoshian S. Sobel A. Knossow M. Cell. 2000; 102: 809-816Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 6Steinmetz M.O. Jahnke W. Towbin H. Garcia-Echeverria C. Voshol H. Muller D. van Oostrum J. EMBO Rep. 2001; 2: 505-510Crossref PubMed Scopus (51) Google Scholar, 7Charbaut E. Curmi P.A. Ozon S. Lachkar S. Redeker V. Sobel A. J. Biol. Chem. 2001; 276: 16146-16154Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 8Segerman B. Larsson N. Holmfeldt P. Gullberg M. J. Biol. Chem. 2000; 275: 35759-35766Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 9Jourdain L. Curmi P. Sobel A. Pantaloni D. Carlier M.F. Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar, 10Curmi P.A. Andersen S.S. Lachkar S. Gavet O. Karsenti E. Knossow M. Sobel A. J. Biol. Chem. 1997; 272: 25029-25036Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Stathmin has also been found to act on GTP-tubulin at MT ends to stimulate the hydrolysis of GTP and to promote the dissociation of GTP-tubulin from the growing ends of MTs (8Segerman B. Larsson N. Holmfeldt P. Gullberg M. J. Biol. Chem. 2000; 275: 35759-35766Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 11Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (159) Google Scholar). Based on these results, two models were proposed to account for the stathmin-dependent destabilization of MTs: 1) sequestration of tubulin dimers to prevent their assembly or 2) stimulation of MT catastrophe at MT tips by removing a "GTP cap" that protects the MT from catastrophe. SCG10 family proteins, including SCG10, SCLIP, and RB3, show a high degree of sequence homology to stathmin in their C terminus, termed the "stathmin-like domain." The various stathmin-like domains including the predicted α-helix display 65–75% amino acid identity with stathmin (7Charbaut E. Curmi P.A. Ozon S. Lachkar S. Redeker V. Sobel A. J. Biol. Chem. 2001; 276: 16146-16154Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 12Beilharz E.J. Zhukovsky E. Lanahan A.A. Worley P.F. Nikolich K. Goodman L.J. J. Neurosci. 1998; 18: 9780-9789Crossref PubMed Google Scholar). They have properties in common with of stathmin, although they differ slightly in activity (7Charbaut E. Curmi P.A. Ozon S. Lachkar S. Redeker V. Sobel A. J. Biol. Chem. 2001; 276: 16146-16154Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 13Brannstrom K. Segerman B. Gullberg M. J. Biol. Chem. 2003; 278: 16651-16657Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In addition to their stathmin-like domain, family members possess unique N-terminal domains, which contain two Cys residues that serve as palmitoylation sites (14Lutjens R. Igarashi M. Pellier V. Blasey H. Di Paolo G. Ruchti E. Pfulg C. Staple J.K. Catsicas S. Grenningloh G. Eur. J. Neurosci. 2000; 12: 2224-2234Crossref PubMed Scopus (66) Google Scholar). These sites mediate the association to intracellular membranes and the transport to growth cones (15Di Paolo G. Lutjens R. Osen-Sand A. Sobel A. Catsicas S. Grenningloh G. J. Neurosci. Res. 1997; 50: 1000-1009Crossref PubMed Scopus (57) Google Scholar, 16Grenningloh G. Soehrman S. Bondallaz P. Ruchti E. Cadas H. J. Neurobiol. 2004; 58: 60-69Crossref PubMed Scopus (141) Google Scholar). In contrast to the ubiquitous cytosolic stathmin, these proteins, excluding SCLIP (17Bieche I. Maucuer A. Laurendeau I. Lachkar S. Spano A.J. Frankfurter A. Levy P. Manceau V. Sobel A. Vidaud M. Curmi P.A. Genomics. 2003; 81: 400-410Crossref PubMed Scopus (49) Google Scholar), are highly expressed in the nervous system (18Ozon S. El Mestikawy S. Sobel A. J. Neurosci. Res. 1999; 56: 553-564Crossref PubMed Scopus (43) Google Scholar) and localized to the Golgi apparatus and the growth cone (14Lutjens R. Igarashi M. Pellier V. Blasey H. Di Paolo G. Ruchti E. Pfulg C. Staple J.K. Catsicas S. Grenningloh G. Eur. J. Neurosci. 2000; 12: 2224-2234Crossref PubMed Scopus (66) Google Scholar, 15Di Paolo G. Lutjens R. Osen-Sand A. Sobel A. Catsicas S. Grenningloh G. J. Neurosci. Res. 1997; 50: 1000-1009Crossref PubMed Scopus (57) Google Scholar, 16Grenningloh G. Soehrman S. Bondallaz P. Ruchti E. Cadas H. J. Neurobiol. 2004; 58: 60-69Crossref PubMed Scopus (141) Google Scholar, 19Di Paolo G. Lutjens R. Pellier V. Stimpson S.A. Beuchat M.H. Catsicas S. Grenningloh G. J. Biol. Chem. 1997; 272: 5175-5182Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Stathmin is also abundant in the developing nervous system, but its regional expression in the brain is quite distinct from that of SCG10 (20Himi T. Okazaki T. Wang H. McNeill T.H. Mori N. Neuroscience. 1994; 60: 907-926Crossref PubMed Scopus (65) Google Scholar). The level of SCG10 protein is very low in native PC12 cells but is strongly increased upon the nerve growth factor (NGF)-dependent induction of differentiation (21Di Paolo G. Pellier V. Catsicas M. Antonsson B. Catsicas S. Grenningloh G. J. Cell Biol. 1996; 133: 1383-1390Crossref PubMed Scopus (66) Google Scholar, 22Stein R. Mori N. Matthews K. Lo L.C. Anderson D.J. Neuron. 1988; 1: 463-476Abstract Full Text PDF PubMed Scopus (199) Google Scholar). Additionally, PC12 cells that constitutively expressed SCG10 showed a dramatic increase in the tendency to form elongated neurites upon NGF-induced neuronal differentiation, although no neurite outgrowth was observed in the absence of NGF (23Riederer B.M. Pellier V. Antonsson B. Di Paolo G. Stimpson S.A. Lutjens R. Catsicas S. Grenningloh G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 741-745Crossref PubMed Scopus (170) Google Scholar). On the other hand, Di Paolo et al. (21Di Paolo G. Pellier V. Catsicas M. Antonsson B. Catsicas S. Grenningloh G. J. Cell Biol. 1996; 133: 1383-1390Crossref PubMed Scopus (66) Google Scholar) showed in PC12 cells that a selective blockade of stathmin expression by phosphorothioate antisense oligonucleotides prevented the differentiation-promoting actions of NGF. Stathmin-depleted PC12 cells produce SCG10 protein normally upon NGF treatment but do not differentiate, suggesting that SCG10 cannot compensate for the function of stathmin (23Riederer B.M. Pellier V. Antonsson B. Di Paolo G. Stimpson S.A. Lutjens R. Catsicas S. Grenningloh G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 741-745Crossref PubMed Scopus (170) Google Scholar). These results have led to the proposal that the functions of stathmin family proteins are considered to be diverged during neural evolution (1Mori N. Morii H. J. Neurosci. Res. 2002; 70: 264-273Crossref PubMed Scopus (107) Google Scholar). The stable ternary complex with αβ tubulin heterodimers is formed via the stathmin-like domain (4Gigant B. Curmi P.A. Martin-Barbey C. Charbaut E. Lachkar S. Lebeau L. Siavoshian S. Sobel A. Knossow M. Cell. 2000; 102: 809-816Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 6Steinmetz M.O. Jahnke W. Towbin H. Garcia-Echeverria C. Voshol H. Muller D. van Oostrum J. EMBO Rep. 2001; 2: 505-510Crossref PubMed Scopus (51) Google Scholar, 24Steinmetz M.O. Kammerer R.A. Jahnke W. Goldie K.N. Lustig A. van Oostrum J. EMBO J. 2000; 19: 572-580Crossref PubMed Scopus (88) Google Scholar), but N- and C-terminal flanks of the domain are required for cooperative binding to the tubulin heterodimers (8Segerman B. Larsson N. Holmfeldt P. Gullberg M. J. Biol. Chem. 2000; 275: 35759-35766Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This suggests that the terminal flanks in addition to the binding motifs in contact with tubulin heterodimers play important roles in the regulation of the tubulin-binding and/or MT dynamics. Since the unique N-terminal domain is one of the most important characteristics of SCG10 family proteins, it is possible that it too affects the interaction with tubulin and/or MT dynamics. Although studies of the SCG10 family have provided important information on their stathmin-like actions, the effects of full-length neural SCG10 proteins on the regulation of MTs are still unresolved. In this paper, we investigated the role of the N-terminal domain of RB3, which is the longest N-terminal domain among member of the SCG10 family, to examine whether the unique N-terminal domain of SCG10 proteins is involved in the regulation of MT dynamics. We compared the effects of full-length RB3 and an N-terminally truncated RB3 in vitro using MT assays and found that the full-length RB3 plays a unique role in regulating MT dynamics. DNA Construction, Protein Expression, and Purification—The isolation and manipulation of DNA were performed using standard techniques. Three RB3 gene transcripts have been identified, but since the gene expression of RB3 splicing variants is similar in rat brain and in PC12 cells stimulated with NGF (12Beilharz E.J. Zhukovsky E. Lanahan A.A. Worley P.F. Nikolich K. Goodman L.J. J. Neurosci. 1998; 18: 9780-9789Crossref PubMed Google Scholar), we used the longest form. Bacterial expression plasmids for the rat full-length RB3 (RB3-f) and an N-terminally truncated mutant (RB3-s, comprising residues 76–217) were constructed by subcloning the cDNAs into the SalI and EcoRI sites of the HAT fusion vector pHAT10 (Clontech) (see Fig. 1). The RB3 derivatives were expressed with a HAT tag at the N terminus and purified from Escherichia coli as follows. The collected cells were resuspended in a lysis buffer (8 m urea, 50 mm sodium phosphate (pH 7.0), and 0.3 m NaCl) and incubated for 15 min at room temperature. Then the samples were centrifuged at 30,000 × g for 30 min at 10 °C. After adding TALON metal affinity resins (Clontech) were added to the supernatants, and samples were incubated for 1 h at room temperature. The HAT-RB3-bound resins were washed once with a wash buffer (50 mm sodium phosphate, pH 7.0, and 0.3 m NaCl) containing different concentrations of urea (8, 4, 2, 1, 0.5, and 0 m, in that order) and once with 50 mm sodium phosphate, pH 7.0, containing 5 mm imidazole. Finally, protein was eluted from the resins in 50 mm sodium phosphate, pH 7.0, containing 100 mm imidazole. The buffer was changed to PM buffer (0.1 m PIPES, pH 6.9, 1 mm EGTA, and 0.5 mm MgSO4) by centrifugal filtration. Tubulin was isolated from porcine brain by two cycles of polymerization and depolymerizatin and was purified by DEAE-Sepharose column chromatography using fast protein liquid chromatography (Amersham Biosciences) as described previously (25Itoh T.J. Hisanaga S. Hosoi T. Kishimoto T. Hotani H. Biochemistry. 1997; 36: 12574-12582Crossref PubMed Scopus (67) Google Scholar). Protein concentrations were determined by SDS-PAGE with Coomassie Brilliant Blue staining using bovine serum albumin as a standard. Purified tubulin and RB3 recombinant proteins were stored at –80 °C prior to use. Cosedimentation Assay of MTs—The MT destabilizing activity and binding with RB3 derivatives were examined in a co-sedimentation experiment as follows. Tubulin (10 μm) was assembled in PM buffer containing 1 mm GTP in a final volume of 20 μl in the presence of various concentrations of RB3-f or RB3-s (0–10 μm) for 15 min at 37 °C. The polymerized MTs were sedimented by ultracentrifugation at 70,000 rpm for 60 min at 36 °C. The resulting supernatants and pellets were examined with 4–20% gradient SDS-PAGE. After Coomassie Brilliant Blue staining, the band signals were analyzed using the program NIH Image. The data were fit by linear or nonlinear regression using the standard binding equation for a macromolecule to obtain the apparent dissociation equilibrium constant. Curve fitting was performed using a Kaleida Graph (Synergy Software). Quantitative Analysis of the Binding between Tubulin and RB3 Derivatives—The binding affinity between RB3 derivatives and tubulin was determined by Scatchard analysis. RB3 derivatives (1 μm) and purified tubulin (0.5–10 μm) were mixed in a final volume of 35 μl in a reaction buffer (0.1 m PIPES, pH 6.9, 0.5 mm MgSO4, 1 mm GTP, 5% Tween, and 1 mg/ml bovine serum albumin) and were incubated for 15 min on ice. RB3-tubulin complexes were captured with 5 μl of TALON metal affinity resin for 30 min at 4 °C. In the present study, we used a low concentration of tubulin heterodimers and an incubation temperature of 4 °C, because these conditions favor the formation of tubulin heterodimers rather than polymers (26Detrich III, H.W. Williams R.C. Biochemistry. 1978; 17: 3900-3907Crossref PubMed Scopus (145) Google Scholar). To confirm the amount of tubulin bound to RB3, the bound proteins were separated by 4–20% gradient SDS-PAGE, subjected to silver staining, and analyzed using NIH Image. The data were normalized by subtraction of the nonspecific binding signals. Optical Microscopic Observation of MT Dynamics—To determine the exact effects of RB3s on MT dynamics, individual MTs were monitored using a dark field microscope with several modifications (27Ichihara K. Kitazawa H. Iguchi Y. Hotani H. Itoh T.J. J. Mol. Biol. 2001; 312: 107-118Crossref PubMed Scopus (19) Google Scholar, 28Ookata K. Hisanaga S. Bulinski J.C. Murofushi H. Aizawa H. Itoh T.J. Hotani H. Okumura E. Tachibana K. Kishimoto T. J. Cell Biol. 1995; 128: 849-862Crossref PubMed Scopus (240) Google Scholar, 29Horio T. Hotani H. Nature. 1986; 321: 605-607Crossref PubMed Scopus (409) Google Scholar). First, 24 μm of tubulin was added to allow the polymerization of MTs in PM buffer containing 1 mm GTP at 37 °C for 5 min. An equal volume of prewarmed RB3 recombinant protein solution (0–9 μm) was added to the MT solution, the mixture was transferred to a glass slide, and the slide was immediately sealed. The final tubulin concentration was 12 μm. The optical microscopy was performed at 24 °C. During observations, bovine serum albumin (1.8 mg/ml) was added to prevent the nonspecific attachment of proteins to the glass surface. The dynamic behavior of the MTs was observed using a dark field microscope (ECLIPSE E600; Nikon) with a plan fluor × 100 objective lens and an oil immersion dark field condenser equipped with a 100-watt high pressure mercury lamp as the illumination source. Images were observed with a CCD camera (IR-1000; DAGE-MTI) and were recorded onto a one-half inch SVHS videotape with a Panasonic model AG 3750 videocassette recorder for 40 min. Changes in MT length were measured from the video images incorporated into a Power Mac G4 with the aid of an image capture board (LG3; Scion) in real time using NIH Image. The change in length at each end of a given MT was followed by determining the distance between each end and an arbitrary fixed point on the MT as a function of time. The two ends of MTs indicated the different polymerization rates; the faster growing end is referred to as the plus end and the slower growing end as the minus end (30Allen C. Borisy G.G. J. Mol. Biol. 1974; 90: 381-402Crossref PubMed Scopus (152) Google Scholar). Whereas the minus ends of MTs are relatively stable (31Baas P.W. Black M.M. J. Cell Biol. 1990; 111: 495-509Crossref PubMed Scopus (285) Google Scholar), the plus ends undergo variable phases of assembly and disassembly, also referred to as dynamic instability (32Mitchison T. Kirschner M. Nature. 1984; 312: 237-242Crossref PubMed Scopus (2369) Google Scholar). The dynamic instability of minus ends is probably not physiologically relevant, because minus ends in cells are either capped by other proteins (e.g. at centrosomes) or depolymerized when free in the cytoplasm (33Desai A. Mitchison T.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (1978) Google Scholar). Therefore, we examined only the MT plus ends. Data were shown as means ± S.D., and an unpaired t test was used to make comparisons between the control value and each means with the Macintosh software Statview (SAS Institute Inc.). MT Destabilizing Activity and Tubulin Binding Affinity Were Weakened in Full-length RB3 as Compared with RB3 Stathmin-like Domain—The activity of the recombinantly produced RB3 derivatives in MT assembly and the direct interaction of RB3 with MTs were tested using an MT sedimentation assay. Both recombinant proteins effectively inhibited tubulin assembly in a dose-dependent manner (Fig. 2A). This indicated that both proteins have MT destabilization activity. As shown in Fig. 2B, the linear regression analysis of the sedimented MTs and the concentration of RB3 used indicated that RB3-s reduces the concentration of MTs with a slope of –1.95, suggesting that the depolymerization was mainly due to the formation of the T2S complex and its sequestering activity. On the other hand, the slope of RB3-f, –1.37, is smaller than that of RB3-s, and complete inhibition of 10 μm tubulin polymerization required a higher concentration of RB3-f (about 9 μm) as compared with RB3-s (about 5 μm) (Fig. 2, A and B). These results suggest that the MT depolymerization activity is suppressed in RB3-f. Most of the tubulin and RB3 proteins were recovered in the supernatants when the polymerization of MTs was completely inhibited by a sufficient amount of RB3 protein. Neither RB3-f nor RB3-s was sedimented in the absence of tubulin (Fig. 2A), indicating that the "T2S" complex with RB3 or RB3 itself did not form large aggregates in the present conditions. However, both RB3-f and RB3-s were found in pellets at low concentrations when MTs were present (Fig. 2A). This suggests that the sedimented RB3 is bound to MTs. As shown in Fig. 2C, the molar ratios of both RB3 proteins to MT increased as the MT concentration decreased (i.e. as the amount of RB3 used increased) and was higher for RB3-f at all concentrations. Although all unbound RB3 is not practically "free" due to the formation of T2S complex, we further plotted the molar ratio against unbound RB3 concentrations, which was recovered in supernatant, as in Fig. 2D. This plot is not a real Langmuir isothermal plot, but we found apparent saturation of binding for each RB3 construct. From these curves, we obtained apparent equilibrium dissociation constants to MTs for RB3-f (0.43 μm) and for RB3-s (0.81 μm). These values suggest that the interaction between RB3 and MTs is enhanced by the existence of the unique N-terminal domain. It has been shown that the tubulin heterodimer-stathmin affinity in vitro is dependent on pH (10Curmi P.A. Andersen S.S. Lachkar S. Gavet O. Karsenti E. Knossow M. Sobel A. J. Biol. Chem. 1997; 272: 25029-25036Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 11Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (159) Google Scholar, 34Amayed P. Pantaloni D. Carlier M.F. J. Biol. Chem. 2002; 277: 22718-22724Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and there is relatively tight interaction between them at pH <7.0 (11Howell B. Larsson N. Gullberg M. Cassimeris L. Mol. Biol. Cell. 1999; 10: 105-118Crossref PubMed Scopus (159) Google Scholar). Hence, the tubulin binding activity of the stathmin-like domain may also play an important role in the MT regulatory mechanism of RB3 in the present buffer at pH 6.9. Thus, a Scatchard analysis was performed to determine the affinity between tubulin heterodimers and RB3 derivatives. As shown in Fig. 3, the dissociation constants (Kd values) for RB3-f and RB3-s were 0.57 ± 0.10 and 0.33 ± 0.10 μm, respectively. In previous studies (8Segerman B. Larsson N. Holmfeldt P. Gullberg M. J. Biol. Chem. 2000; 275: 35759-35766Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 35Larsson N. Segerman B. Howell B. Fridell K. Cassimeris L. Gullberg M. J. Cell Biol. 1999; 146: 1289-1302Crossref PubMed Scopus (60) Google Scholar), the Scatchard conversion of the binding between tubulin dimers and stathmin was shown to be a complex process with a nonlinear data point distribution typical of two-site positive cooperativity in binding. However, the data point distributed along a linear regression in the present study. This would not be due to the difference in the mode of binding between stathmin and our RB3 constructs. The difference is rather due to a weaker sensitivity for the detection of bound proteins in our experiments than in previous ones using a radioisotope as the protein tracer (8Segerman B. Larsson N. Holmfeldt P. Gullberg M. J. Biol. Chem. 2000; 275: 35759-35766Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Nevertheless, we emphasize that the Kd values were found to be suppressed in RB3-f as compared with RB3-s in this study. The present results suggested that the affinity of RB3-f for tubulin heterodimers is somehow suppressed, but that for MTs is enhanced as compared with RB3-s. The effects of RB3-f could be attributed to the unique N-terminal domain. Full-length RB3 Stabilizes MTs by Enhancing the Rescue from MT Depolymerization but Simultaneously Promotes MT Catastrophe—We used dark field microscopy to examine whether the N-terminal domain of RB3 alters the characteristic properties of the stathmin-like domain on MT assembly. The number of elongation or shortening phases and the total time observed in the present study were shown in Table I. Four parameters of MT dynamics were assessed: parameter 1, rate of elongation (Fig. 5A); parameter 2, rate of shortening (Fig. 5B); parameter 3, elongation length required for a catastrophe (the transition from elongation to shortening) (Fig. 6A); and parameter 4, shortening length required for a rescue (the transition from shortening to elongation) (Fig. 6B). Elongation or shortening length required for a transition event (catastrophe or rescue) was calculated by dividing the summed length of elongation or shortening phases by the total number of transition events, respectively (28Ookata K. Hisanaga S. Bulinski J.C. Murofushi H. Aizawa H. Itoh T.J. Hotani H. Okumura E. Tachibana K. Kishimoto T. J. Cell Biol. 1995; 128: 849-862Crossref PubMed Scopus (240) Google Scholar). These parameters were calculated to express how the level of ease (or difficulty) that ended each phase in the given condition, and the values might be rather comparable with the dynamicity that reflects how dynamic each microtubule is. Parameter 5 (mean length of elongation phase) (Fig. 6C) and parameter 6 (mean length of shortening phase) (Fig. 6D) were also calculated to know the variance of the data. Mean length is the simple average of the lengths of each phase lying between the starting transition event and ending transition event. Since each phase does not always end with the event and since the number of the transition event is smaller than the number of corresponding phases, it is impossible to obtain the S.D. value in obtaining the phase (elongation or shortening) length required for an event (catastrophe or rescue). In addition, the number of phases shown in Fig. 6, C and D, is usually smaller than that shown in Fig. 6, A and B, respectively. Especially, the number of the phase used in the calculation of the mean length of the shortening phase in the presence of 1.5 μm RB3-s (Fig. 6D) is obviously smaller than that of the shortening length required for a rescue (Fig. 6B). Parameter 7 (catastrophe or rescue frequency) was obtained by dividing the total number of catastrophe or rescue events by the total time spent in elongation or shortening phases, respectively (25Itoh T.J. Hisanaga S. Hosoi T. Kishimoto T. Hotani H. Biochemistry. 1997; 36: 12574-12582Crossref PubMed Scopus (67) Google Scholar), and was shown in Table I. In the present study, a clear difference in the dynamics profiles of MTs was detected between RB3-f and RB3-s, although the MT elongation rate seemed to be low throughout the experiment.Table IEffects of RB3-f and RB3-s on catastrophe and rescue frequency of individual MTsRB3 concentrationNeNcTimeCatastrophe frequencyμmminmin-1Control4333246.30.134RB3-s0.752116138.10.1161.5151365.40.199RB3-f0.752323133.00.1731.53528171.40.16333631147.90.2104.54638176.20.216RB3 concentrationNsNrTimeRescue frequencyμmminmin-1Control383225.51.253RB3-s0.75252015.01.3381.517915.80.569RB3-f0.75231613.51.1831.5322917.41.6693332516.21.5464.5463620.01.799 Open table in a new tab Fig. 6Elongation and shortening length of individual MTs in the presence of RB3 derivatives. Elongation length required for a catastrophe (A), shortening length required for a rescue (B), mean length of elongation phase (C), and mean length of shortening phase (D). Phase number (n) indicated the number of phases used in these calculations. Data shown are means ± S.D. C, control (without RB3 derivatives). Statistical significance was as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus control.View Large Image Figure ViewerDownload (PPT) Typical examples of micrographs and the dynamics profiles of single MTs are shown in Fig. 4.

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