Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

The establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process. Here, we identify a novel PAR-1-binding protein, which we call microtubule crosslinking factor 1 (MTCL1), that crosslinks MTs through its N-terminal MT-binding region and subsequent coiled-coil motifs. MTCL1 colocalized with the apicobasal MT bundles in epithelial cells, and its knockdown impaired the development of these MT bundles and the epithelial-cell-specific columnar shape. Rescue experiments revealed that the N-terminal MT-binding region was indispensable for restoring these defects of the knockdown cells. MT regrowth assays indicated that MTCL1 was not required for the initial radial growth of MTs from the apical centrosome but was essential for the accumulation of non-centrosomal MTs to the sublateral regions. Interestingly, MTCL1 recruited a subpopulation of PAR-1b (known as MARK2 in mammals) to the apicobasal MT bundles, and its interaction with PAR-1b was required for MTCL1-dependent development of the apicobasal MT bundles. These results suggest that MTCL1 mediates the epithelial-cell-specific reorganization of non-centrosomal MTs through its MT-crosslinking activity, and cooperates with PAR-1b to maintain the correct temporal balance between dynamic and stable MTs within the apicobasal MT bundles.

DOI： 10.1242/jcs.127845

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

TubZ is a structural homologue of tubulin and FtsZ GTPases, which are involved in the type III plasmid-partitioning system. TubZ assembles into polymers in a GTP-dependent manner and drives plasmid segregation as 'cytomotive' filaments. In this study, C-terminally truncated TubZ from Bacillus cereus was crystallized in the presence or absence of GDP by the hanging-drop vapour-diffusion method. The crystal of TubZ in complex with GDP belonged to the monoclinic space group P2(1), with unit-cell parameters a = 67.05, b = 84.49, c = 67.66 angstrom, beta = 92.92 degrees, and was non-isomorphous with GDP-bound TubZ previously crystallized in the presence of the slowly hydrolysable GTP analogue GTP gamma S. TubZ was also crystallized in the free form and the crystal belonged to space group P2(1), with unit-cell parameters a = 53.91, b = 65.54, c = 58.18 angstrom, beta = 106.19 degrees. Data were collected to 1.7 and 2.1 angstrom resolution for the free and GDP-bound forms, respectively.

DOI： 10.1107/S1744309112045551

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Stable maintenance of low-copy-number plasmids requires partition (par) systems that consist of a nucleotide hydrolase, a DNA-binding protein, and a cis-acting DNA-binding site. The FtsZ/tubulin-like GTPase TubZ was identified as a partitioning factor of the virulence plasmids pBtoxis and pXO1 in Bacillus thuringiensis and Bacillus anthracis, respectively. TubZ exhibits high GTPase activity and assembles into polymers both in vivo and in vitro, and its "treadmilling" movement is required for plasmid stability in the cell. To investigate the molecular mechanism of pXO1 plasmid segregation by TubZ filaments, we determined the crystal structures of Bacillus cereus TubZ in apo-, GDP-, and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S)-bound forms at resolutions of 2.1, 1.9, and 3.3 angstrom, respectively. Interestingly, the slowly hydrolyzable GTP analog GTP gamma S was hydrolyzed to GDP in the crystal. In the post-GTP hydrolysis state, GDP-bound B. cereus TubZ forms a dimer by the head-to-tail association of individual subunits in the asymmetric unit, which is similar to the protofilament formation of FtsZ and B. thuringiensis TubZ. However, the M loop interacts with the nucleotide-binding site of the adjacent subunit and stabilizes the filament structure in a different manner, which indicates that the molecular assembly of the TubZ-related par systems is not stringently conserved. Furthermore, we show that the C-terminal tail of TubZ is required for association with the DNA-binding protein TubR. Using a combination of crystallography, site-directed mutagenesis, and biochemical analysis, our results provide the structural basis of the TubZ polymer that may drive DNA segregation.

DOI： 10.1074/jbc.M112.373803

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER INC  

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a +TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure.

DOI： 10.1016/j.yexcr.2011.11.006

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Language：English   Publishing type：Research paper (scientific journal)  

XH/pi hydrogen bonds have been predicted to make important contributions to protein structure and function. NMR evidence is presented for an OH/pi interaction between a highly conserved threonine and phenylalanine pair found specifically in CAP-Gly domains associated with mictrotubule plus ends. The functional contribution of this nonclassical hydrogen bond in target peptide recognition is demonstrated via subtle point mutagenesis. The OH/pi interaction is part of a TxFxxxxW motif that comprises a conserved "threonine clasp" that defines function in CAP-Gly domains.

DOI： 10.1021/ja805576n

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

CLIP170 and p150(Glued) localize to the plus ends of growing microtubules. Using crystallography and NMR, we show that autoinhibitory interactions within CLIP170 use the same binding determinants as CLIP170's intermolecular interactions with p150(Glued). These interactions have both similar and distinct features when compared with the p150(Glued)-EB1 complex. Our data thus demonstrate that regulation of microtubule dynamics by plus end-tracking proteins (+TIPs) occurs through direct competition between homologous binding interfaces.

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jmb.2007.06.037

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The end-binding protein 1 (EB1) family is a highly conserved group of proteins that localizes to the plus-ends of microtubules. EB1 has been shown to play an important role in regulating microtubule dynamics and chromosome segregation, but its regulation mechanism is poorly understood. We have determined the 1.45-A resolution crystal structure of the amino-terminal domain of EB1, which is essential for microtubule binding, and show that it forms a calponin homology (CH) domain fold that is found in many proteins involved in the actin cytoskeleton. The functional CH domain for actin binding is a tandem pair, whereas EB1 is the first example of a single CH domain that can associate with the microtubule filament. Although our biochemical study shows that microtubule binding of EB1 is electrostatic in part, our mutational analysis suggests that the hydrophobic network, which is partially exposed in our crystal structure, is also important for the association. We propose that, like other actin-binding CH domains, EB1 employs the hydrophobic interaction to bind to microtubules.

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