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Cell Reports, Volume 3, Issue 3, 759-768, 21 February 2013
Structural Model for Tubulin Recognition and Deformation by Kinesin-13 Microtubule Depolymerases
Ana B. Asenjo1, Chandrima Chatterjee1, Dongyan Tan1, Vania DePaoli1, William J. Rice2, Ruben Diaz-Avalos2, Mariena Silvestry2 and Hernando Sosa1, ,
1 Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2 New York Structural Biology Center, New York, NY 10027, USA
Graphical Abstract
► Kinesin-13 tubulin complex structure reported at nanometer resolution ► Inter- and intradimer tubulin heterodimer interfaces are unambiguously identified ► A distinct curved-sheared tubulin conformation is described ► Insights provided into how kinesin-13s recognize and depolymerize microtubule ends
- To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.
Kinesins are a superfamily of microtubule (MT)-associated ATPases characterized by the presence of a highly conserved catalytic or "motor" head domain (HD, ∼340 residues long) containing MT- and ATP-binding sites (Goldstein and Philp, 1999). Although most kinesins work as motile molecular motors generating force and movement along MTs, some kinesins act as MT depolymerases. The latter group includes the kinesin-13 family members, which were the first to be clearly recognized as nonmotile kinesins with MT depolymerase activity (Desai et al., 1999).
Kinesin-13s have been reported to play important roles in various cellular processes, including mitosis (Manning et al., 2007; Rogers et al., 2004), cytokinesis (Rankin and Wordeman, 2010), axonal branching (Homma et al., 2003), and ciliogenesis (Kobayashi et al., 2011). Kinesin13s have also attracted considerable attention as potential targets for anticancer therapy due to their effects on MT dynamics and their involvement in mitosis (Sanhaji et al., 2011).
Although much work has been devoted to elucidating the mechanism of action of kinesin motors, it is still not fully clear why some are motile and others are MT depolymerases. The molecular mechanism of kinesin-13 MT depolymerization likely involves the stabilization of a curved tubulin structure, which is incompatible with the formation of lateral interprotofilament contacts in the MT lattice (Desai et al., 1999). However, how kinesin-13s induce tubulin curvature is not well understood. Catalytic activity is coupled to ATP hydrolysis (Hunter et al., 2003) as in other kinesins, but the interaction of kinesin-13s with MTs is very different from that of motile kinesins. Motile kinesins alternate between strong and weak interactions with tubulin as they translocate along the MT lattice. On the other hand, kinesin-13s bind to the MT lattice weakly and undergo unbiased one-dimensional (1D) diffusion until they reach the end of the MT, where they induce depolymerization (Helenius et al., 2006). How kinesin-13 distinguishes tubulin in the MT lattice or at the MT ends is unknown.
As with motile kinesins, many key kinesin-13 functions, such as ATP hydrolysis and tubulin binding, are located in the kinesin-13 HD, but additional areas outside the HD are also functionally important (Hertzer et al., 2006; Maney et al., 2001). A positively charged, ∼60-residue-long sequence N-terminal to the HD, known as the neck, promotes MT binding (Cooper et al., 2010) and is critical for normal in vivo MT depolymerization (Ovechkina et al., 2002). The neck may also increase MT depolymerization efficiency by inducing the two HDs of a full-length kinesin-13 dimer to bind adjacent protofilaments (Mulder et al., 2009). However, the kinesin-13 HD alone has the ability to induce tubulin curvature and depolymerize MTs (Moores et al., 2002; Tan et al., 2008). Thus, elucidating how the kinesin-13 HD in particular binds to and bends tubulin is crucial for understanding the mechanism of kinesin-13-mediated MT depolymerization.
Previous lower-resolution electron microscopy (EM) studies have indicated that the kinesin-13 HD binds to curved tubulin in a configuration that is similar to other kinesin HDs bound to straight tubulin in the MT lattice (Tan et al., 2008). This similarity makes it difficult to explain how the kinesin-13 HD binds to and bends tubulin. To address this problem, we analyzed the structure of several complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10AHD using EM and fluorescence polarization microscopy (FPM). Our data identify the kinesin-13 HD binding site on the tubulin intradimer interface, reveal a novel tubulin conformation, and provide an intriguing explanation for the distinct modes of interaction of kinesin-13s with straight or curved tubulin. We propose that the kinesin-13 HD induces a curved-sheared (CS) tubulin configuration by a crossbow-type bending mechanism. In this mechanism, the kinesin-13 HD bends the tubulin dimer by pulling the two tubulin subunits relative to the intradimer interface.
KLP10AHD Binds to the Intradimer Interface
A critical missing piece of information regarding the molecular mechanism of kinesin-13-induced MT depolymerization is the location of the kinesin-13 HD binding site on tubulin. Due to the similarity between α- and β-tubulin, it has not been possible to distinguish whether the kinesin-13 HD binds the tubulin interdimer or intradimer interface. The structures of α- and β-tubulin are only distinguishable at near-atomic resolution (Nogales et al., 1998), which makes it impossible to distinguish between the intra- and interdimer interfaces in medium-resolution EM studies. Additional experiments are needed to make this distinction unambiguously, and, to our knowledge, this has been done only for conventional kinesin (Marx et al., 2006). However, given the sequence similarities between the two tubulin interfaces (Wang and Nogales, 2005), and the completely different functionalities associated with kinesin-1 and kinesin-13s, it cannot simply be assumed that these two kinesin types bind to the same tubulin site.
To distinguish between α- and β-tubulin in the kinesin-13-tubulin complexes, we sought to visualize the complex at the protofilament ends where the tubulin interdimer interface is exposed (Figure 1A). Figure 1B shows a negative-staining electron micrograph of a field with many tubulin open rings produced by incubating KLP10AHD with tubulin in the presence of the nonhydrolyzable ATP-analog AMP-PNP. Similar rings are observed as depolymerization products when MTs are incubated with KLP10AHD in the presence of ATP (Figure 1C). We observed open and closed rings, as well as longer spirals, but concentrated our analysis on the open ones obtained in the presence of AMP-PNP because they were more numerous and homogeneous. We selected thousands of individual ring images (Table 1) and subjected them to single-particle image analysis to produce class averages with improved signal-to-noise ratio. The classification procedure separated image groups according to the number and curvature of tubulin heterodimers included in the ring, as well as other nonstructure-related image differences (Figures S1 and S2). Figure 1D shows three class averages of particles with distinct numbers of tubulin heterodimers (n = 5, 6, and 7), giving six independent, well-aligned protofilament end averages. In all cases, a density corresponding to KLP10AHD is centered at the intradimer interface, as in the model shown at the top of Figure 1A. Therefore, we conclude that the kinesin-13 HD binds at the tubulin intradimer interface.
Table 1 |
Ring Curvature (Deg/Tub Dimer) | ||||||||
Avg | SD | Min | Max | N Part. | KinRing/Tot | |||
ADP-ALlF4− | 25.6 | 1.8 | 20.2 | 28.33 | 3,662 | 0.434 | ||
Ring curvature was measured in the class-average images. The given statistics are weighted according to the number of particles in each class group. N part. is the total number of particles selected. KinRing/Tot is the ratio of particles that were positively identified as KLP10AHD-tubulin ring complexes to the total number of particles. See also Experimental Procedures and Figures S1 and S2. |
Nucleotide Dependence of the KLP10AHD–Curved Tubulin Interaction
To investigate possible changes in the KLP10AHD-tubulin complex related to the kinesin ATPase activity, we analyzed rings formed in the presence of ATP analogs (Figure 2). In contrast to a previous study (Moores and Milligan, 2008), the tubulin rings analyzed here were induced by kinesin only. No additional tubulin drugs to promote curved tubulin protofilaments were used. We investigated three conditions related to the kinesin-13 ATPase cycle: (1) the presence of AMP-PNP (to mimic the ATP state), (2) the presence of ADP-AlF4− (to mimic the ADP-Pi transition state; Wittinghofer, 1997), and (3) the absence of ATP or ADP (the no-nucleotide condition [NN]). We also investigated the effect of guanine nucleotide analogs. In the presence of AMP-PNP, we found similar ring structures in the presence of guanosine diphosphate (GDP), guanosine triphosphate (GTP), or the nonhydrolyzable analogs GTP-ϒ-S and GMPCPP (not shown). This indicates that the ability of KLP10AHD to bind and induce tubulin curvature is not dependent on whether tubulin has GDP or GTP in its exchangeable nucleotide site.
The most obvious difference found between the different adenine nucleotide conditions tested was in the number of ring-type oligomers observed (Table 1). Many ring-like structures were observed in the presence of AMP-PNP, with decreasing frequency in the presence of ADP-AlF4− followed by ADP and then NN, where none were observed. AMP-PNP was also the condition in which the averaged images showed the best-defined KLP10AHD density, followed by ADP-AlF4− and then ADP (Figure 2). These results indicate that KLP10AHD is best oriented relative to tubulin in the presence of AMP-PNP. Increased relative disorder between protein subunits and less KLP10AHD occupancy produced blur in the average density images, particularly in the ADP condition. A comparison of the average structures in the AMP-PNP and ADP-AlF4− conditions (Figure 2G) also reveals some small changes in the orientation of the KLP10AHD and tubulin subunits. Overall, these results indicate that the ATP state stabilizes the tightest interaction of KLP10AHD with curved tubulin, followed by the transition ADP-Pi state. Nucleotide-dependent conformational changes result in a weakening of the interaction until the complex is dissociated, likely after ATP hydrolysis product release (in the ADP or NN condition, or during ADP/ATP exchange).
Nanometer-Resolution Structure of a Kinesin-13-Tubulin Depolymerization Intermediate
To gain molecular-level insight into the mechanism by which kinesin-13 induces tubulin curvature, we used cryo-EM and image analysis to obtain a three-dimensional (3D) structure of KLP10AHD in complex with curved tubulin. We took advantage of the discovery that when MTs are incubated in the presence of kinesin-13 constructs and nonhydrolyzable ATP analogs, spirals consisting of a curved tubulin protofilament and bound kinesin-13s can wrap around MTs (Moores et al., 2006; Tan et al., 2006). These spirals can form very uniform helical arrays suitable for 3D reconstruction. By increasing the number of images and applying single-particle analysis procedures, we increased the resolution to ∼11 Å (Figure S3) from a previous 28 Å resolution map (Tan et al., 2008). At this improved resolution, some exposed secondary structure elements can be resolved and a more precise fitting of the different protein subunits constituting the complex can be achieved (Figure 3; Movie S1. KLP10HD MT Ring Complex Cryo-EM 3D Map with Fitted Atomic Structures, Related to Figure 3).
The KLP10AHD in the spirals has two areas of interaction with tubulin positioned at opposite sides of the HD (Figure 3C). We refer to these areas as Kin-Tub-1 and Kin-Tub-2 (Tan et al., 2008). We will focus our description here on the Kin-Tub-1 interface because this is the one that is present in the isolated ring and spiral complexes (Figures 1 and 2) and is involved in stabilizing tubulin curvature. The Kin-Tub-1 interface can be separated into three distinct areas going from the tubulin plus end to the minus end: area 1, containing kinesin loop 8 (L8); area 2, containing kinesin α-helix 4 (α4); and area 3, containing kinesin loop 2 (L2). Close-ups of these three areas, indicating likely interacting secondary structure elements in KLP10AHD and tubulin, are shown in Figures 3D–3F. Area 2, in addition to α4, also includes kinesin loop 11 (L11), which is disordered and not resolved in most kinesin crystal structures. This region is one of two regions commonly found in kinesins, myosins, and G-proteins that change conformation depending on the nucleotide species that is present on the active site (Kull and Endow, 2002). L2, in the third area, is a loop that is elongated in the kinesin-13 family and contains the class-conserved KVD sequence at the tip. Mutating these three residues severely disrupts depolymerization activity (Ogawa et al., 2004; Shipley et al., 2004). We found that KLP10AHD constructs with the KVD residues mutated to AAA were unable to form tubulin ring complexes, highlighting the critical role that this area plays in kinesin-13's ability to bend tubulin and induce MT depolymerization. The elongated L2 characteristically sticks out from the core of the kinesin-13 HD and is very well resolved in the cryo-EM density map (Figure 3F). Thus, its location when bound to curved tubulin is very well determined in the fitted model of the KLP10AHD-tubulin ring complex. The tip of L2 is at the interdimer interface and is likely to form binding interactions to residues in the α-tubulin subunit of the dimer, to which the rest of the HD is bound, and to the β-tubulin subunit of the next heterodimer toward the minus end of the protofilament (Figures 3C and 3F).
The structure of straight tubulin does not fit well into the density of the outside curved protofilament (Figure 4A), as would be expected. A good fit is only achieved when α- and β-tubulin monomers are allowed to fit independently. In this fitted tubulin structure, there is a bend between monomers relative to the configuration of straight tubulin (Figure 4; Movie S2. Straight versus CS Tubulin (End-On View), Related to Figure 4). The fitting also reveals an unexpected displacement (shear) between the tubulin subunits (Figure 4, right; Movie S3. Straight versus CS Tubulin (from Outside the Complex View), Related to Figure 4). This curved and sheared heterodimer is a tubulin structure that is considerably different from previously described straight or curved tubulin structures (Figure S4). We will refer to this structure as CS tubulin.
A detailed comparison of the 3D map with the fitted atomic structures also reveals small areas with a less than perfect fit. In these areas, there is density in the 3D EM map with no corresponding atomic structures from the fitted atomic structures or atoms with no corresponding density (Figure 3; Movie S1. KLP10HD MT Ring Complex Cryo-EM 3D Map with Fitted Atomic Structures, Related to Figure 3). At the present resolution, it cannot be fully established whether such differences correspond to local conformational changes or areas that are disordered in the crystal structures becoming ordered in the complex. In the tubulin atomic structures, there are 12–16 residues missing (disordered) at the C-terminal end and most residues in kinesin L11 and L12 are also missing. Despite the small fitting mismatches, the overall positions and orientation of all the fitted protein subunits can be determined with high accuracy.
In order to estimate the accuracy with which our cryo-EM 3D map and molecular fitting procedure support the CS-tubulin structure, we made 18 independent fits to maps calculated from separate data sets or different asymmetric units in a nonhelically averaged map. The relative positions of all the individual subunits in the resulting 18 models were very similar, and all tubulins in the outside ring showed curvature and shear (Figure S5). We estimated an accuracy in determining the relative position between protein subunits of better than 2.3 Å, which is much smaller than the distance between corresponding tubulin monomers (16 Å) when we compare CS-tubulin with straight or curved tubulin. Thus, the 3D map and fitting procedure provide strong evidence for the CS-tubulin conformation.
A consequence of the curve and shear between tubulin monomers in CS-tubulin is that the Kin-Tub-1 interface is very different from the analogous interface between other kinesin HDs and straight tubulin (Figures 4B and 4C; Movie S4. Straight versus CS Tubulin (Kinesin-13-Binding Site), Related to Figure 4). KLP10A and kinesin-1 interact with some of the same residues of α- and β-tubulin, with the notable exception of the residues toward the α-tubulin minus end that interact with the kinesin-13-specific elongated L2. Despite including similar residues, the kinesin-binding sites are different due to the different displacement between the α- and β-tubulin subunits in straight and CS-tubulin. We propose that this structural difference forms the basis of the distinct interactions of kinesins with straight tubulin at the MT lattice or curved tubulin at MT ends. Motile kinesins are adapted to interact with straight tubulin, whereas kinesin-13s are adapted to interact with curved tubulin. This adaption in the case of kinesin-13s includes the elongated L2, which binds to α-tubulin and orients the motor domain in such a way that the other interacting areas (α-4, L11, L12, and L8) match the corresponding binding areas in CS-tubulin but not in straight tubulin.
The three interacting areas of the Kin-Tub-1-binding site suggest a crossbow-type mechanism for inducing tubulin curvature (see Figure 6A) where the regions at the extreme of the interface (kinesin L8 and L2) pull the plus and minus ends of the tubulin heterodimer relative to the center region (kinesin α4-L11). One can visualize this kinesin-13-induced tubulin curvature process by comparing images of KLP10AHD-protofilament complexes with distinct curvature, or by comparing the atomic models of the KLP10AHD-CS-tubulin and kinesin-1-tubulin complexes (Movie S5. KLP10AHD Tubulin Complexes with Distinct Curvature, Related to Figure 1 and Movie S6. KLP10AHD-CS-Tubulin versus Kinesin-1-Straight-Tubulin Complex, Related to Figures 3 and 4).
Interactions Involving Kinesin-13 L2 and α4 Mediate Weak Interactions with the MT Lattice
To investigate how kinesin-13 interacts with straight tubulin, we used FPM of fluorescently labeled KLP10AHD constructs bound to MTs. FPM allows one to obtain structural information (e.g., orientation and mobility) about proteins bound to the MT even with low occupancy or in disordered states, as may be expected from the relatively weaker interaction of kinesin-13 with the MT lattice (Wang et al., 2012).
We made several cys-light bis-((N-iodoacetyl)piperazinyl) sulfonerhodamine (BSR)-labeled KLP10A constructs (KLP10A-BSR). Figure 5A shows the location and orientation of the fluorescence transition dipole on KLP10AHD. This location is equivalent to the one used in previous work on the kinesin-1 HD (Asenjo et al., 2003; Sosa et al., 2001), which allows us to make comparisons between these different kinesins. We measured the linear dichroism (LD0) of ensembles of KLP10A-BSR molecules on MTs. The LD0 reports on the orientation and disorder (static or dynamic) of the probes relative to the MT axis (Sosa et al., 2010). We determined the LD0 of KLP10A-BSR constructs with or without mutations in the previously identified putative binding areas of KLP10AHD with CS tubulin. The cys-light construct with no additional mutations is named WT. The L2 mutant in which the kinesin-13-specific KVD residues were mutated to AAA is named the KVD mutant, and the L8 mutant is named D444A,K446A according to the residue substitutions made. We also investigated a kinesin α4 mutant in which the residues KEC were mutated to alanines. This KLP10A-BSR mutant did not decorate MTs well enough even for FPM analysis. This is consistent with a previous study (Shipley et al., 2004) indicating that this mutation severely disrupts MT depolymerization activity and MT binding.
Figure 5B shows typical FPM images with KLP10A-BSR-decorated MTs. Anisotropy can be clearly seen in the top images (corresponding to WT KLP10A-BSR), whereas the bottom ones (corresponding to the KVD mutant) show much less anisotropy. We measured the LD0 values of all the constructs in the presence of ADP or absence of nucleotides (NN) (Figure 5C). In these conditions, very few or no curved tubulin ring complexes should form, and thus the reported values should correspond mainly to KLP10A-BSR molecules interacting with the MT lattice.
The LD0 in most conditions is positive, indicating that the dipole probe is oriented on average nearly perpendicular to the MT axis (Figure 5C). This is similar to kinesin-1 labeled with the BSR probe in an equivalent location. However, the values are significantly lower for the NN state and higher for ADP. For conventional kinesin, ADP produces a weak, highly mobile state with an LD0 value near zero, whereas nucleotide depletion produces a strong rigor binding state with relatively low mobility (LD0 = 0.5 ± 0.06; Asenjo et al., 2003). The relative similarity in the LD0 values between different nucleotide conditions for KLP10A-BSR (0.2 and 0.3 versus 0 and 0.5 for conventional kinesin) indicates that the interaction of KLP10A-BSR with the MT lattice is less dependent on the nucleotide state than in the case of conventional kinesin. The lower LD0 value of KLP10A-BSR relative to conventional kinesin in the NN state indicates a slightly different orientation (probe closer to 54.7° from the MT axis) and/or a more disordered binding configuration for KLP10A-BSR (multiple conformations and/or mobility). We favor the second interpretation because it is consistent with the diffusive nature of kinesin-13's interaction with the MT lattice. The simplest interpretation of the data is that KLP10A-BSR cannot bind to the MT lattice in a well-defined orientation as does conventional kinesin. Instead, it binds weakly, with higher mobility and less dependence on the nucleotide state.
A weakly diffusive state could be the result of kinesin-13 switching between alternative binding sites as the molecule diffuses along the MT lattice. The effect of mutations on the putative CS-tubulin-binding site on the LD0 values provides further insights into how these regions participate in the interaction with straight tubulin. As explained above, the kinesin α4 KEC mutant did not bind well to MTs, indicating that α4 interactions are critical for KLP10A-BSR MT lattice binding. Mutating residues D444 and K446 to alanines in KLP10A L8 produced small changes in the binding configuration of KLP10A-BSR to the MT, as indicated by the lower LD0 values of this mutant relative to the control WT (Figure 5C). Mutations on equivalent residues on conventional kinesin reduce its affinity for MTs (Woehlke et al., 1997). Mutating the kinesin-13 class-conserved residues KVD to alanines in L2 does not abolish binding to the MT (Figure 5B, bottom panels), but produces a large effect on the configuration of the KLP10A-BSR MT complex. The LD0 in all nucleotide conditions falls to near zero for this mutant, consistent with a major increase in mobility or change in the average orientation of KLP10A-BSR relative to the MT. Therefore, kinesin-13 L2, in addition to being critical for stabilizing the CS-tubulin configuration (see previous section), also determines the binding orientation of the HD when bound to straight tubulin.
The FPM results are consistent with a model in which the KLP10A binds to straight tubulin in the MT lattice using the same structural elements (kinesin L2, α4, and L8) as when it is bound to CS tubulin (Figures 3C–3F), but with more mobility, perhaps alternating between the three binding areas. The contacts that dominate the interaction (in time and/or strength) would be the ones in areas 2 and 3 (Figures 3D–3F), because mutations in these areas (kinesin α4 and L2) have the strongest effect on binding of KLP10A-BSR to the MT lattice. The tubulin-bending crossbow model (Figure 6A) would also predict a change in the average orientation/mobility of the motor domain relative to tubulin, consistent with the observed changes in LD0 when key residues in L2 or L8 are mutated (Figure 5C). Disrupting one of the two interaction points at the extremes of the binding interface (diagonal red arrows in Figure 6A) would alter the balance of forces and produce a change in the orientation of the HD relative to tubulin.
We have produced a structural model for the kinesin-13 MT depolymerization mechanism based on EM and FPM data. In this model, binding interactions in three main areas of the kinesin-13 HD tubulin interface generate relative forces akin to a crossbow to stabilize or induce curvature between the α- and β-subunits of the tubulin heterodimer (Figure 6A). A nanometer-resolution structure of the kinesin-13 HD tubulin complex also revealed that in addition to curvature, there is considerable relative displacement (shear) between the tubulin subunits. We call this tubulin configuration CS-tubulin.
The CS-tubulin structure suggests a novel mechanism by which the kinesin-13 HD discriminates between straight and curved tubulin conformations. A previous model proposed that a more convex shape for the kinesin-13 HD was better suited to interact with the concave binding site of a curved tubulin (Ogawa et al., 2004). However, we find this concave-convex complementary model unsatisfactory because the curvature between tubulin subunits is relatively modest and the tubulin-binding site of kinesin 13s is no more convex than that of other kinesins (Figure S6). In our model, the key to the curved versus straight tubulin distinction lies in the presence of kinesin-binding sites at both sides of the tubulin intradimer interface. With this split binding surface, displacement between α- and β-tubulin results in a different binding site (Figures 4B and 4C). The elongated kinesin-13-specific L2 plays a critical role here because it creates a new contact point at the interdimer interface and orients the HD for proper matching of the other interacting areas with CS tubulin.
The formation of long, multimeric tubulin protofilaments with the KLP10AHD, as observed here (Figures 1, 2, and 3), indicates that the KLP10AHD stabilizes tubulin interdimer contacts in addition to inducing intradimer curvature. This effect may also be mediated by kinesin L2, because its tip is at the interdimer interface (Figures 3C and 3F). This interaction may give kinesin-13s a higher affinity for isolated protofilaments at the end of the MT compared with free tubulin, where interdimer interactions would be missing. This would provide a structural explanation for why kinesin-13s are efficient MT catastrophe promoters rather than polymerization inhibitors (Gardner et al., 2011).
A simple explanation for how kinesin-13s specifically recognize and depolymerize MT ends is that they recognize tubulin protofilaments with missing lateral contacts. In the absence of capping proteins, the MT ends will always have at least one tubulin subunit missing lateral contacts due to their helical arrangement. Also, in many conditions the MT ends are thought to have one or more protofilaments missing lateral contacts with their neighbors due to the formation of open sheets, protofilament curling, or different lengths of the protofilaments (Nogales and Wang, 2006). In the absence of lateral contacts, tubulin may be free to adopt the CS conformation and form a strong interaction with the HD of kinesin-13s. An alternative hypothesis is that kinesin-13 recognizes some structural feature of GTP-tubulin, which is thought to cap the end of MTs (Zanic et al., 2009). However, the fact that we observed similar KLP10AHD tubulin rings in the presence of GDP, GTP, or nonhydrolyzable GTP analogs argues against this possibility.
The process of kinesin-13-induced protofilament bending at the end of the MTs could occur by an induced fit or a conformational selection-type mechanism (Changeux and Edelstein, 2011). In the first case, interactions of the kinesin-13 HD in the ATP state with straight tubulin would promote a transition from straight to CS-tubulin concomitantly with the formation of a strong binding complex. This transition would only occur on isolated protofilaments where kinesin-13 could overcome the straightening force created by lateral tubulin interactions. In the second view, the kinesin-13 HD would stabilize a pre-existing CS-tubulin configuration. The probability of finding this pre-existing configuration would be higher in isolated protofilaments where the lack of stabilizing lateral protofilament contacts would allow tubulin to fluctuate between different conformations. In favor of a conformational selection-type mechanism is the fact that tubulin with curvature exists in the absence of kinesin-13s (Nogales and Wang, 2006). However, proving this mechanism will require determining whether the CS-tubulin structure in particular can exist outside a kinesin-13-tubulin complex. Currently, we favor a model that incorporates elements of both mechanisms (induced fit and conformational selection). A plausible sequence of events is that the kinesin-13 HD initially forms a complex with slightly curved protofilaments at the MT ends, and this interaction triggers further tubulin bending and shearing. The fact that KLP10AHD binds to tubulin protofilaments with a range of curvatures (Table 1; Movie S4. Straight versus CS Tubulin (Kinesin-13-Binding Site), Related to Figure 4) supports this view.
Our structural data can be combined with previously reported kinetic data (Friel and Howard, 2011; Wang et al., 2012) to obtain a mechanochemical cycle model for kinesin-13 (Figure 6B). In this model, the distinct effects of the MT ends or lattice on kinesin-13 activity are explained by the ability/inability to form all the three kinesin-13-tubulin interactions, as seen in the Kin-Tub1 interface of the KLP10AHD CS-tubulin complex (Figures 3C–3F). As indicated by our FPM data, the kinesin-13 HD binds to the MT lattice in a relatively mobile state mediated, at least in part, by interactions with the kinesin-13 L2 and α4. In this weakly bound state, kinesin-13 could undergo 1D diffusion toward either MT end (Helenius et al., 2006). At the MT ends, a kinesin-13 could arrive in either the ATP state (from solution) or the ADP state (by 1D diffusion along the MT lattice). Lack of lateral protofilament contacts at the MT ends would allow tubulin curvature and the kinesin-13 to form complementary binding at the three regions of the Kin-Tub-1 interface. ADP/ATP exchange of kinesins arriving in the ADP state from the MT lattice (Friel and Howard, 2011) may also be triggered during this process. The resulting stable kinesin-13-CS-tubulin complex would not be able to straighten spontaneously and reform lateral interprotofilament contacts, leading to MT end depolymerization. Following ATP hydrolysis and product release, the complex would dissociate, as suggested by the very small number or lack of KLP10AHD-curved tubulin complexes observed in the ADP and NN conditions (Table 1). Separation of individual tubulin dimers from the MT end protofilament could occur at this step by a yet to be defined additional conformational change. Alternatively, it could occur as the curved protofilaments grow longer and become prone to breakage.
Experimental Procedures
Protein Constructs
D. melanogaster KLP10A protein constructs were expressed and purified as previously described (Tan et al., 2006, Tan et al., 2008). The KLP10AHD construct includes KLP10A residues I279–I615. For FPM, we made several new constructs that include the KLP10A head and neck domains (residues T198–I615). Inclusion of the neck residues improved MT decoration. For site-specific labeling, all of the surface-exposed cysteines were replaced with alanines or other residues based on naturally occurring substitutions within the kinesin-13 family. Accordingly, we made the following substitutions: C282A, C339A, C389S, C548S, and C594V. Two residues were mutated to cysteines (T455C and D460C) for labeling with the bifunctional thiol-reactive fluorescence probe BSR (Molecular Probes). We call this basic construct WT. We verified that this WT construct retained kinesin-13's ability to induce tubulin curvature. We prepared three mutant versions of this construct by replacing the following class-conserved residues: K317A, V318A, and D319A triple mutant (residues located in L2); D444A and K446A double mutant (residues located in L8); and K546A, E547A, C548A triple mutant (residues located in kinesin α4). All of the mutations were introduced into the plasmid vector using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies/Stratagene). The attachment location of the probe (crosslinking residues 455–460) was verified by trypsin digestion followed by mass spectrometry. Pig brain tubulin was purchased (Cytoskeleton) or purified in the laboratory according to standard protocols (Miller and Wilson, 2010).
Single-Particle Negative-Staining EM
Tubulin (0.15 μM) was first mixed with the nucleotide species to be tested and then with an excess amount of KLP10AHD in cold BRB80 buffer (1 mM MgCl2; 1 mM EGTA; 80 mM PIPES, pH = 6.8). The final concentrations of nucleotide depending on the experimental condition to be tested were as follows: AMP-PNP, 1 mM AMP-PNP (Sigma Aldrich); ADP, 1 mM ADP (Sigma Aldrich); NN, 5 U ml−1 apyrase (Sigma Aldrich) and no added nucleotides; and ADP-AlF4−, 4 mM ADP, 2 mM AlCl3, and 10 mM KF. The mixture was incubated on ice for 1 min and then loaded onto carbon-coated copper grids. We also tested the effect of including different GTP analogs (GTP: 1 mM GTP [Sigma Aldrich]; GDP, 1 mM GDP [Sigma Aldrich]; GMPCPP: 1 mM GMPCPP [Jena Bioscience]; and GTP-ϒ-S, 1 mM GTP-ϒ-S [Cytoskeleton]). The grids with the KLP10AHD and tubulin mixtures were negatively stained with 1% uranyl acetate (Electron Microscopy Sciences). Electron micrographs were recorded on a TVIPS F224HD digital camera in an FEI Tecnai-20 microscope operated at 120 kV with a nominal magnification of 50KX (pixel size: 0.27 × 0.27 nm2).
A suspension of the KLP10AHD-MT complex was applied onto freshly glow-discharged EM grids and flash frozen as previously described (Tan et al., 2008). Electron micrographs were recorded on film under low-dose conditions at a nominal magnification of 50,000 or 45,000 with a nominal defocus range of 1–2.5 μm on a Tecnai-F20 FEG cryo-electron microscope operated at 200 kV or on a JEOL 3200FSC operated at 300 kV. Electron micrographs were recorded on film under low-dose conditions and digitized for further analysis.
Image Analysis and 3D Reconstruction
Negatively stained particle images of KLP10AHD tubulin complexes were digitized and subjected to multivariate statistical analysis, classification, and averaging (see Extended Experimental Procedures).
We carried out 3D reconstruction of helical 15-pf MTs with wrapped-around KLP10AHD tubulin protofilament spirals using a single-particle approach (see Extended Experimental Procedures).
Molecular Fitting
We created atomic models by fitting the atomic structures of tubulin (Protein Data Bank ID code [PDB]: 1JFF; Löwe et al., 2001) and KLP10AHD into the asymmetric unit of the 3D map using the fitmap function of UCSF-Chimera (Pettersen et al., 2004). The atomic coordinates of each protein subunit were fitted independently as rigid bodies (for further details see Figure S5 and corresponding legend). An atomic model of KLP10AHD was generated with MMM (Rai and Fiser, 2006) using the sequence of KLP10A and the crystal structure of the kinesin-13 KIF2C (PDB: 1V8K (Ogawa et al., 2004)).
Taxol-stabilized MTs were attached to glass coverslip flow chambers, and solutions with KLP10A fluorescent constructs were then flowed through the chamber to decorate the MTs as previously described (Asenjo and Sosa, 2009). Nucleotides were added in accordance with the experimental condition to be interrogated: ADP (2 mM) and NN (5 U ml−1 apyrase with no nucleotide added) in BRB12 buffer (12 mM PIPES, 2 mM MgCl2, 1 mM EGTA, and 20 μM Taxol, pH 6.8). Flow chambers were placed in a custom-modified microscope for fluorescence polarization observation and data recording. Ensemble fluorescence polarization data were recorded and analyzed as previously described (Sosa et al., 2010).
Single-Particle Image Analysis
Individual particles were selected manually using EMAN boxer (Ludtke et al., 1999). We identified and selected in the raw images two group of particles for each experimental condition: 1) the ones that appeared like tubulin protofilaments ring like structures and 2) the ones that could not be easily identified as ring structures (they appeared straighter or shorter). We could not identify ring like particles in the NN state, so all particles were placed in a single group in this case. Each particle group was then subjected to reference free alignment. The resulting reference free averages were then used as initial references in 20 rounds of iterative reference alignment. Each of the aligned data sets groups were then separated into 20 distinct classes using correspondence analysis followed by k-means classification. These procedures were carried out using the SPIDER suite of image analysis programs (Frank et al., 1996) and custom Python scripts and wrapper functions. Particles were counted as KLP10AHD-ring like tubulin complexes depending on the appearance of the average image of the class to which they belong (Figures S1 and S2). Curvature radius and angles of the protofilament complexes (Table 1) were calculated by marking equivalent positions on the tubulin monomers on the class-average images and fitting the resulting XY coordinate points to a circle equation. Reported statistics (Table 1) are weighted according to the number of particles in each class.
Helical 3D Reconstruction
Electron micrographs were recorded on film under low dose conditions at a nominal magnification of 50000 or 45000 with a nominal defocus range of 1-2.5 μm on a Tecnai-F20 FEG cryo-electron microscope operated at 200 kV or on a JEOL 3200FSC operated at 300 kV. Cryo-electron micrographs were digitized in a flatbed scanner (Creo Eversmart Supreme) with step size of 12.5 μm for a nominal final pixel size of 0.25 or 0.28 nm. Digitized images with MTs were inspected and manually selected first based on the presence of clear decoration with KLP10AHD wrapped around the MTs. From these MTs only the ones with 15 protofilaments in which the tubulin lattice forms a helical 2-start helix were selected. MTs with this symmetry were selected based on their diameter and characteristic layer line pattern visible in their corresponding power spectrum (Sosa and Milligan, 1996). From the power spectrum we also estimated the helical symmetry parameters corresponding to each filament, selection rule, rise and rotation per subunit.
We aligned all the filament images and performed a 3D reconstruction using a single particle approach based on previously used methods for particles with helical symmetry (Egelman, 2000; Fujii et al., 2010; Li et al., 2002; Sosa and Milligan, 1996). The contrast transfer function (CTF) function, defocus and astigmatism parameters for each filament image were estimated using the program CTFFIND3 (Mindell and Grigorieff, 2003). The determined CTF parameters were used to create versions of each filament image phase flip corrected or CTF multiplied and to CTF correct the final 3D map. Images were also interpolated to a common pixel size of 2 Å using the 4 nm tubulin axial periodicity on the MTs as a calibration standard. Points along the axis of each filament were first selected manually using the program tkir from the software package SUPRIM (Schroeter and Bretaudiere, 1996) package. The manually determined filament axis points were refined by excising 300x300 pixel box images centered at each axis point and performing a multi-reference alignment against projections of a reference filament. The intensities of each of the boxed images were corrected for intensity gradients and normalized to a common average and standard deviation. The refined axis point coordinates in the original images were then determined from the alignment parameters. For this refinement step we used the phase-flipped corrected version of the experimental images. This alignment produced the first estimate of the filament particles in-plane rotation. A new set of equally spaced coordinate points along the filament axis was then created from the refined axis coordinates by interpolation. The spacing between axis coordinate points was chosen to be equal to the average subunit spacing determined from the power spectrum analysis (see above). The newly set of equally spaced axis coordinate points was used to generate a new set of boxed images ("single particles") from the CTF multiplied filament images. All the box-image particles were then subjected to seven rounds of iterative projection matching multireference alignment. Reference projections were created by rotating the 3D reference map 360° around the helical axis and ± 15° from it (out of plane tilt) at 1° degree intervals. The first 3D map used for alignment was a 2.8 nm resolution map of the KLP10HD-MT helical ring complex previously determined by the Fourier-Bessel method (Tan et al., 2008). At each alignment step a new 3D map was created by back-projection. Each newly calculated 3D map was then used as reference for the following alignment step. Before alignment, the 3D reference map was low pass filtered and masked with a soft mask. The soft mask was used to eliminate noisy areas outside the filament and the low pass filter to avoid over-fitting high resolution noise. A 1/18 Å−1 to 1/10 Å−1 frequency top-hat low pass filter function was progressively used in subsequent refinement steps. At each refinement step images were eliminated and not included for 3D back-projection if: their assigned in plane rotation angles were > 40° from the average axis orientation of the filament to which they belong; their translational alignment parameters placed them less than half the helical subunit spacing from another box-image with a higher alignment cross-correlation coefficient; their out of plane-tilt values were at the limit of the search range (±15°). Refinement was stopped when no further changes were detected in the Fourier Shell Correlation (FSC) between two half of the data set (Figure S3). The 3D map of the last refinement cycle (obtained by back-projecting the CTF multiplied filament images) was multiplied by 1/∑ (CTF2+1/SNR) (where SNR is signal-to-noise ratio, a SNR = 1 was used) to obtain a CTF corrected density. The 3D map was also sharpened with a B factor of −300Å2 and low pass filtered with a soft-edged cosine function in Fourier space going from 1 to 0 between 1/10.8 and 1/9 Å−1. Helical symmetry was determined and imposed in the final 3D density map but not in the intermediate maps used for refinement. To determine and impose helical symmetry we used the programs HSEARCH and HIMPOSE (Egelman, 2000) as modified in Ge and Zhou, 2011. The final 3D map included data from 111 filaments and 54584 asymmetric units. Multi-reference alignment and Fourier space interpolation back-projection were performed using the routines AP SHC and BP32F of the SPIDER software package (Frank et al., 1996). All the steps in the procedure were controlled by a custom package of Python routines and wrapper functions for SPIDER and other image processing software packages.
UCSF Chimera (Pettersen et al., 2004) was used for display of 3D density maps. Noise in the final 3D map was eliminated for display purposes in some of the views using the UCSF-Chimera "eliminate dust" utility. Quantitative fitting of atomic structures into the 3D densities was performed using the UCSF-Chimera fitmap utility as explained in the legend to Figure S5.
We thank J. Hargitai and S. Wang for help with installing and running software in the high-performance computer cluster at the Albert Einstein College of Medicine (AECOM); F. Macaluso, L. Gunther, and members of the AECOM Analytical Imaging Facility for help with EM; and D. Sharp and G. Gerfen for discussions and critical reading of the manuscript. Part of this investigation was conducted in a facility constructed with support from the Research Facilities Improvement Program of the National Center for Research Resources, National Institutes of Health (NIH, grant C06 RR017528-01-CEM). The 300 keV microscope at the New York Structural Biology Center was purchased with funds from NIH grant S10 RR17291. This project was supported by NIH grant R01-GM083338 to H.S.
Accession Numbers
The electron density map has been deposited in the EMDataBank (EMDB) under accession code EMD-5565, and the atomic model of the complex has been deposited in the Protein Data Bank under ID code 3J2U.
Supplemental Information
- Movie S1. KLP10HD MT Ring Complex Cryo-EM 3D Map with Fitted Atomic Structures, Related to Figure 3 (MP4 8802 kb)
- Mesh isosurface representation of three asymmetric units of the 3D density map, shown with ribbon representations of the fitted atomic models. The movie starts with an end-on view of the 3D map and rotates 360° around an axis perpendicular to the MT filament long axis. Yellow, MT α-tubulin; gold, MT β-tubulin; blue-cyan, KLP10HD; magenta, curved protofilament α-tubulin; purple, curved protofilament β-tubulin.
- Movie S2. Straight versus CS Tubulin (End-On View), Related to Figure 4 (MP4 799 kb)
- The movie goes back and forth between a straight- and CS-tubulin protofilament as fitted inside the cryo-EM electron density map. This view highlights the bending of tubulin by KLP10AHD. The movie was created by interpolating between the structures of the straight- and CS-tubulin protofilaments using UCSF-Chimera (Pettersen et al., 2004). Both structures are aligned at the topmost α-tubulin subunit. Gray, MT tubulin; yellow, α-tubulin in the outside protofilament; orange, β-tubulin in orange. Cryo-electron density is shown as a surface representation in semitransparent gray. The outside protofilament plus end is at the top.
- Movie S3. Straight versus CS Tubulin (from Outside the Complex View), Related to Figure 4 (MP4 1204 kb)
- The movie goes back and forth between a straight- and CS-tubulin protofilament as fitted inside the cryo-EM electron density map. This view highlights the shearing of tubulin by KLP10AHD. The movie was created by interpolating between the structures of the straight- and CS-tubulin protofilaments using UCSF-Chimera. Both structures are aligned at the topmost α-tubulin subunit. Yellow, α-tubulin in the outside protofilament; orange, β-tubulin in orange. Cryo-EM density is shown as a surface representation in semitransparent gray. The outside protofilament plus end is at the top.
- Movie S4. Straight versus CS Tubulin (Kinesin-13-Binding Site), Related to Figure 4 (MP4 2073 kb)
- The movie goes back and forth between a straight- and CS-tubulin protofilament as fitted inside the cryo-EM electron density map. Two views are shown simultaneously (left: end-on view; right: from KLP10AHD). Tubulin residues that are very close to kinesin (≤5 Å) in the KLP10AHD-CS-tubulin complex are shown as cyan atom spheres. Yellow, α-tubulin; orange, β-tubulin. The straight- and CS-tubulin protofilament structures are aligned at the topmost α-tubulin subunit. The protofilament plus end is at the top. Note how the relative position between putative kinesin-13-binding areas (cyan) in α- and β-tubulin changes between the straight- and CS-tubulin configurations.
- Movie S5. KLP10AHD Tubulin Complexes with Distinct Curvature, Related to Figure 1 (MP4 136 kb)
- The movie goes back and forth between two class-average images of the KLP10AHD and tubulin in the presence of AMP-PNP.
- Movie S6. KLP10AHD-CS-Tubulin versus Kinesin-1-Straight-Tubulin Complex, Related to Figures 3 and 4 (MP4 868 kb)
- The movie was created by interpolating between the structure of KLP10AHD-CS tubulin complex (as fitted into the cryo-EM electron density map) and the structure of the kinesin-1 MT complex (PDB: 2P4N; Sindelar and Downing, 2007). The two structures were positioned to align the kinesin HD of both structures. The tubulin plus end is at the top. Blue, kinesin HD; yellow, α-tubulin; orange, β-tubulin. The KLP10AHD-CS tubulin complex can be distinguished in the movie sequence by the tubulin curvature and the extended L2 on the kinesin HD reaching toward the minus end of α-tubulin.
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Publication Information
Received: July 24, 2012
Revised: December 12, 2012
Accepted: January 24, 2013
Published: February 21, 2013


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The subject of intentional breeding or meat rabbits is prohibited. The answers provided on this board are for general guideline purposes only. The information is not intended to diagnose or treat your pet. It is your responsibility to assess the information being given and seek professional advice/second opinion from your veterinarian and/or qualified behaviorist.
LEADERS: Beka27 BinkyBunny Elrohwen KokaneeandKahlua LittlePuffyTail MoveDiagonally RabbitPam Sarita
Hi my name is Debbie im from the UK, i live in the countryside with 3 dogs, 1 cat & sadly now only 1 bunny as my Thumper sadly passed away last week
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OBITS - Incomplete obituaries are marked with a *
Juanita Quinn Sluder
Dec 30, 2012
Dec. 30, 2012
Juanita Quinn Sluder, 79, went home to be with the Lord on Sunday, Dec. 30, 2012, at Sweetwater Nursing Center.
She was a member of Faith Baptist Church in Decatur, and was preceded in death by her parents, Roy and Margaret James Quinn; and one brother, Bobby Quinn.
Survivors include her loving husband of 58 years, the Rev. Winfred Sluder of Ten Mile; three sisters, Margie Skidmore of Brewton, Ala., Mary Jones of Dalton, Ga., and Polly of Cohutta, Ga.; one granddaughter, Sarah Guffee; and a host of good Christian Friends.
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Year-RoundA spectacular boat parade, more than 50 restored classic and antique boats. A beach bar with music, delicious luncheon. Cast your vote for your favorite boat!
Annually, the fourth Saturday in July.


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Quotation Marks
Quotation Marks
Punctuating Quotations
Quotation marks are used to set off a person's words, whether spoken or written. They are placed at both the beginning and end of the quote.
Ex: Sue remarked, "I'll meet you at the movies,"
A comma is used to set off the quote from the rest of the sentence (as above) unless the quotation is blended into the sentence.
Ex: Sue said that the choir will "chant an ancient tribal song."
If a quote is interrupted by explanatory words, use commas to set off the explanation.
Ex: "We'll go back to living in caves," said Old Man Warner to the crowd, "and we'll be eating chickweed and acorns."
A colon may be used if the quotation is long and begins with a full sentence of introduction.
Ex: The following speech was made by Presient Lincoln at Gettysburg: "Four score and seven years ago..."
Use an ellipsis (...) when you want to drop words out of a quote.
Ex: Lincoln said, "Four score and seven years ago, our fathers brought forth...a new nation."
Single quotation marks are used to set off a quotation within a quotation.
Ex: Mary heard the cleaning woman mutter, "In the words of Lacy Macbeth, 'Out! Out! Damn spot!'" (Note that the single quotes and double quotes are used together at the end.)
Rules for Placement of Puncuation Marks with Quotations
Periods and commas are always placed inside quotation marks.
Ex: In "The Lottery," Tessie Hutchinson protests, "It isn't fair."
Exception: In MLA citations, the period follows the parentheses.
Ex: Tessie Hutchinson protested, "It isn't fair" (Jackson 346).
Colons and semicolons are always placed outside quotation marks.
Ex: John wrote, "I'm sorry that I won't be able to come home for Christmas"; however, he managed to get some time off.
Question marks and exclamation points are placed inside UNLESS they apply to the whole sentence and not just the quotation.
Ex: Did you hear the teacher say "Please bring your texts tomorrow"? (sentence is a question)
Ex: His mother asked, "Do you plan to come with us?" (quote is a question)
Other Uses for Quotation Marks
Use quotation marks around the titles of short works: newspaper and magazine articles, poems, short stories, songs, television and radio programs, chapters and subdivisions of books.
Ex: Many Americans enjoy Walt Whitman's poem "Leaves of Grass."
titles of longer works, such as books, films, plays, names of magazines and newspapers and television series, are put in italics or underlined.
Quotation marks may be used to set off words used as words.
Ex: The words "its" and "it's" are often confused.
Do NOT use quotation marks in an attempt to justify the use of slang or cliched expressions. Avoid the use of those types of phrases; find something better.
Ex: Her attitude was "sweet as sugar." (try ingratiating)
Ex: That job was "a piece of cake." (try easy)


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Senator Wright takes leave from Senate
There is No Denying Bradley Although Marquez is Crying
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17 October 2013
CLEARLY UNDENIABLE: WBO welterweight champion Tim Bradley exalts in victory with his father and co-trainer (Tim Bradley Sr., right) pointing skyward. Bradley outpointed Juan Marquez last Saturday at ...
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No Concern for Canelo as 'Money Talks'
Written by Kenneth D. Miller, Sentinel Sports Editor
05 September 2013
Earning his money: Floyd 'Money' Mayweather during his final tune up at Mayweather Boxing Club in Las Vegas. Mayweather will take on undefeated young redhead Mexican star Canelo Alvarez at the MGM ...
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Decking Out Nevada Hall Holmes, King, Tyson, Leonard and 'Money
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15 August 2013
The long wait is over for the state of Nevada officially welcomed its first boxing hall of fame class at the inaugural Nevada Boxing Hall of Fame (NVBHOF) Saturday August 2 at the Monte ...
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The Cinco de Mayo King Returns to Ring
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03 May 2013
Floyd 'Money' Mayweather storms Mexican holiday again The rivalry of Mexicans and Blacks is a long and bitter feud that has not just manifested itself in the boxing ring, but also has ...
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Bradley dished it as much as he took it in dramatic victory
Written by Jason Lewis (Sports Editor)
20 March 2013
Timothy Bradley (right) and Rusian Provodnikov dropped bombs on each other for 12 rounds in what may end up as the fight of the year. Photo by Jae C. Hong/Associated Press By Jason Lewis Sentinel ...
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Hopkins is still going at it at age 48
Written by Jason Lewis (Sentinel Sports Editor, email@example.com)
20 March 2013
Bernard Hopkins was supposed to retire years ago, but instead, at age 48, he is taking the younger fighters to school. Photo by Frank Franklin II/Associated Press By Jason Lewis Sentinel Sports ...
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Emanuel Steward: Detroit Fight Icon Was A Real Hero
Written by Kenneth Miller (Entertainment Editor)
29 October 2012
The sport of boxing has often been associated with the greed of promoters to the adversity that its combatants endure only to fall into the abyss of its slimy mitts. However, there is one figure ...
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Around the Ring: Terrel "Tyger" Williams remains undefeated; Pacquiao puts ball in Mayweather's court
Written by Jason Lewis (Sports Editor, email@example.com)
26 September 2012
Terrel 'Tyger' Williams, left, hurt his opponent bad with a right hand and the fight was quickly over, giving Williams another first round knockout. Photo by Jason Lewis Terrel "Tyger" ...
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Around the Ring: Andre Ward is on his way to stardom; Terrel Williams fights in Hollywood
Written by Jason Lewis (Sports Editor, email@example.com)
12 September 2012
Los Angeles native Terrel Williams (left) is a rising star in the welterweight division. He is 6-0 with five knockouts. He will fight at Florentine Gardens in Hollywood on September 20. Photo ...
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Around the Ring: 50 Cent and Money May's promotion company; Deotay Wilder is a rising star
Written by Jason Lewis (Sports Editor, email@example.com)
09 August 2012
Deotay Wilder made short work of another opponent. That brings his record to 24-0 with 24 knockouts. Time for rising star Deotay Wilder to step it up If you like knockouts, watch a ...
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Around the Ring: Broner looking to be the next 'Money May' and Thurman creates his own controversy
Written by Jason Lewis (Sports Editor, email@example.com)
26 July 2012
Adrien Broner (left) had an easy night against a much smaller opponent. Photo by Joseph Fuqua/AP Adrien Broner looking to be the next Floyd Mayweather Adrien "The Problem" Broner is ...
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Around the Ring: Khan KO'd; Broner on HBO; and boxing in Hollywood
Written by Jason Lewis (Sports Editor, email@example.com)
19 July 2012
Cruiserweight boxer Paul Parker traveled to Hollywood for his first professional fight, and he put on a show…that lasted about two minutes. A big right hand punch and this fight was over. ...
read more >>
Around the Ring: Boxing action from Home Depot Center
Written by Sentinel News Service
06 June 2012
Winky Wright, right, takes a punch from Peter Quillin in the ninth round of a middleweight boxing match. Quillin won by unanimous decision in the 10-round bout. Photo by Jae C. HongAntonio Tarver, right, ...
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Ringside Update: Tragedy hits boxing
Written by Mohammed Mubarak
30 May 2012
Paul Williams was one of the best and most avoided welterweight champions over the past 10 years, but his career has been cut short because of a motorcycle accident. Photo by Eric Jaminson (AP)Johnny ...
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Ringside Update 5/16/12
Written by Mohammed Mubarak
16 May 2012
Lamont Peterson (right) defeated Amir Khan in December, but that was after he was injected with a testosterone pellet, which is a banned substance. Photo by The Associated PressPeterson vs. Khan bout ...
read more >>
Mayweather Earns His Bloody Money
Written by Kenneth Miller Sentinel Correspondent & Jason Lewis Sports Editor
09 May 2012
Miguel Cotto (left) challenged Floyd Mayweather, which only brought out the best in Mayweather, who landed 179 punches to Cotto's 105. Photo by The Associated Press Undefeated Mayweather cashes in ...
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Follow the Mayweather/Cotto fight on Facebook and Twitter
Written by Sentinel News Service
04 May 2012
Floyd Mayweather (left) looks to stay undefeated when he takes on Miguel Cotto, one of his toughest opponents to date. Photo by Hoganphotos/Golden Boy PromotionsGoing to miss the big fight? No problem. ...
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All-American Heavyweights looks to save the boxing division
Written by Jason Lewis (Sports Editor)
02 May 2012
Dominic Breazeale's athletic career was nearly over when he did not make it to the NFL as a quarterback out of the University of Northern Colorado, but his 6-7, 270 pound frame is perfect for the sport ...
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Ringside Update 5/2/12
Written by Mohammed Mubarak
02 May 2012
47-year-old Bernard Hopkins (left) has been a legend, but after Saturday night it was clear that Chad Dawson is the best light heavy weight in the world. Photo by Mel Evans/AP Age catches up to Hopkins, ...
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Ringside Update 4/4/12
Written by Mohammed Mubarak
04 April 2012
Timothy Bradley packs a punch, and he's hoping to knock around Manny Pacquiao when they fight in June. Photo by Julie Jacobson/APJean Pascal is looking to take on Tavoris Cloud, but a negotiation issues ...
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Ringside Update 3/22/12
Written by Mohammed Mubarak
22 March 2012
Sugar Shane Mosley (Left) and WBC Super Welterweight World Champion Canelo Alvarez are introduced to an estimated crowd of 15,000 people in Mexico City at a press conference for their world championship ...
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Mayweather takes on Cotto
Written by Mohammed Mubarak
07 March 2012
Before Floyd Mayweather (left) can get to a potential fight with Manny Pacquiao, he has to face Miguel Cotto. Photo by Hoganphotos/Golden Boy PromotionsMayweather says that he is looking to add another ...
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Ringside Update 2/22/12
Written by Mohammed Mubarak
22 February 2012
This past Saturday night Showtime Network broadcasted an exciting night of matches in Corpus Christi, Texas. The night featured bouts between Paul Williams and Nobuhiro Ishida, Tavoris Cloud and Gabriel ...
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Ringside Update 2/9/12
Written by Mohammed Mubarak
09 February 2012
Floyd Mayweather is set to get back in the ring on May 5, but it will not be against Manny Pacquiao. Photo by Jae C. Hong (AP) Oscar De La Hoya's Golden Boy Promotions found a fill in for the May 5 date ...
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L.A. Matadors taking on all comers… worldwide!
Written by Jason Lewis (Sports Editor)
27 January 2012
Terrell Gausha (left) of the LA Matadors throws the left jab, but his opponent should be more worried about Gausha's right. Gausha hopes to make the 2012 Olympic team before turning pro later this ...
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Mississippi Civil Rights Hero Clyde Kennard Honored in D.C.
Second District Steps into the Fight Towards a Cure for Triple Negative Breast Cancer
Clayton Powell & Wyatt Tee Walker
who fought for the 'Right to Vote'- a weekly series counting down to the election"
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Keck Medicine of USC's New Downtown Los Angeles Location Ribbon Cutting and Open House
Leads a Safer Walk on King Blvd
Entertainment & ESSENCE Magazine Celebrate the "Images of Black Women in Media and Entertainment"
Victorie Celebrates Famous Grandmother Aretha Franklin at BET Honors
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Over Dessert Dress
- Shell: 100% Polyester. Lining: 95% Polyester, 5% Spandex.
- Runs small at bust. Fabric does not provide stretch. Length measurement taken to shortest point of hem.
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Cash is King You have many things to do to properly manage your small business, but nothing is more important than properly managing your cash. In this workshop... | Register |
Mar 12, 2014 5:45pm EDT - Mar 13, 2014 9:00pm EDT Jeffersontown, KY |
Own Your Own Business Seminar Interested in starting your own business or know anyone who is? SCORE and the Louisville SBDC are holding the Own Your Own Business Seminar.... | Register |
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Aspose.BarCode is a .NET component for generation and recognition of Linear and 2D barcodes on all kinds of .NET applications. It supports WPF with 29+ Barcode symbologies like OneCode, QR, Aztec, MSI, EAN128, EAN14, SSCC18, Code128, Code39, Postnet, MarcoPDF417, Datamatrix, UPCA, etc. Other features include barcode insertion in PDF, Word, and Excel documents. It can also take image input in BMP, GIF, JPEG, PNG, and WMF formats. You can also control image styles such as background color and bar color.
Aspose.Diagram is a class library for working with MS Visio files. It is a pure .NET alternative for MS Visio Object Model. It enables developers to work with VSD and VDX files on ASP.NET Web applications, Web services, and desktop applications. It makes use of the advanced functionality of Visio's services to manipulate Visio documents on a server. A developer can open files and manipulate the elements of the diagram, from lines and fills to more complex elements, and then export to native Visio formats or XML.
Aspose.BarCode for Reporting Services is a .NET solution for rendering barcodes images in SQL Server 2000, 2005, and 2008 Reporting Services. It supports over 29 linear (1D) and 2D barcode symbologies including MacroPdf417, Australia Post, OneCode, Code128, Code39, PDF417, UPCA, Codabar, MSI, and QR. It can also render barcode images on reports in BMP, JPEG, PNG, and GIF formats. Other features include EAN-128 application identifiers, DPI resolution settings, barcode size, and location adjustments.
Aspose.Report is a .NET query building and charting control. It allows developers to quickly add ad hoc capabilities to their .NET applications as well as create 21 popular chart types with some stylish effects like 3D rendering, transparency, gradients, and anti-aliasing. Simply drag and drop the Aspose.Report component on Web or Win form, assign a DataTable object as data source, and abstract a variety of outputs including SQL, Select, Where, and Order By statements and many more.


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ARLINGTON, Texas — The right fielder for the Texas Rangers has purchased a fire truck for his hometown, which lacks proper firefighting equipment.
AP Photo/Tony Gutierrez Texas Rangers right fielder Nelson Cruz poses with Arlington FD chief and the Las Matas Santa Cruz fire chief.
Nelson Cruz is originally from San Francisco de Macoris, the rural capital of the Dominican Republic. The city has 245,000 residents, but firefighters do not have a rugged pumper truck and few ambulances, according to the Star-Telegram.
With assistance from the Arlington Fire Department, Cruz was able to buy an all-terrain apparatus.
"In my hometown, we don't have ambulances or fire trucks, so I decided to buy one," he said.
American Medical Response, inspired by Cruz's purchase, is also donating two ambulances. The Arlington Fire Department is also throwing in hand tools, hoses, and nozzles.
Cruz presented the keys to the new truck to Chief Luis Esmurdoc on Wednesday at a game against the Oakland A's, the Star-Telegram reported.
Esmurdoc's department services three neighboring provinces and three outlying communities.
"They cover a lot of territory with just a few trucks. Having an additional truck will help them out tremendously," Arlington Assistant Fire Chief David Carroll said.
Cruz hopes all the donated equipment will be shipped by November.


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The thunderous results of Thailand's general election on July 3 will seem familiar to anyone attuned to the political upheaval in the Middle East and North Africa.
Entrenched incumbent regimes everywhere are under severe stress from advances in information technology, shifts in demographics, rising expectations, and the obsolescence of Cold War exigencies. In the absence of a willingness and ability to use violent repression, regime survival can be achieved only through concessions, accommodation, and periodic reinvention.With 47 million voters and turnout at 75 per cent, Thailand's latest election results pose a decisive challenge to the country's long-established regime. The Pheu Thai party, led by Yingluck Shinawatra, the youngest sister of exiled fugitive former Prime Minister Thaksin Shinawatra, secured a resounding triumph, winning 265 seats in the 500-member assembly, while the ruling Democrat Party mustered just 159.The return to power of Pheu Thai is extraordinary - and not only because Yingluck will be Thailand's first female prime minister. The establishment-aligned courts dissolved the party's two previous governments, and banned scores of its leading politicians from office for five years.Pheu Thai's victory thus suggests that a previously marginalised electorate has been permanently awakened.
A similar majority of the Thai electorate voted for Thaksin's parties and their pro-poor populist platforms in January 2001, February 2005, April 2006, and December 2007, defying a military coup, a coup-induced constitution, judicial interventions, and army coercion and repression.The recent election marked a profound break from the past. In the second half of the twentieth century, Thai elections seemed to alternate with military coups. Voters were bought and sold like commodities.
After elections, voters hardly ever saw or heard from their MPs, who typically went on to engage in corruption and graft in Bangkok - eventually losing legitimacy and paving the way for military coups. A new constitution and elections invariably ensued. This vicious cycle of coups, constitutions, and elections defined Thai politics for decades.That pattern reflected Cold War imperatives. The pillars of the Thai state - nation, religion, and the king - struck a unifying, collective chord, and the resulting stability enabled economic development. While growth was so concentrated that popular resentment simmered, communism was kept at bay.
Challenges to the established order, with the military-monarchy-bureaucracy triumvirate as its anchor, were repeatedly put down.Back then, Thai schoolchildren sang martial songs each morning, and Thais knew their place in the rigidly elitist pecking order, which was reinforced by socialisation and indoctrination in classrooms and living rooms, where only state-controlled media could enter. Thais were more like obedient subjects than informed citizens. Dissenting views found little traction.
Thai Rak ThaiThe rise of Thaksin's Thai Rak Thai party in 2001 changed all that.
The party pursued a scientific approach to elections, relied on expensive polling by foreign experts, and offered a clear agenda and strong leadership. It was the first post-Cold War party to capture Thais' collective imagination.
The voices of neglected swaths of the electorate, particularly in the rural north and northeast of the country, began to count. Vote-buying became increasingly insufficient. A bond between party and voters - a bond based on policy - took root.By 2001, the Cold War was long over. Political leaders who dissented from the status quo could no longer easily be jailed on communism-related charges. The advent of the internet had made it harder for the authorities to shape Thai minds, as media sources multiplied and the resulting diffusion of information undermined the effectiveness of state propaganda. Moreover, new international norms had come to the fore: external powers that previously turned a blind eye to coups, military dictatorships, and repression now rallied around democracy and human rights.Thailand's demographics also changed. The Cold War curriculum of induced unity and stability has no relevance for today's schoolchildren; indeed, most university students nowadays were born after the Cold War ended.These factors fostered a new political environment, and Thaksin, who was a telecommunications tycoon at the time, was well positioned to seize the opportunity. He overhauled the bureaucracy, delivered on his promises to the poor, mapped out an industrial strategy, and re-designed an overstretched foreign policy agenda, among other innovative measures.Of course, Thaksin's rule had a dark underside: corruption, legislated conflicts of interest, cronyism, human rights violations, and abuse of power, among other evidence of misrule.Such is Thaksin's mixed legacy. The opportunities, hopes, and dreams delivered to the downtrodden and the vision and plans for Thailand's future were enmeshed with his own venality. But, while Thaksin committed many infractions, his gravest "sin" was to have changed the way Thais think and behave. Some see this change as usurpation; others view it as Thailand's deliverance into the twenty-first century.Thaksin's adversaries in Thailand's entrenched regime were unwilling to accede to his policy innovations and populism. For them, doing so would be tantamount to admitting that most people in this hospitable, well-endowed kingdom had been kept poor by design all along.For his part, Thaksin has sought to portray the recent election results as being all about him. But he is best viewed as a self-serving, unwitting agent of political modernisation.
It is these twenty-first-century dynamics and changes, underpinned by an increasingly assertive citizenry, with which the Thai establishment must come to terms if the country is to move forward.Thitinan Pongsudhirak is Director of the Institute of Security and International Studies at Chulalongkorn University.
The views expressed in this article are the author's own and do not necessarily represent Al Jazeera's editorial policy.
A version of this article was previously published on the Project Syndicate.
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Dragonair (Base Set 18)
From Bulbapedia, the community-driven Pokémon encyclopedia.
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Dragonair (Japanese: ハクリュー Hakuryu) is a Colorless-type Stage 1 Pokémon card. It is part of the Base Set.
Card text
Pokédex data
Dragonair - Dragon Pokémon | ||
No. | Height | Weight |
148 | 13'1" (4.0 m) | 36 lbs. (16.5 kg) |
Pokédex entry | ||
A mystical Pokémon that exudes a gentle aura. Has the ability to change climate conditions. | ||
海や湖などに住むという。羽を持たないが、たまに空を飛ぶ姿が目撃される。 |
Release information
This card was reprinted in the Base Set 2 reprint set, which was only released outside of Japan.
Slam and Hyper Beam are both moves in the Pokémon games that Dragonair can learn. This card's English Pokédex entry comes from Pokémon Red and Blue, whereas the Japanese entry comes from Pokémon Red and Green.
This article is part of Project TCG, a Bulbapedia project that aims to report on every aspect of the Pokémon Trading Card Game. |


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Synthesizing music, film, and an unforgettable live experience, this innovative indie band, X: THC is evolving and redefining how a live show is presented and experienced within the model of the music industry.Musically, X: THC combines chilled, shattered beats, s
The work of Australian artist Georgie Roxby Smith challenges the relationship between analog and digital systems, questions materiality and explores new possibilities of virtual reality software. In her recent Watermill Center residency, Smith collaborated with new media and theater
Paul Clay is a musician and video artist whose music blends carelessness and glitz with concern and radicalism. Drawing on his convictions, life experience, and influences as varied as Paul Simon, Enya, and Peaches, he is creating exciting music with a vibrant flavor and a sound all his own.


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Boredom can run, but it can't hide! That's because the colorfully caped crusader wearing this crown will hunt it down and transform it into a fun-filled fantasy. This powerful topper is the perfect finishing touch to any little crime fighter's superhero ensemble.


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Less than 5 minute walk from the train and bus station. Trains take you directly to Venice in 10 minutes and run very regularly. The bus brought us from Treviso airport to the bus station, and then back again on our day of departure, in about 50 minutes and we bought the return tickets for the bus on the RyanAir... More
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The answer is some degree of "very."
The second wave of British lawsuits is coming.
Both marquee hosts are set to stick around through 2016.
Criminal charges could be next.
Sky News said they intercepted e-mails, but it was "in the public interest."
The phone-hacking scandal that launched a thousand resignations.
The News Corporation son had made a final written plea about phone hacking to Parliament.
This scandal is logging some serious frequent-flier miles.
The media mogul will likely be called again to a judicial inquiry in the spring.
Rupert's scandal-plagued son is finally taking that "breather."
The iPad newspaper threatened legal action against a disparaging former employee.
The former News Corp. editor was quite cozy with Scotland Yard.