kinesin is a molecular motor that transports organelles along a microtubule ( mt ) using the energy of atp hydrolysis .
conventional kinesin is highly processive and can move across hundreds of tubulin dimers ( > 1 m ) without dissociating from the mt ( howard et al . , 1989 ; block et al . , 1990 ) .
this processive movement has been explained by a  hand - over - hand  model in which the two heads of a kinesin alternately interact with the mt ( schnapp et al . , 1990 ; hackney , 1994 ; howard , 1996 ) . a nucleotide - dependent conformational change in the  neck - linker  , a small peptide emerging from the cooh terminus of the catalytic core , is considered crucial for the  walking  mechanism of two - headed kinesin ( rice et al . , 1999 ) .
the model attributes the movement to structural change in the kinesin , whereas mts are regarded as merely a passive track . on the other hand , in actomyosin , the motor system in muscle
, several reports have indicated that the structure and dynamics of actin filaments may play an active role in motility . when actin filaments interact with heavy meromyosin in the presence of atp , a rapid and significant bending motion or accelerated diffusion of actin filaments
was observed , indicating that some kind of conformational change may be occurring in actin filaments ( yanagida et al .
moreover , an experiment using intrastrand cross - linked actin demonstrated that the velocity of actin filaments in an in vitro motility assay was reduced with an increase in actin cross - linking , whereas the rigor myosin subfragment ( s1 ) binding to actin filaments and the acto - s1 atpase activity were virtually unaffected by the cross - link ( kim et al . , 1998 ) .
this indicated that the dynamic property of actin filaments that is essential for motility might be inactivated by the intrastrand cross - link .
these results in the actomyosin system raise the possibility that in the corresponding mt  kinesin system ,
mts are more than simply a passive track . to study the dynamic properties of mts during motility , we directly observed the binding and motility of kinesin - coated beads along a single mt .
we find that in the presence of atp , a kinesin - coated bead binds to a mt in a cooperative manner with a large cooperative range , in the order of micrometers in length .
the result suggests that the mt has both high and low affinity states for kinesin , and binding of kinesin induces the high affinity state over an extended range .
in contrast to this cooperative binding in the presence of atp , in the presence of adp or 5-adenylylimidodiphosphate ( amp - pnp ) , binding was random along the mt , indicating that this state transition is closely related to motility .
mt interactions , native kinesin purified from bovine brain was first coated onto the surface of fluorescent latex beads ( 0.2 m in diameter ) at a molar ratio of 17 to 1 .
these kinesin - coated beads were then combined with rhodamine - labeled mts in the presence of atp , and their binding to a mt was directly observed by conventional fluorescence microscopy . in this experimental condition , the kinesin beads bound to the mt at an average binding frequency of 0.018 m s and moved toward the mt plus end at a velocity of 0.90  0.15 m / s ( mean  sd ) . due to the high density of bound kinesin , most of the beads that had bound to the mt ( 97  4% ) did not dissociate from the mt until reaching the plus end ( block et al . ,
1 a shows sequential images of kinesin beads moving on a single mt , taken at 1-s intervals . new bindings
quite unexpectedly , the binding of kinesin beads was not completely random , but occurred preferentially in the vicinity of beads that were already moving along the mt .
consequently , a cluster of kinesin beads was formed over several micrometers of mt filament , and longer observation times revealed new clusters being repeatedly formed ( video 1 , available at http://www.jcb.org/cgi/content/full/jcb.200409035/dc1 ) .
interaction of kinesin - coated beads with a mt in various nucleotide conditions . ( a ) sequential images of kinesin beads interacting with a mt in the presence of 1 mm atp , taken at 1-s intervals .
( b ) diagrams illustrating how the windows ( represented by rectangles of various colors ) were defined for the statistical analysis of the binding frequency .
the example is taken from the images shown in a. starting from the position of the preexisting kinesin beads ( yellow dots ) , a window of 1 m was shifted along a mt in both plus- and minus - end directions until the entire length of mt was covered .
( c ) the binding frequency calculated as a function of distance from the preexisting kinesin beads .
binding was measured in the presence of 1 mm atp ( blue dot ) , amp - pnp ( gray dot ) , and adp ( green dot ) at a kinesin - bead concentration of 90 pm .
total number of binding events , number of mts , and total observation time were 934 binding events , 5 mts ( length = 12.9018.55 m ) , 61 min for atp ; 734 binding events , 65 mts ( length = 11.9418.10 m ) , 114 min for adp ; and 669 binding events , 72 mts ( length = 12.1818.87 m ) and 115 min for amp - pnp , respectively .
when the position of the kinesin bead was recorded at a higher temporal resolution ( 10 frame / s ) , the binding frequency was not affected , indicating that the temporal resolution of 3 frames / s is adequate . to test the statistical validity of the observed preferential binding , the binding frequency of kinesin beads
first , the positions of the beads interacting with a single mt were determined using the image analysis software , nih image , at 0.33-s intervals ( at a rate of 3 frame / s ) over an observation period of 815 min , which corresponded to between 159 and 205 new binding events .
based on the instantaneous position of each preexisting , moving kinesin bead , a 1-m window was shifted along the mt in both the plus- and minus - end directions ( fig . 1 b , colored rectangles ) , and for each window with a distinct color , the total number of new binding events , n , and the total length of mt , l , was measured .
n and l were thus measured in five randomly selected mts and the results were combined . finally , the frequency of new binding events in each window was obtained by dividing the combined n by the product of combined l and t ( = n/(l*t ) ) , where t is the observation time ( 0.33 s ) .
1 c is a result of such a calculation , showing that binding was actually enhanced in the vicinity of preexisting kinesin beads except for the area immediately surrounding the preexisting kinesin bead . in that area ( within 1 m distance )
, the binding was slightly suppressed due to physical obstruction of the binding site by the preexisting bead .
statistical significance of the preferential binding was confirmed by the kolmogorov - smirnov test of probability density distributions ( pdds ; fig .
this calculation of binding frequency does not depend on any statistical assumptions , because it simply divides the mt into zones depending on the distance from the moving kinesin beads , and calculates the binding frequency in each zone .
binding was less frequent and random along the mt in the presence of both adp and amp - pnp ( fig .
1 c ) . it should be noted that the kinesin concentration used here ( 1.5 nm ) was significantly lower than that used previously ( 610 m ) to examine the cooperative binding of kinesin dimers in the presence of amp - pnp ( vilfan et al . , 2001 ) .
the cooperative binding observed in the previous study was most likely a result of attractive interaction between kinesin molecules .
although the binding frequencies were similar for adp and amp - pnp , the behavior of the kinesin beads after the binding event was different , reflecting the weak and strong binding states in adp and amp - pnp conditions , respectively ( crevel et al . , 1996 ) . in the presence of adp ,
the position of the kinesin beads changed slightly with time along the mt ( diffusion coefficient = 380 nm / s ) , and in some rare occasions , they even dissociated from the mts ( koff = 0.004/s ) .
in contrast , in the presence of amp - pnp , the kinesin beads neither changed their position ( diffusion coefficient 26 nm / s ) nor dissociated from the mt once bound .
we next wished to determine if there is any bias in the direction of cooperative binding . in the analysis of binding frequency described above ,
directionality was not explicitly considered because in situations where multiple beads were simultaneously moving along a mt , it was difficult to determine whether it was the forward or the rear bead that facilitated the new binding . to simplify the analysis ,
directionality was examined only in cases where a single kinesin bead was moving on the mt before the analysis ( fig .
the distribution of subsequent new binding events relative to the position of the moving beads was then examined . to guarantee equal opportunity for new binding in either direction ,
new bindings were included in the statistics only when the available area of mt exceeded 6 m in length on both sides of the moving kinesin beads .
situation was relatively rare , records obtained over a total observation period of 4 h ( corresponding to 20 mts ) were scrutinized and 76 new bindings were counted ( fig .
the result revealed a biased distribution in the plus - end direction ; within a 6-m distance , binding in the plus - end direction was 2.2 times higher than the binding in the minus - end direction ( 52 plus - end events vs. 24 minus - end events ) .
assuming no particular preference in the direction of cooperative binding , the probability of such an uneven distribution within statistical fluctuation is < 0.2% ( chi - square test ) .
distribution of the lone binding summed for 20 mts . in both a and b , an arrowhead indicates the position of the preexisting , moving kinesin bead . as multiple kinesin beads were simultaneously moving along the mt most of the time ,
only 2% of the total binding events was counted for this lone - binding analysis .
directional , cooperative binding was also confirmed in another experiment where a kinesin bead was first fixed on the mt and then the subsequent binding of other kinesin beads was examined ( fig .
this experiment was designed to more efficiently analyze the directionality of cooperative binding . to make a kinesin bead stationary on the mt in the presence of atp
, we used a previously constructed heterodimeric kinesin , wt / e236a , which can hydrolyze atp , and yet remain fixed on the mt ( table s1 , available at http://www.jcb.org/cgi/content/full/jcb.200409035/dc1 ; kaseda et al . , 2002 ) .
in addition to this refinement , compared with the previous experiment shown in figs . 1 and 2 , we lowered both the kinesin density on the beads and the concentration of beads used in the binding assay , thereby increasing the chance of lone bindings ( see materials and methods for details ) .
( b ) three representative example distributions of binding frequency for a mt with a bound wt / e236a - bead .
total binding number and observation time is ( from top to bottom ) 324 , 1,174 s ; 158 , 1,200 s ; 292 , 1,193 s. ( video 2 ) .
( c ) three example distributions for a mt with a bound e236a / e236a - bead .
total binding number and observation time is ( from top to bottom ) 99 , 1,026 s ; 78 , 1,200 s ; 119 , 1,200 s. in both b and c , binding was suppressed in the immediate vicinity of the reference bead due to physical obstruction of the binding site by the reference bead .
length of the mt on either side of the reference bead , measured from the center of the bead , is indicated in the graph .
bin size = 1 m . ( d ) distribution of the binding frequency averaged for 10 mts within a 6-m distance either side of the reference bead .
the red line is for mts with a bound wt / e236a - bead ( 1,429 bindings were counted over a total observation period of 187 min ) and the blue line is for mts with a bound e236a / e236a - bead ( 699 bindings were counted over a total observation period of 196 min ) .
asterisks indicate that the difference in mean binding frequencies between the two groups was significant ( p < 0.01 ; t test ) .
bin size = 1 m . note that the absolute value of the binding frequency in this experiment can not be directly compared with the binding frequency shown in fig .
kinesin beads were different between the two experiments ( see materials and methods ) . before the binding assay ,
fluorescent latex beads with a diameter of 0.5 m were first coated with wt / e236a via a streptavidin  biotin interaction , and these wt / e236a - beads were bound to mts in the absence of atp ( fig .
3 a , step 1 , fixing a reference bead to an mt ) . subsequently ,
binding of other kinesin beads ( 0.2 m in diameter , coated with wild - type kinesin ) to these mts was tested in the presence of atp ( fig .
3 a , step 2 , binding assay using test beads ) . to guarantee equal opportunity for binding in either direction , again , only those mts with a single reference bead bound and having > 6 m of free mt on either side of the reference bead were analyzed .
a homodimer of e236a showing no atpase activity ( table s1 ) was used as a control to coat the reference bead .
first , to validate the usage of wt / e236a , we measured the distribution of binding frequency along the mts with a bound wt / e236a - bead .
the result showed that binding of the test beads was enhanced in the vicinity of the wt / e236a - bead , most prominently in the plus - end direction ( fig .
in contrast , for mts with a bound e236a / e236a - bead , the binding frequency was low and no significant bias was detected ( fig .
3 c ) . when the binding frequency averaged for 10 mts was compared between the two groups , the frequency was significantly higher for mts with a wt / e236a - bead ( fig .
. 1 c , which indicated atp hydrolysis is essential for cooperative binding . now to evaluate the directionality of cooperative binding , based on the same binding data , only those new bindings occurring in the absence of any other beads moving on the mt were counted .
although the experimental conditions were optimized here to increase the chance of lone bindings occurring , the value for total bindings shown in fig .
3 b may still include those bindings that are facilitated by the presence of other test beads on the mt ( fig .
4 a ) . the distribution of the lone bindings revealed that for mts with a bound wt / e236a - bead , the binding was significantly higher in the plus - end than the minus - end direction ( 205 plus - end events and 115 minus - end events , p < 0.01 ; chi - square test ; fig . 4 b ) .
in contrast , for mts with a bound e236a / e236a - bead , distribution of lone bindings was not significantly biased ( 203 plus - end and 211 minus - end events , p = 0.694 ; chi - square test ; fig . 4 c ) .
we conclude that cooperative binding induced genuinely by the wt / e236a - bead was biased toward the plus - end direction .
3 b may include those that are genuinely induced by the wt / e236a - bead and those that are facilitated by the presence of other test beads .
even if the cooperative binding induced genuinely by the wt / e236a - bead was symmetric , the movements of these bound beads toward the mt plus end might lead to the asymmetric distribution of the subsequent binding .
( b ) distribution of the lone bindings summed for 10 mts with a bound wt / e236a - bead ( total 320 bindings ) .
( c ) distribution of the lone bindings summed for 10 mts with a bound e236a / e236a - bead ( total 414 bindings ) . in both b and c ,
bin width is 1 m . with a similar observation time ( 190 min ) , more lone bindings were counted for mts with a bound e236a / e236a - bead as compared with the mts with a bound wt / e236a - bead .
this result is not surprising given that less frequent binding means a larger fraction of lone bindings in the total binding count . in an independent series of experiments , the directionality of lone binding was also examined in the presence of either adp or amp - pnp for mts with a bound wt / e236a - bead . in both conditions ,
no significant directional bias was detected ( for adp , 57 plus - end and 59 minus - end events , p = 0.853 ; for amp - pnp , 56 plus - end and 53 minus - end events , p = 0.774 ; chi - square test ) .
together , the results show that binding of kinesin beads to a mt is cooperative in the presence of atp and that it is biased toward the mt plus end .
it is unlikely that cooperative binding over micrometers in mt length is the result of electrostatic attraction between kinesin beads ( israelachvili , 1985 ) .
rather , the results can be explained if mts have more than two binding - affinity states for kinesin beads . under this model , in the presence of adp or amp - pnp , mts remain in a low affinity state along their entire length , while in the presence of atp , binding of a kinesin bead to a mt might induce transition of the mt to a state with higher affinity for other kinesin beads over a range of micrometers .
as the e236a / e236a - bead was unable to induce cooperative binding , atp hydrolysis by kinesin is essential for inducing the state transition in the mt .
quantitative analysis of the number of vesicles associated with individual mt revealed that mts in axons exist in two populations , each having a different affinity for vesicles ( miller et al . , 1987 ) .
in vivo , organelle transport could be regulated by alteration of the mt affinity states .
furthermore , our result that the cooperative binding was biased in the plus - end direction suggests a possibility that anisotropy in the potential of kinesin  mt interaction might contribute to the directional movement of kinesin ( okada and hirokawa , 1999 ) .
structural studies of mts and tubulin sheets have revealed that the binding of kinesin in the presence of amp - pnp induces a conformational change in tubulin dimers ( hoenger and milligan , 1997 ; krebs et al . , 2004 ) .
it is of interest to know how the structure of the kinesin  mt complex changes during atp hydrolysis . because the movement of motors should involve an intimate and concerted partnership between the motor and the cytoskeletal filaments , understanding
the role of cytoskeletal filaments could be essential to the complete elucidation of the motility mechanism .
tetramethylrhodamine - labeled mts were prepared as described previously ( miyamoto et al . , 2000 ) . to prepare kinesin - coated beads , fluorescent latex beads ( carboxylate modified , 0.2 m in diameter , 10% wt / vol ; f-8811 ; molecular probes )
were first incubated with an equal volume of casein solution ( 5 mg / ml in 10 mm tris , 0.1 m nacl ) for 30 min on ice .
casein mixture was subsequently combined with a 0.63 m kinesin solution and 1 m nacl at a volume ratio of 2:1:0.3 , and incubated for 4 h on ice . using this protocol , virtually all the kinesin molecules in solution
were adsorbed onto the surface of the latex beads ( determined by folin ; lowry et al . , 1951 ) , and thus the molar stoichiometry of kinesin to bead
was estimated to be 17 molecules per bead . for binding assays in the presence of atp , mts ( 10 g / ml ) in buffer
a ( 20 mm pipes , 10 mm k - acetate , ph 6.8 , 4 mm mgso4 , 2 mm egta , 0.2 mm edta ) supplemented with 10 m taxol ( t-7402 ; sigma - aldrich ) were introduced to a flow chamber made of two coverslips ( dimensions 10 mm  10 mm  110 m ) .
the bottom coverslip was pretreated with sigmacote ( sl2 ; sigma - aldrich ) and the mts tended to stick to its surface for their entire length . in the following procedure ,
after a 3-min incubation with the mt solution , 20 l of casein solution was introduced to the chamber .
after another 5-min incubation , the chamber was extensively washed with 40 l of buffer a and loaded with 20 l of kinesin - bead solution ( kinesin - coated beads prepared as above and diluted to a concentration of 90 pm in buffer a including 1 mm atp and an oxygen scavenger [ miyamoto et al . , 2000 ] ) .
the flow chamber was sealed with vaseline and observed under an inverted fluorescence microscope ( tmd-300 ; nikon ) equipped with an oil immersion objective lens ( neofluar , 100 , na = 1.3 ; carl zeiss microimaging , inc . ) and a sit camera ( c2400 - 8 ; hamamatsu photonics ) .
the observation was made at 26  1c and the image was recorded using a digital video recorder ( dsr20 ; sony ) . for binding assays in the presence of adp or amp - pnp ,
as kinesin beads are difficult to dissociate once bound to mts in these nucleotide conditions , the number of kinesin beads on the mt increased with time . to observe this process from the start
, mts were first allowed to attach to the bottom of the flow chamber , and then viewed under a fluorescence microscope .
the kinesin - bead solution including 1 mm adp or amp - pnp was subsequently introduced to the chamber so that the process of their binding to the mts could be monitored in real time by fluorescence microscopy .
images of the binding were recorded until the average bead density on the mt approached 0.58 beads/m , which was the maximum bead density achieved in the experiment in the presence of atp . in the case of amp - pnp , the solution was supplemented with apyrase ( a-6132 , 1 u / ml ; sigma - aldrich ) .
statistical significance of cooperativity was examined by comparing the pdd expected for random binding and the pdd of actual binding by the kolmogorov - smirnov test ( fig .
if the binding was random along the mts , n should be a linear function of l in each window .
then , the pdd of actual binding , \documentclass[10pt]{article } 
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 \begin{equation*}{l_{i}}/{{{\sum^{9}_{i=2}}}l_{i}}{\mathrm{,}}\end{equation*}\end{document}where ni and li are the total number of bindings and the total length of mt in the ith window from the preexisting kinesin bead , respectively .
the window closest to the preexisting kinesin bead ( i = 1 ) was omitted from the calculation , because in this area , binding was suppressed due to physical obstruction of the binding site . the homodimeric and heterodimeric e236a mutants were prepared as described previously ( kaseda et al . , 2002 ) .
steady - state mt - activated atpase was measured using malachite green ( kodama et al . , 1986 ) .
to make the wt / e236a- and e236a / e236a - beads , carboxylated fluorescent latex beads with a diameter of 0.5 m ( l-5261 ; molecular probes ) were coated with streptavidin and subsequently incubated with wt / e236a or e236a / e236a as described previously ( kaseda et al . , 2003 ) .
labeling stoichiometry of the reference beads , estimated from their probability of movement in optical nanometry ( kojima et al . , 1997 ) , was 100 homodimers and 25 heterodimers to a bead on average . to prepare test beads for the binding assay , 0.2-m diam fluorescent latex beads were coated with native kinesin as described previously ( binding assay under various nucleotide conditions ) , except that the stoichiometry of kinesin to a bead was 12 and the final concentration of the beads used in the binding assay was 80 pm . for the binding assay ,
mts were made attached to the bottom of the flow chamber and 20 l of casein solution including 10 m taxol was introduced to the chamber , and incubated for 10 min . in the following procedure , 10 m taxol was always included .
the chamber was washed with 30 l of buffer a and then loaded with 20 l of wt / e236a- or e236/e236a - bead solution ( 3 pm ) .
after an 8-min incubation , the chamber was extensively washed with 60 l of buffer a to remove the beads not bound to mts .
finally , 15 l of test - bead solution including 1 mm atp and an oxygen scavenger was introduced into the chamber , and the binding was observed under fluorescence microscope .
mts with only one reference bead attached and having > 6 m of mt length on either side of the reference bead were imaged for 20 min . for binding assays in the presence of adp or amp - pnp for mts with a bound wt / e236a - bead ,
the protocol was same as above except that the nucleotide included in the final test - bead solution was 1 mm adp or amp - pnp .
because the test beads only showed minimal dissociation from the mts in the presence of these nucleotides , we counted only one lone binding from a single mt . in each experiment , after the observation of the initial binding event , test - bead solution including 1 mm atp was freshly introduced into the chamber and the polarity of this single mt was determined by the direction of the movement of the beads .
s1 showing statistical analysis of kinesin - bead binding ; table s1 showing kinetic and motile properties of heterodimeric and homodimeric e236a kinesin ; and videos 1 and 2 , corresponding to data in figs . 1 a and 2 b , respectively .
tetramethylrhodamine - labeled mts were prepared as described previously ( miyamoto et al . , 2000 ) . to prepare kinesin - coated beads , fluorescent latex beads ( carboxylate modified , 0.2 m in diameter , 10% wt / vol ; f-8811 ; molecular probes )
were first incubated with an equal volume of casein solution ( 5 mg / ml in 10 mm tris , 0.1 m nacl ) for 30 min on ice .
casein mixture was subsequently combined with a 0.63 m kinesin solution and 1 m nacl at a volume ratio of 2:1:0.3 , and incubated for 4 h on ice . using this protocol , virtually all the kinesin molecules in solution
were adsorbed onto the surface of the latex beads ( determined by folin ; lowry et al . , 1951 ) , and thus the molar stoichiometry of kinesin to bead
was estimated to be 17 molecules per bead . for binding assays in the presence of atp , mts ( 10 g / ml ) in buffer
a ( 20 mm pipes , 10 mm k - acetate , ph 6.8 , 4 mm mgso4 , 2 mm egta , 0.2 mm edta ) supplemented with 10 m taxol ( t-7402 ; sigma - aldrich ) were introduced to a flow chamber made of two coverslips ( dimensions 10 mm  10 mm  110 m ) .
the bottom coverslip was pretreated with sigmacote ( sl2 ; sigma - aldrich ) and the mts tended to stick to its surface for their entire length . in the following procedure ,
after a 3-min incubation with the mt solution , 20 l of casein solution was introduced to the chamber .
after another 5-min incubation , the chamber was extensively washed with 40 l of buffer a and loaded with 20 l of kinesin - bead solution ( kinesin - coated beads prepared as above and diluted to a concentration of 90 pm in buffer a including 1 mm atp and an oxygen scavenger [ miyamoto et al . , 2000 ] ) .
the flow chamber was sealed with vaseline and observed under an inverted fluorescence microscope ( tmd-300 ; nikon ) equipped with an oil immersion objective lens ( neofluar , 100 , na = 1.3 ; carl zeiss microimaging , inc . ) and a sit camera ( c2400 - 8 ; hamamatsu photonics ) .
the observation was made at 26  1c and the image was recorded using a digital video recorder ( dsr20 ; sony ) . for binding assays in the presence of adp or amp - pnp ,
as kinesin beads are difficult to dissociate once bound to mts in these nucleotide conditions , the number of kinesin beads on the mt increased with time . to observe this process from the start
, mts were first allowed to attach to the bottom of the flow chamber , and then viewed under a fluorescence microscope .
the kinesin - bead solution including 1 mm adp or amp - pnp was subsequently introduced to the chamber so that the process of their binding to the mts could be monitored in real time by fluorescence microscopy .
images of the binding were recorded until the average bead density on the mt approached 0.58 beads/m , which was the maximum bead density achieved in the experiment in the presence of atp . in the case of amp - pnp , the solution was supplemented with apyrase ( a-6132 , 1 u / ml ; sigma - aldrich ) .
statistical significance of cooperativity was examined by comparing the pdd expected for random binding and the pdd of actual binding by the kolmogorov - smirnov test ( fig .
if the binding was random along the mts , n should be a linear function of l in each window .
then , the pdd of actual binding , \documentclass[10pt]{article } 
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 \begin{equation*}{n_{i}}/{{{\sum^{9}_{i=2}}}n_{i}}{\mathrm{,}}\end{equation*}\end{document}is expected to coincide with the pdd of the mt length , \documentclass[10pt]{article } 
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 \begin{document } 
 \begin{equation*}{l_{i}}/{{{\sum^{9}_{i=2}}}l_{i}}{\mathrm{,}}\end{equation*}\end{document}where ni and li are the total number of bindings and the total length of mt in the ith window from the preexisting kinesin bead , respectively .
the window closest to the preexisting kinesin bead ( i = 1 ) was omitted from the calculation , because in this area , binding was suppressed due to physical obstruction of the binding site .
the homodimeric and heterodimeric e236a mutants were prepared as described previously ( kaseda et al . , 2002 ) .
steady - state mt - activated atpase was measured using malachite green ( kodama et al . , 1986 ) .
to make the wt / e236a- and e236a / e236a - beads , carboxylated fluorescent latex beads with a diameter of 0.5 m ( l-5261 ; molecular probes ) were coated with streptavidin and subsequently incubated with wt / e236a or e236a / e236a as described previously ( kaseda et al . , 2003 ) .
labeling stoichiometry of the reference beads , estimated from their probability of movement in optical nanometry ( kojima et al . , 1997 ) , was 100 homodimers and 25 heterodimers to a bead on average . to prepare test beads for the binding assay , 0.2-m diam fluorescent latex beads were coated with native kinesin as described previously ( binding assay under various nucleotide conditions ) , except that the stoichiometry of kinesin to a bead was 12 and the final concentration of the beads used in the binding assay was 80 pm . for the binding assay ,
mts were made attached to the bottom of the flow chamber and 20 l of casein solution including 10 m taxol was introduced to the chamber , and incubated for 10 min . in the following procedure , 10 m taxol was always included .
the chamber was washed with 30 l of buffer a and then loaded with 20 l of wt / e236a- or e236/e236a - bead solution ( 3 pm ) .
after an 8-min incubation , the chamber was extensively washed with 60 l of buffer a to remove the beads not bound to mts .
finally , 15 l of test - bead solution including 1 mm atp and an oxygen scavenger was introduced into the chamber , and the binding was observed under fluorescence microscope .
mts with only one reference bead attached and having > 6 m of mt length on either side of the reference bead were imaged for 20 min . for binding assays in the presence of adp or amp - pnp for mts with a bound wt / e236a - bead ,
the protocol was same as above except that the nucleotide included in the final test - bead solution was 1 mm adp or amp - pnp .
because the test beads only showed minimal dissociation from the mts in the presence of these nucleotides , we counted only one lone binding from a single mt . in each experiment , after the observation of the initial binding event , test - bead solution including 1 mm atp was freshly introduced into the chamber and the polarity of this single mt was determined by the direction of the movement of the beads .
s1 showing statistical analysis of kinesin - bead binding ; table s1 showing kinetic and motile properties of heterodimeric and homodimeric e236a kinesin ; and videos 1 and 2 , corresponding to data in figs . 1 a and 2 b , respectively .
 interaction of kinesin - coated latex beads with a single microtubule ( mt ) was directly observed by fluorescence microscopy . in the presence of atp , binding of a kinesin bead to 
 the mt facilitated the subsequent binding of other kinesin beads to an adjacent region on the mt that extended for micrometers in length . 
 this cooperative binding was not observed in the presence of adp or 5-adenylylimidodiphosphate ( amp - pnp ) , where binding along the mt was random . 
 cooperative binding also was induced by an engineered , heterodimeric kinesin , wt / e236a , that could hydrolyze atp , yet remained fixed on the mt in the presence of atp . 
 relative to the stationary wt / e236a kinesin on a mt , wild - type kinesin bound preferentially in close proximity , but was biased to the plus - end direction . 
 these results suggest that kinesin binding and atp hydrolysis may cause a long - range state transition in the mt , increasing its affinity for kinesin toward its plus end . 
 thus , our study highlights the active involvement of mts in kinesin motility . 
Introduction
Results and discussion
Materials and methods
Binding assay under various nucleotide conditions
Analysis of cooperativity
Preparation of kinesin constructs and ATPase assays
Binding assay using MTs with a bound WT/E236A- or E236A/E236A-bead
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