Flexibility of the neck-linker during docking is pivotal for function of bi-directional kinesin

The role of the neck-linker (NL) element in regulating the functions of bi-directional kinesins is unknown. We report that replacing the NL of the bi-directional kinesin-5 Cin8 with sequences from plus-end directed kinesins produces non-functional Cin8 with defective spindle localization and abolished minus-end directionality and microtubule-crosslinking in vitro. Mutation of a single glycine in the NL of Cin8 to asparagine (proposed to serve as an N-latch that stabilizes the docked conformation of the NL in the plus-end directed kinesins) causes defects in the functions of Cin8. Strikingly, in a non-functional Cin8 containing the NL of the plus-end directed kinesin-5 Eg5, a single mutation of the N-latch asparagine back to glycine rescues the in vivo and in vitro defects. Since such replacement eliminates stabilizing interactions between the docked NL and the motor domain, we conclude that flexibility of NL during docking is pivotal for the function of bi-directional kinesin motors.


Introduction
Kinesin-5 motor proteins perform essential mitotic functions by providing the force that separates the spindle poles apart during spindle assembly, maintenance and elongation [reviewed in [1][2][3][4][5][6] ]. These motors are homotetramers, with two pairs of catalytic domains located on opposite sides of a central minifilament [7][8][9][10] . This unique structure enables kinesin-5 motors to crosslink the antiparallel microtubules (MTs) of the spindle and to slide them apart by moving in the plus-end direction of the two MTs that they crosslink [11][12][13] . Because of the MT architecture within the mitotic spindles, plusend directed motility of kinesin-5 motors is essential to separate the spindle poles 1,5,6 . Moreover, since the catalytic domains of kinesin-5 motors are located at the N-terminus, it was previously believed that such N-terminal motors are exclusively plus-end directed. Recently, however, several studies have demonstrated independently that three kinesin-5 motors -the Saccharomyces cerevisiae Cin8 and Kip1 and the Schizosaccharomyces pombe Cut7 -move processively towards the minus-end of the MTs in single-molecule motility assays and switch directionality under different experimental conditions [14][15][16][17][18][19][20][21] . The above-described body of work notwithstanding, the molecular mechanism and physiological implications of such bi-directional motility remain elusive.
The majority of kinesin motors share a mechanical structural element of 10-18 amino acids, termed the neck-linker (NL), which connects the two catalytic domains of the kinesin dimers, enabling them to step on the same MT. For N-terminal plus-end directed kinesins, the NL is located at the C-terminal end of the catalytic domain, between the last helix of the catalytic domain (6) and the neck helix (7) that is required for dimerization 22,23 . It has been proposed that during "handover-hand" stepping in the plus-end direction, the NL isomerizes between a disordered "undocked" conformation in the presence of ADP and an ordered, motor-domain-bound "docked" conformation in the presence of ATP [24][25][26][27][28] . It is believed that the docked conformation is stabilized by N-terminal sequences upstream to the motor domain, termed the cover strand (CS), which form a β-sheet with the docked NL [29][30][31][32][33] . Additional stabilization of the docked NL conformation is provided by an asparagine residue in the middle of the NL that is predicted to serve as a latch (N-latch) holding the docked NL along the core motor domain and is conserved in processive plus-end directed kinesins 33 .
During plus-end directed stepping of pairs of motor domains connected via the NL, the transition to, and stabilization of, the docked NL conformation bring the trailing head forward in the plus-end direction and are thus critical for plus-end directed motility. Since bi-directional kinesin motors can move in both plus-and minus-end directions on the MTs, the dynamics of the NL in these kinesins has to allow stepping in both directions. Thus far, however, the role of the NL in regulating the motor functions of bi-directional kinesins has not been reported. To address this issue, we designed a series of NL variants of the bi-directional kinesin-5 Cin8 motor and examined their functions in vivo and in vitro. We showed that partial replacement of the NL of Cin8 with homologous sequences from the NLs of plus-end directed kinesins resulted in the generation of nonfunctional Cin8 variants that could neither move in the minus-end direction nor crosslink MTs in vitro. In contrast, partial replacement of the NL of Cin8 with NL sequences from the bi-directional kinesin Cut7 produced a partially active Cin8 variant that was minus-end directed in vitro, indicating that the NL regulates the directionality and intracellular functions of a bi-directional kinesin.
Strikingly, in the non-functional Cin8 variant containing sequences from the NL of the plus-end directed vertebrate kinesin-5 Eg5, a single replacement of the conserved N-latch asparagine with glycine rescued the majority of the defects of the nonfunctional variant, both in vivo and in vitro. The replacement of the N-latch asparagine with glycine is predicted to eliminate stabilizing interactions and increase the flexibility of NL during docking 33 . Moreover, examination of the structure of the bidirectional kinesin-5 Cut7 reveals that in the docked conformation, its homologous N-latch asparagine is not stabilized in the same manner as that in the plus-end directed kinesins, due to differences in residues at a key position within the motor domain. Thus, we propose that flexibility of NL during docking is a common trait of bi-directional kinesin motors and is crucial for their functions.

Results
To examine the role of the NL in regulating the functionality of Cin8, we generated a series of variants in which some of the amino acids of the Cin8 NL sequence were replaced with homologous sequences from other kinesin motors ( Fig. 1A and B). Based on amino acid alignment, we created Cin8 variants containing NL sequences from two exclusively plus-end directed motors, human kinesin 1 KHC (designated here as Cin8NLKHC) and Xenopus laevis kinesin-5 Eg5 (Cin8NLEg5) or sequences from the bi-directional S. pombe kinesin-5 Cut7 (Cin8NLCut7). In addition, we created Cin8 NL variants with single amino acid replacements with amino acids from the above-mentioned kinesin motors (Fig. 1B). These variants were examined in a series of in vivo and in vitro assays to study the effect of the above-described replacements on the functionality of Cin8.   46 . The amino acids are color coded by percentage identity with a 55% threshold. (C) Viability of S. cerevisiae cells expressing NL variants of Cin8, indicated on the left, as a sole source of kinesin-5. Temperatures ( o C) at which cell growth was examined are indicated on the top. Amino acids of the NL are indicated in the middle; positions in the sequence of Cin8 are indicated on the top. The amino acids highlighted in green are those of the wt Cin8 sequence. Amino acids in the NL of Cin8 that were mutated to amino acids from other kinesin motors are highlighted in pink. Asterisks indicate the reduced growth of cells expressing the Cin8-G522N variant at 35°C and 37°C.

Cin8 containing NL sequences from plus-end directed kinesin motors is not functional in cells
To assess the intracellular functionality of the NL variants, we first examined their ability to support yeast viability as the sole source of kinesin-5 function. Since at least one of the two S. cerevisiae kinesin-5 motors, Cin8 and Kip1, is essential for cell viability 47,48 , the NL variants were examined on a low-copy centromeric plasmid in cells carrying chromosomal deletions of CIN8 and KIP1, following the shuffling-out of the original Cin8 plasmid that covered the double-deletion (Fig.   1C) [49][50][51] . We found that NL variants carrying a single amino acid replacement at positions where the consensus of most bi-directional kinesin-5 motors differs from the consensus of the exclusively plusend directed kinesin-5 motors (variants Cin8-K516M, Cin8-Q520E, Cin8-M526T, and Cin8-D528K) were mostly viable (Fig. 1C), thereby indicating these replacements do not significantly affect the functionality of Cin8 in vivo.
In contrast to the single amino acid replacements, Cin8 variants containing partial NL sequences from exclusively plus-end directed kinesin motors (Cin8NLKHC and Cin8NLEg5) failed to grow at all the examined temperatures (Fig. 1C), thereby indicating that these variants were not functional in cells.
In addition, cells deleted for Cin8 (but with functional Kip1) and expressing the Cin8NLEg5 variant exhibited significantly longer doubling times than cells expressing wild-type (wt) Cin8 (Table 1), as is consistent with the impaired functionality of this variant. Importantly, cells expressing the Cin8 variant with the NL sequence of the bi-directional Cut7 (Cin8NLCut7) were viable at 23⁰C and 26⁰C, but not at temperatures above 30⁰C (Fig. 1C), thereby indicating that at least some of the Cin8 functions are preserved in the Cin8 variant containing the NL sequence from a bi-directional kinesin.
Doubling time of cells expressing the Cin8NLCut7 variant was significantly longer than those of cells expressing wt Cin8 in both the presence and the absence of Kip1 (Table 1), which is consistent with the partial functionality of this variant. Moreover, in cells with functional Kip1, variants containing the NL sequence from the plus-end directed Eg5 and bi-directional Cut7 accumulated with monopolar spindles (Fig. 2), indicating that both variants were defective in the spindle assembly function in S. cerevisiae cells.  Moreover, in contrast to wt Cin8 and Cin8NLCut7, Cin8NLEg5 appeared to be attached to the nuclear MTs ( Fig. 2C, white arrow). Such localization, similar to the previously reported pattern of the tailless Cin8 mutant that had lost its minus-end directionality preference 20,52 , suggests that the minus-end directed motility is impaired in the Cin8NLEg5 variant, although it is maintained in the partially functional Cin8NLCut7. This is probably the reason for the ability of Cin8NLCut7, but not Cin8NLEg5, to function as a sole source of kinesin-5 function in cells.
In summary, the above experiments indicate that replacement of the Cin8 NL sequence with the sequence from plus-end directed kinesin motors produced variants that are not functional in cells, probably due to decreased affinity to MTs and abolished minus-end directed motility on nuclear MTs prior to spindle assembly. In contrast, replacement of the Cin8 NL sequence with a sequence from the bi-directional Cut7 probably maintained minus-end directed motility, resulting in a partially functional variant of Cin8 that can support cell viability as a sole source of kinesin-5.

Glycine at N-latch position is crucial for the intracellular functions of Cin8
Cin8 contains a glycine at position 522 in the NL, whereas the majority of plus-end directed kinesin motors contain an asparagine at this position ( Fig. 1B), which is predicted to serve as a latch (N-latch) stabilizing the docked NL along the core motor domain 33 . Importantly, we found that the variant in which this glycine had been replaced with an asparagine, namely, Cin8-G522N, exhibited reduced viability at 35⁰C and 37⁰C, when expressed as the sole source of kinesin-5 ( Fig. 1C asterisk).
Consistently, cin8 kip1 cells expressing the Cin8-G522N variant exhibited longer doubling times than cells expressing wt Cin8 (Table 1), thereby indicating that the functionality of Cin8 was undermined by the replacement of glycine at position 522 with asparagine. To examine the role of this glycine, we generated a mutant of Cin8 containing the NL sequence of the plus-end directed Eg5, with the asparagine at position 522 mutated back to glycine; this mutant is designated Cin8NLEg5-NG. Strikingly, reinstating the original glycine rescued the non-viable phenotype of the Cin8NLEg5 variant, supporting the viability of cin8 kip1 cells at temperatures up to 30℃ (Fig. 1C).
The doubling time of cin8 kip1 cells expressing Cin8NLEg5-NG was longer than that of cells expressing wt Cin8 (Table 1)

Glycine at the N-latch position regulates the motile properties of Cin8 in vitro
To understand the intracellular phenotypes of the NL variants, we examined their motor function in vitro. In these assays, we characterized the activity of GFP-tagged full-length NL variants, overexpressed and purified from S. cerevisiae cells, on fluorescently labeled GMPCPP-stabilized MTs 13 . We first compared the MT affinities of the NL variants by determining the average number of MTattached motors per MT length at equal motor protein concentrations. We found that all the NL variants exhibited significantly lower levels of MT-bound motors than wt Cin8 (Fig. 3A), thereby indicating that NL affects the MT-affinity of Cin8. Moreover, we also found that the Cin8NLEg5 variant, which was unable to support yeast cell viability as a sole source for kinesin-5, exhibited the lowest levels of MT-bound motors, indicating that its affinity to MTs is greatly reduced. The majority of Cin8NLEg5 motors bound to MTs at 140 mM KCl were not motile (not shown). At a lower salt concentration, at which the affinity of the motors to MTs is increased 19 , Cin8NLEg5 exhibited only bidirectional diffusive motility, with no minus-end directed bias, in contrast to wt Cin8, which exhibited processive minus-end directed motility (Fig. 3B). These results indicate that, consistent with its intracellular localization ( Fig. 2C-E), the Cin8NLEg5 variant exhibited very low MT affinity and was unable to move towards the minus-end of the MTs.
We next examined the motility of the functional Cin8 NL variants at a saturating ATP concentration and at a high ionic strength (140 mM KCl). We found that under these conditions, consistent with previous reports 13,14,52 , all Cin8 NL variants moved processively in the minus-end direction of the MTs (Fig. 3C). We have previously demonstrated that one of the factors that affects the directionality and velocity of Cin8 is its accumulation in clusters on MTs 13 . Thus, to examine the effects of mutations in the NL sequence on motility but not on motor clustering, we sorted out single molecules of Cin8 from a total population of moving Cin8 particles, based on their intensities (see Materials and Methods). By following the fluorescence intensity of Cin8 particles as a function of time, we observed single events of intensity decrease, of ~45 arbitrary intensity units (a.u.), most probably originating from the photobleaching of single GFP molecules (Fig. S1A of the Expanded Vie section). Since single Cin8 motors are tetramers comprised of four identical subunits 53 , the maximal fluorescence intensity of a single Cin8 molecule containing four GFP molecules is expected to be ⁓180 a.u. The intensity distribution of the total Cin8-GFP population was consistent with this notion, in that it exhibited a major intensity peak containing ~65% of Cin8 particles with intensity <180 a.u.
( Fig. S1B). The maximal intensity of the peak was ~120 a.u., consistent with an average intensity of single Cin8 molecules containing one, two, three or four fluorescent GFP molecules (Fig. S1B). Based on this analysis (see Materials and Methods), we defined "single Cin8 molecules" as particles of intensity lower than 180 a.u. and analyzed the motile properties only of those Cin8 molecules, thereby ensuring analysis of mainly single molecules of tetrameric Cin8 NL-variants.  Consistent with previous reports [13][14][15]19 , we found that under high ionic strength conditions, single molecules of wt Cin8 exhibited fast processive movements towards the minus ends of MTs, with a high average velocity of -318±4 nm/s ( Fig. 3C and D).
Remarkably, in contrast to the diffusive motility of the Cin8 variant containing the NL sequence of the plus-end directed Eg5 (Cin8NLEg5) (Fig. 3B, orange arrows), mutating the asparagine at position 522 to glycine (Cin8NLEg5-NG) dramatically changed the motile behavior, inducing processive minus-end directed motility (Fig. 3B-D). Although the average velocity of the Cin8NLEg5-NG variant was lower than that of wt Cin8 (Fig. 3D) Consistently, in wt Cin8, mutation of the glycine at this position to asparagine resulted in considerably slower minus-end directed motility, compared with that of the original wt Cin8 (Fig.   3D). Furthermore, Cin8NLCut7, the Cin8 variant with the NL sequence of the bi-directional Cut7 containing asparagine at position 522 ( Fig. 1C) also exhibited processive minus-end directed motility, but with reduced velocity compared with that of wt Cin8 (Fig. 3D). These results further support the notion that glycine at position 522 is important for maintaining fast and processive minus-end directed motility of Cin8.

The NL of Cin8 regulates its MT-crosslinking function
The intracellular functions of kinesin-5 motors are attributed mainly to their ability to crosslink the interpolar MTs of the spindle 1,5,6 . Thus, to establish additional correlations between intracellular phenotypes and the motor activity of the NL variants, we examined their MT-crosslinking activity in vitro. In this assay, purified Cin8 variants were mixed with fluorescently labeled GMPCPP-stabilized MTs in solution, followed by imaging and quantitation of fluorescence intensity of the MTs (see Materials and Methods). Consistent with previous reports 54 , we found that Cin8 induced the accumulation of MT bundles, thereby increasing the apparent fluorescence intensity of these bundles, compared with that of single MTs (Fig. 4). The MT-bundling activity of wt Cin8 was concentration dependent, inducing MT bundling at a minimal molar ratio of ~1:40 Cin8 motors to tubulin dimer (Fig. 4D).  Color-coded asterisks for each NL variant indicate comparison to average MT intensity in the absence of motors. *p < 0.05; **p < 0.005.
With the exception of Cin8NLEg5, all NL variants (Fig. 1C) also induced MT bundling in a concentration-dependent manner, with the fluorescence intensity of bundles induced by high concentrations of motors being significantly higher than that of MTs without the addition of motors (Fig. 4D). Moreover, at high motor protein concentrations, the functional NL variants, Cin8-G522N, Cin8NLCut7 and Cin8NLEg5-NG, induced MT bundling to a significantly greater extent, compared with the non-functional Cin8NLEg5. This result indicates that the MT-bundling activity of the NL variants is strongly correlated with the ability of these variants to support cell viability as a sole source for kinesin-5 function (Fig. 1C). However, the functional NL variants exhibited reduced ability to bundle MTs, compared with wt Cin8, a finding that is consistent with the reduced affinity to MTs of these variants (Fig. 3A). In summary, data obtained from the in vitro experiments indicates that the NL of Cin8 regulates both its affinity for MTs and the minus-end directed motility of single Cin8 molecules and their ability to crosslink MTs. In addition, the data showed that glycine at position 522, which is equivalent to a conserved asparagine that stabilizes the NL docking in plus-end directed processive kinesins 33 , is crucial for the fast minus-end directed motility of Cin8 and its ability to crosslink MTs in vitro.

Discussion
The role of the NL dynamics in regulating the motor functions of plus-end directed kinesins has been addressed in a number of studies. However, the current study is the first to demonstrate that mutations in the NL modulate the motile properties and intracellular functions of a bi-directional Nterminal kinesin motor. Data presented here indicates that mutations in the NL regulate MT affinity, minus-end directed motility, and MT crosslinking functions of the bi-directional kinesin-5 Cin8. In turn, these motor functions affect the ability of Cin8 to localize at the spindle poles prior to spindle assembly, to form mitotic spindles, and to support cell viability as a sole source of kinesin-5.

Minus-end directed motility is important for the intracellular function of a bi-directional kinesin-5
The minus-end directed and switchable directionality of fungal kinesin-5 motors was discovered nearly a decade ago. Although a recent study, based on theoretical simulations, suggests that the minus-end directed motility of the bi-directional S. pombe kinesin-5 is necessary for spindle assembly 55 , experimental support for this notion is still missing. We have recently proposed that in fungi cells, which divide via closed mitosis, minus-end motility of kinesin-5 motors is needed to localize these motors near the spindle poles prior to spindle assembly. At this location, kinesin-5 motors capture and crosslink MTs emanating from the neighboring SPBs and promote SPB separation via antiparallel sliding of the crosslinked MTs 13 . Analysis of in vivo and in vitro functions of the NL variants of Cin8 presented here support this model. Our data demonstrates that, compared with wt Cin8, all the examined NL variants exhibited reduced affinity to MTs (Fig. 3A) and an impaired ability to crosslink MTs in vitro (Fig. 4C-D). Importantly, our data also shows that all NL variants that can support the viability of yeast cells exhibit two common traits: a) they are able to move processively in the minus-end direction in vitro (Fig. 3), and b) they localize near the SPBs prior to spindle assembly (Fig. 2C-E), indicating a connection between intracellular functionality, minusend directed motility, and localization at the SPBs. Differences in minus-end directed velocity per se ( Fig. 3C and D) seem to be less important for the intracellular function, as long as the variants can move processively in the minus-end direction. Thus, we conclude that consistent with our model 5,13 , processive minus-end directed motility that localizes the motors near the SPBs prior to spindle assembly is necessary for mitotic functions of the kinesin-5 Cin8.

Flexibility of the NL during docking and movement in two directions
The profound difference between the activity of the Cin8NLEg5 and Cin8NLEg5-NG variants indicates that the glycine at position 522 is crucial for the functions of Cin8. The majority of kinesin motors contain asparagine at the position equivalent to glycine 522 of Cin8 (Fig. 1B). It has been suggested that this asparagine plays a central role in the docking of the NL to the catalytic motor domain of the plus-end directed kinesin-1 motors and serves as an N-latch that holds the NL firmly in the motor-domain bound state in the presence of ATP 33 . Based on the crystal structure of Eg5 in the ATP-like bound state with a docked NL, it appears that this conserved NL asparagine is stabilized by a series of hydrogen bonds with residues within the motor domain (Fig. 5A).  A and B). The secondary structure of the α-helix and β-strands in the motor domain are annotated by squares and arrows, respectively. In each panel, the orientation of the NL, from the N-terminus to the C-terminus, is represented by a purple arrow.
These residues include asparagine 98 in β3 (equivalent to asparagine 159 in Cin8), glycine 96 in the loop between α1 and β3 (equivalent to glycine 157 in Cin8), and isoleucine 258 in β7 (equivalent to isoleucine 414 in Cin8) (Fig. 5A, C and Fig. S2). Plus-end directed kinesin-5 motors are highly homologous at these positions (Fig. S2), indicating that stabilization of NL docking is conserved in these kinesin motors. Although these amino acids are also conserved in the motor domain of Cin8 (Fig. S2), the hydrogen bonds that stabilize NL docking in Eg5 are probably missing (Fig. 5C), due to the presence of glycine rather than asparagine at position 522, which probably results in more flexible NL in the docked conformation in Cin8. In contrast, it is likely that such stabilizing hydrogen bonds are present in the non-functional Cin8NLEg5 variant and in the partially active Cin8NLCut7 and Cin8-G522N variants, which contain asparagine at position 522 (Fig. 1). Replacing this asparagine with the original glycine in the Cin8NLEg5-NG variant probably abolishes these stabilizing hydrogen bonds, thereby partially reinstating the original flexibility of the NL during docking that is required for Cin8 functions. Moreover, based on a recently published CryoEM reconstructed structure, the NL of the bi-directional S. pombe kinesin-5 Cut7 is not stabilized by the same interactions as those in Eg5 (Fig. 5B); this difference is probably due to the absence of isoleucine at the position equivalent to I414 in Cin8 ( Fig. 5B and C, S2B), indicating that the NL docking of the bi-directional Cut7 is also more flexible than NL docking of the plus-end directed Eg5. Based on these structures, we suggest that flexibility of the NL during docking may be a common trait of bi-directional kinesin-5 motors.
Recent studies indicate that processive stepping in the plus-end direction of kinesin-1 motors is achieved, in part, by a gating mechanism that prevents simultaneous strong binding of the two kinesin-1 motor-domain heads to MTs 56 . It is believed that this gating is achieved by the coupling between the orientation of the NL, the inter-head tension, and ATP binding and hydrolysis, such that ATP binding in the leading head is prevented until the rear head assumes a weakly bound state or detaches 27,36,57-61 . However, a different gating mechanism probably takes place during motility of plus-end directed kinesin-5 motors 44 , which allows for simultaneous strong MT-binding of two motor domains of a dimer in a rigor state with no nucleotides 62,63 . The two-head strongly bound state has been attributed to the longer and more flexible NL of kinesin-5 motors 41,59,64 and may be necessary for force production during antiparallel sliding of MTs in spindle assembly and elongation 47,[65][66][67][68] . The bi-directional kinesin-5 motors have the ability to move in both plus-end and minus-end directions, in addition to their ability to crosslink and slide apart antiparallel MTs. Thus, their NL dynamics has to be adapted for bi-directional stepping. Since inducing flexibility by extending the length of the NL was previously shown to enable minus-end directed back-stepping of kinesin-1 dimers 36 , we propose that increased flexibility during docking of the NL of Cin8, compared to Eg5 (Fig. 5), provides the necessary adaptation to allow bi-directional motility.

Requirement for flexibility of the NL during docking for the functioning of bi-directional kinesins
In a recent study, the role of NL-docking stabilization in regulating the motility of kinesin-1 was experimentally examined 42 . That study reported that mutating the N-latch asparagine in the NL of kinesin-1 (equivalent to glycine 522 in Cin8) to alanine, which eliminates stabilizing interactions, increased the plus-end directed motor velocity in a single-molecule motility assay 42 . Strikingly, we observed the same effect on the velocity of single-molecules of Cin8 during processive movement in the minus-end direction. The velocity of Cin8 variants that contain asparagine in position 522 was significantly slower in the minus-end direction than that of wt Cin8 and Cin8NLEg5-NG, which contain glycine at this position (Fig. 3D). These results are consistent with recent CryoEM reports indicating that in the ATP-bound state the NLs of bi-directional kinesin-5 motors assume the docked conformation 18,29,69 , similarly to kinesin-1 motors 70 Cin8, reflected in cell viability, localization to the spindle and doubling times, are defective in cells expressing Cin8 variants containing asparagine at position 522 ( Fig. 1-3, and Table 1). Furthermore, intracellular functions as well as minus-end directed motility and MT crosslinking were drastically improved by introducing glycine to replace asparagine at position 522 of a non-functional Cin8 variant containing sequences from the NL of Eg5 (Figs. 1-4). Since, as mentioned above, in the presence of glycine, stabilizing interactions of NL docking are considerably reduced (Fig. 5), we conclude that in contrast to plus-end directed kinesin-1, flexibility of NL in the docked conformation is critical for the intracellular functions of bi-directional kinesin-5 motors.

Molecular cloning and strains
NL variants were generated using standard molecular cloning techniques. All mutations were confirmed by sequencing. Plasmids and S. cerevisiae strains used in this study are listed in Tables S1 and S2 of the Expanded View section.

Multiple sequence alignments (MSAs)
MSAs were calculated by the MUSCLE algorithm via Unipro UGENE program 45 , color coded by percentage identity with a 50-55% threshold. Sequences of the organisms presented in Fig. 1B

Viability assay
The viability of S. cerevisiae cells expressing NL variants of Cin8 as the sole source of kinesin-5 was determined as described previously 13,20,49,51 (Fig. 1). S. cerevisiae strains used for this assay were deleted for their chromosomal copies of CIN8 and KIP1 and contained an endogenic recessive cycloheximide resistance gene (cin8Δkip1Δcyh r ), containing a plasmid (pMA1208) encoding for wt Cin8 and a wt dominant cycloheximide sensitivity gene (CYH); see Table S2 for the list of S. cerevisiae strains. Following transformation with a plasmid encoding for Cin8 NL variants, the pMA1208 plasmid was shuffled out by growth on yeast extract-peptone-dextrose (YPD) medium containing 7.5 µg/mL cycloheximide at 23 o C and at elevated temperatures.

Imaging of S. cerevisiae cells
Imaging was performed as previously described 49

Purification of Cin8-GFP
Overexpression and purification of Cin8-GFP from S. cerevisiae cells was performed as previously described 13,19 . Cells expressing Cin8-TEV-GFP-6HIS in a protease-deficient S. cerevisiae strain under the GAL1 promoter on a 2μ plasmid were grown in liquid medium supplemented with

Single molecule motility assay
The single molecule motility assay was performed on piranha-cleaned salinized coverslips as previously described 13

Image and data analysis of the motility assay
Kymographs were generated using ImageJ software for MTs with both ends visible.
Directionality of the MTs was assigned on the basis of bright plus-end labeling or by the direction of fast-moving Cin8 particles under high salt conditions 13,14 . To distinguish between single Cin8-GFP tetramers and clusters, we followed the intensity of stationary Cin8-GFP particles on background- Therefore, Cin8-GFP particles that have the intensity of four GFPs or less are likely to be single Cin8-GFP tetramers 74 . MD analysis was performed as previously described 19,75 . where ν is the velocity and t is time.

Affinity of Cin8 to MTs
The affinity of Cin8 to MTs was measured on stationary GMPCPP-stabilized fluorescently labeled MTs in the presence of 1 mM ATP and 140 mM KCl, as in the single molecule motility assay, keeping the concentration of Cin8-GFP NL variants constant at ~2.7 ng/ml. One frame of MTs was captured, followed by a one frame of Cin8-GFP signal. Background subtraction was performed by

Doubling time of S. cerevisiae cells
The doubling time of S. cerevisiae cells was determined as follows: cin8Δ or cin8Δkip1Δcyh r cells were transformed with a centromeric plasmid expressing one of the Cin8 NL variants and grown overnight. In cin8Δkip1Δcyh r cells, the original plasmid encoding a wt Cin8 and a dominant cycloheximide sensitive gene (CYH) (pMA1208) was shuffled out by addition of 7.5 µg/ml cycloheximide to the growth medium. The cells were diluted to OD600 ~0.2 2 h prior to the start of the experiment. The OD600 of cells then was measured over time for a culture growing at 26°C, and a plot of ( 0 ) as a function of time was generated, where ODt is the OD600 at a given time, and OD0 is the OD600 at the initial time point. Finally, the doubling time was calculated using the slope of the

Statistical analysis
Data was first examined for normality by the Kolmogorov-Smirnov test 76 . If normality was rejected, data was subjected to an appropriate Box-Cox transformation 77 to yield normal distributions. Next, significant differences were assessed using ANOVA, followed by either all pairwise comparisons with Tukey correction 78 , or comparisons to wt Cin8 as a control, according to Dunnett's method 72 .