Polyglutamylation of tubulin’s C-terminal tail controls pausing and motility of kinesin-3 family member KIF1A

The kinesin-3 family member KIF1A plays a critical role in site-specific neuronal cargo delivery during axonal transport. KIF1A cargo is mislocalized in many neurodegenerative diseases, indicating that KIF1A’s highly efficient, superprocessive motility along axonal microtubules needs to be tightly regulated. One potential regulatory mechanism may be through posttranslational modifications (PTMs) of axonal microtubules. These PTMs often occur on the C-terminal tails of the microtubule tracks, act as molecular “traffic signals” helping to direct kinesin motor cargo delivery, and include C-terminal tail polyglutamylation important for KIF1A cargo transport. microtubule C-terminal tails K-loop, a positively charged surface loop of the KIF1A motor domain. K-loop


ABSTRACT
The kinesin-3 family member KIF1A plays a critical role in site-specific neuronal cargo delivery during axonal transport. KIF1A cargo is mislocalized in many neurodegenerative diseases, indicating that KIF1A's highly efficient, superprocessive motility along axonal microtubules needs to be tightly regulated. One potential regulatory mechanism may be through posttranslational modifications (PTMs) of axonal microtubules. These PTMs often occur on the Cterminal tails of the microtubule tracks, act as molecular "traffic signals" helping to direct kinesin motor cargo delivery, and include C-terminal tail polyglutamylation important for KIF1A cargo transport. KIF1A initially interacts with microtubule C-terminal tails through its K-loop, a positively charged surface loop of the KIF1A motor domain. However, the role of the K-loop in KIF1A motility and response to perturbations in C-terminal tail polyglutamylation is underexplored. Using single-molecule imaging, we present evidence that KIF1A pauses on different microtubule lattice structures, linking multiple processive segments together and contributing to KIF1A's characteristic superprocessive run length. Furthermore, modifications of the KIF1A K-loop or tubulin Cterminal tail polyglutamylation reduced KIF1A pausing and overall run length. These results suggest a new mechanism to regulate KIF1A motility via pauses mediated by Kloop/polyglutamylated C-terminal tail interactions, providing further insight into KIF1A's role in axonal transport.
Axonal transport is a critical process for neuronal viability and function involving the highly-choreographed, long-distance trafficking of cargo. This process is facilitated in part by members of the kinesin superfamily of motors that utilize the mechanochemical coupling of ATPhydrolysis [1] to carry motor-specific cargo in the anterograde direction along axonal microtubules.
The neuron-specific kinesin-3 family member KIF1A [2] is a key mediator of axonal transport, delivering neuronal cargo, such as dense core vesicles [3] and synaptic vesicle proteins [2], in a spatiotemporally regulated manner along axonal microtubules [4,5]. Upon cargo-mediated dimerization and release of steric autoinhibition, KIF1A motors are reported to exhibit "superprocessive" motility behavior, traveling comparatively long distances on microtubules when compared to conventional kinesin [6,7]. The discovery of this superprocessive behavior supports KIF1A as an important player in long distance axonal transport, providing further insight into KIF1A's role in many neuronal processes such as synaptogenesis [8] and neurogenesis [9]. The importance of tightly regulated KIF1A cargo delivery is highlighted in neurodegenerative diseases where irregular KIF1A cargo accumulation is observed [10][11][12]. However, our understanding of this disease-state presentation is limited by the gap in knowledge of the mechanisms that regulate KIF1A motility.
Beyond serving as tracks for kinesin motors, microtubules can direct subcellular trafficking by providing directional cues for motor specificity on select microtubule populations. These "traffic signs" are frequently introduced via microtubule posttranslational modifications (PTMs), often on the C-terminal tails of microtubules [13,14]. While there are many recent discoveries of how microtubule PTMs regulate different kinesin families [15][16][17], how this concept can be extended to kinesin-3 motors such as KIF1A is relatively unexplored. One potential PTM regulating KIF1A trafficking is the heavy polyglumylation of both aand btubulin subunits in neurons [18] via the tubulin tyrosine ligase-like family of enzymes [19,20]. KIF1A's dependence on optimal levels of polyglutamylation has been observed in the ROSA22 mouse model, characterized by a loss of neuronal a-tubulin polyglutamylation leading to altered KIF1A cargo trafficking and reduced KIF1A affinity for microtubules [21]. These physiological repercussions of altered KIF1A function highlight the importance of further studying interactions between KIF1A and the polyglutamylation of Cterminal tails of tubulin at the molecular level.
Polyglutamylation may impact KIF1A motility via a lysine-rich surface loop (loop-12) that is characteristic of the kinesin-3 motor family [22,23]. Previous work has demonstrated that this "Kloop" is a critical component for optimal KIF1A function. From a catalytic perspective, it is thought that the K-loop is necessary for KIF1A's structural interaction with the microtubule during the ATPhydrolysis cycle [24]. Furthermore, it is known that the K-loop interacts electrostatically with the glutamic acid-rich C-terminal tail of tubulin [25,26], tethering the motor to the microtubule track [22]. This K-loop/C-terminal tail relationship, combined with the physiological detriment of reduced microtubule polyglutamylation, illustrates that specific interactions between the KIF1A Kloop and C-terminal tails of tubulin are essential for optimal KIF1A function. Therefore, we hypothesized that C-terminal tail polyglutamylation regulates KIF1A motility and behavior on microtubules mediated by interactions with the KIF1A K-loop structure. Using singlemolecule total internal reflection fluorescence microscopy, we first tested our hypothesis by quantifying the motility and behavioral response of KIF1A on various microtubule lattices and then specifically perturbed the K-loop/C-terminal tail interaction. Our findings demonstrate that that KIF1A pauses on the microtubule lattice, and suggest that this behavior is caused by a polyglutamylation-sensitive K-loop/CTT interaction.

KIF1A
exhibits pausing behavior and superprocessive motility on taxol-stabilized and GMPCPP microtubules assembled from brain tubulin.
Initial single molecule TIRF microscopy observations revealed that dimeric KIF1A motors possess the novel ability to pause during a run on taxol-stabilized microtubules ( Figure 1A, Movie S1). While pausing, the dimeric motor appears to be spatially constrained as it does not exhibit the highly diffusive behavior of the KIF1A monomer [26]. While other kinesin motors have been shown to exhibit transient pauses [27,28], the high pause frequency (Table 1) and substantial number of pauses per distance ( Figure 2F) of KIF1A has not been previously characterized, leading us to investigate this behavior further.
The discovery of this novel pausing behavior led us to redefine our nomenclature of KIF1A motility by dissecting KIF1A motility into three segments ( Figure 1A). First, we define the continuous run length as segments where, within a single event, KIF1A is moving at a constant velocity. Next, continuous run lengths are connected by pauses that occur at the beginning of continuous runs, in between continuous runs, or after continuous runs. To characterize KIF1A pausing, we have quantified both the pause frequency (# of pauses/overall run) and pauses per distance (# of pauses/µm microtubule). While representative KIF1A kymographs include long pauses ( Figure 1C, 2A, 2B), it is important to note that the majority of pausing events are less than 1.5 seconds and occur at stochastic locations on the microtubule ( Figure 1C, Table 1). Lastly, we define the overall run length as the sum of continuous run lengths during a single motility event on the microtubule. KIF1A pausing behavior ( Figure 1B, 1C) is best revealed at motor concentrations lower than those utilized in previous work [22] .
To confirm that this behavior was not dependent on the facilitated dimerization of our leucine zipper construct (KIF1A-LZ-3xmCitrine), we repeated our single molecule experiments with a non-leucine zipper construct (KIF1A-GFP). We confirmed that in the absence of the leucine zipper, KIF1A exhibits extensive pausing behavior and superprocessive motility ( Figure S1). Additionally, we observed an increase in purely diffusive events with this construct ( Figure S1A), likely resulting from KIF1A monomers [6,7]. Our results demonstrate that the KIF1A-LZ-3xmCitrine construct is an appropriate model for dimeric KIF1A behavior. As our current investigations are focused on the processive, dimeric KIF1A motor, we performed all other experiments with the LZ construct.
It has been previously reported that the perturbation of KIF1A's interaction with the Cterminal tail reduces the motor's landing rate [22].
Measuring KIF1A landing rate in ADP-containing motility buffer confirms the necessity of this interaction, showing that subtilisin-treatment of microtubules reduces the landing rate of KIF1A by 65% (7.93 ± 1.25 events/µm/min to 2.80 ± 0.42 events/µm/min, Figure 7, Table 1). Taken together, these results expose KIF1A's reliance on the microtubule C-terminal tail to engage with the microtubule, initiate pausing behavior, and generate superprocessive overall run lengths.

Conserved lysine residues of the K-loop are key mediators in KIF1A pausing and motility.
Given the influence of the tubulin Cterminal tails on KIF1A pausing behavior, we further investigated the influence of the electrostatic interaction between the KIF1A K-loop and the microtubule C-terminal tails. First, we assessed the dependence of KIF1A pausing on the local electrostatic environment by increasing the salt concentration of our motility buffer from 12 mM to 80 mM PIPES. We observed a decrease in pause duration (1.23 sec to 0.78 sec, Figure 4C) with no significant changes in pause frequency (0.90 pauses/overall run), pauses per distance (0.14 pauses/µm) or any motility parameters ( Figure 4A, 4B, S2, S3). Furthermore, in 80 mM PIPES we observed a decrease in KIF1A landing rate to 3.28 ± 0.61 events/µm/min [n=592] ( Figure 7, Table 1).
Second, we investigated how positivelycharged residues within the K-loop contribute to KIF1A pausing and motility. It has been previously reported that mutating all six lysines of the K-loop to alanine residues abolishes KIF1A motility and interaction with the microtubule [22], highlighting the importance of the K-loop/microtubule interaction to the intrinsic motility properties of KIF1A. Therefore, we mutated the three lysines most conserved across the kinesin-3 family to maintain the ability of KIF1A motors to engage with the microtubule surface ( Figure 5A). The KIF1A K-loop mutants (Tri-Ala KIF1A) displayed a marked reduction in both motility and pausing behavior ( Figure 5B-E). Specifically, both the overall run length (2.41 ± 1.42 µm [n=173]) and continuous run length (2.00 ± 1.11 µm [n=207]) were significantly reduced by 61% and 32%, respectively, when compared to WT KIF1A on taxol-stabilized microtubules ( Figure 5C, Table 1). Due to fewer pausing events, there was a 23% increase in overall speed (1.66 ± 0.45 µm/s) and a continuous speed of 1.72 ± 0.45 µm/s (Table 1, Figure S2, Figure S3). Tri-Ala KIF1A mutants also exhibited impaired pausing behavior quantified by a 62% reduction in pause duration (1.23 sec to 0.47 sec), a 75% decrease in pause frequency (0.95 pauses/overall run to 0.23 pauses/overall run, and a 64% decrease in pauses per distance (0.14 pauses/µm to 0.05 pauses/µm) ( Figure 5D,E, Table  1). As expected from previous work [22], the Tri-Ala KIF1A mutant also had a dramatic, 92% reduction in landing rate when compared to WT KIF1A on taxol-stabilized microtubules (0.64 ± 0.14 events/µm/min [n=527] for Tri-Ala and 7.93 ± 1.25 events/µm/min [n=1552] for WT) in motility buffer supplemented with ADP ( Figure 7, Figure  S7, Table 1) but no change in microtubule interaction in motility buffer supplemented with AMPPNP (1.47 ± 0.34 events/ m/min [n=574] for Tri-Ala and 2.01 ± 0.49 events/ m/min [n=917] for WT ( Figure S7). These results confirm that the KIF1A K-loop/microtubule C-terminal tail interaction plays a critical role not only in the landing rate as previously described [22], but also for the pausing behavior of KIF1A.

Microtubule C-terminal tail polyglutamylation regulates KIF1A pausing behavior and motility.
We next aimed to identify a specific property of the tubulin C-terminal tail that could influence KIF1A pausing and motility. It has been previously established that reduced levels of atubulin polyglutamylation result in improper KIF1A localization and insufficient cargo delivery in hippocampal and superior cervical ganglia neurons of ROSA22 mice [21]. As neuronal microtubules are subjected to extensive C-terminal tail polyglutamlation [18], we hypothesized that this posttranslational modification is important for regulation of KIF1A motility behavior including pausing. We thus assessed KIF1A's pausing and superprocessivity on tubulin purified from HeLa cells, which are known to contain markedly less polyglutamylation than neuronal tubulin ( Figure  6A) due to low expression of the tubulin tyrosinelike ligase enzymes [33][34][35]. It is important to note that this change in tubulin polyglutamylation alters the local charge of the microtubule surface as compared to neuronal tubulin. KIF1A overall run length was reduced by 34% on taxol-stabilized HeLa microtubules (4.12 ± 1.48 µm [n=164]) when compared to taxol-stabilized neuronal microtubules with no significant reduction in continuous run length ( Figure 6C, Table 1). In regards to pausing behavior, we observed a 58% reduction in pause frequency (0.95 pauses/overall run to 0.40 pauses/overall run), a 50% reduction in pauses per distance (0.14 pauses/µm to 0.07 pauses/µm), and a 51% decrease in pause duration (1.23 sec to 0.59 sec) ( Figure 6B, 6D-E, Table 1). Due to the reduction in pausing events, the overall speed of KIF1A on HeLa tubulin (1.70 ± 0.41 µm/s) increased by 26% when compared to KIF1A on neuronal microtubules, while continuous speed decreased by only 7% (Table 1, Figure S2, Figure  S3). We also observed a substantial, 94% reduction in KIF1A landing rate on HeLa microtubules when compared to neuronal microtubules (7.93 ± 1.25 events/µm/min [n=1552] versus 0.49 ± 0.13 events/µm/min [n=429]) ( Figure 7, Table 1). Taken together, these results confirm that microtubule Cterminal tail polyglutamylation dictates KIF1A's ability to pause and thus mediates superprocessive behavior.

Discussion
The superprocessivity of the kinesin-3 family member KIF1A has been previously established [7], supporting its known role in longdistance axonal transport [3,10]. Here we present a novel molecular mechanism that influences KIF1A's superprocessive motion. First, we demonstrate that KIF1A engages in previously unreported pauses on the microtubule lattice. KIF1A walks a superprocessive distance (~3 µm on average) before initiating a pause, supporting the notion that although pausing is rare, it is related to the superprocessive nature of the motor itself. Pausing has several effects on the overall motility of dimeric KIF1A motors. Pauses enable KIF1A to string together multiple continuous runs to generate a longer overall run. Of note, even in the absence of pauses, KIF1A remains a superprocessive motor, with continuous run lengths of ~ 3 µm. KIF1A's overall speed is also decreased in the absence of pauses, with no effect on continuous speed. This response is expected, considering that a reduction in pause frequency will reduce a slower population of motors undergoing pauses.
Furthermore, we show that pauses are mediated by interactions between the KIF1A Kloop and tubulin C-terminal tails. When the Kloop/C-terminal tail interaction is interrupted (e.g., subtilisin treatment, K-loop mutation), KIF1A shows a reduced pause frequency and duration on the microtubule lattice. Disruption of the K-loop/Cterminal tail interaction also results in a dramatic reduction in the landing rate of dimeric KIF1A motors, consistent with previous work [22]. The Kloop is also critical for monomeric KIF1A motors to diffuse along the surface of microtubules [23]. In addition, cellular work has shown that loop-12 is important for regulation of KIF1A dendritic cargo sorting [36]. As the K-loop is conserved across members of the kinesin-3 family, our newlyidentified pausing mechanism is likely to be a conserved feature of this class of motor.
Finally, we demonstrate that polyglutamylation of the tubulin C-terminal tail is a posttranslational modification that regulates KIF1A pausing behavior and subsequent motility. Along HeLa microtubules, which lack polyglutamylation, KIF1A displayed significantly reduced landing rate, pause frequency, and pause duration. Electrostatic interactions between the K-loop and C-terminal tail play an important role as increasing the salt concentration in our motility buffer resulted in a reduced landing rate and pause duration. That the increased salt concentration did not influence pause frequency suggests that the factor that influence pausing are not completely based on electrostatics. That tubulin C-terminal tail polyglutamylation influences KIF1A behavior at the single molecule level provides a mechanistic understanding of how polyglutamylation can regulate KIF1A cargo trafficking [21]. The levels of tubulin C-terminal tail polyglutamylation are highly tuned during neural development [18,37] and could also influence KIF1A during synaptogenesis [38] and interkinetic nuclear migration [39]. Compartmentalizing neuronal tubulin polyglutamylation into localized hotspots [40] may contribute an advantageous level of regulation to fine-tune KIF1A function during these developmental processes.
With a molecular regulatory mechanism identified in regards to KIF1A pausing and motility, a question still remains: how is KIF1A able to pause in between processive segments (continuous runs)? Our proposed mechanism states that pausing is mediated in part by a KIF1A/C-terminal tails interaction. A structural mechanism responsible for KIF1A pausing can be proposed based on past work examining the flexibility and structural confirmation of the K-loop during ATP hydrolysis [24]. This study demonstrated that when KIF1A is bound to AMPPNP, loop-12 is positioned up and away from the microtubule surface whereas when KIF1A is bound ADP, loop-12 adopts a "downward" position to interact with the Cterminal tails on the microtubule surface. From this, we propose that pauses are initiated by loop-12 interaction with the microtubule C-terminal tail when KIF1A is in the ADP state. Importantly, a pause would happen when both motor domains are in the same nucleotide state at the same time, making this a catalytically rare event and explaining why pausing occurs on the magnitude of every ~ 3 µm. We speculate that with both loop-12's in a downward position, KIF1A exhibits a spatially-constrained form of diffusion close to the resolution limit of our TIRF microscope as compared to the previously observed highly diffusive behavior of the KIF1A monomer [26].
The mechanistic importance of loop-12 positioning is further supported by our data comparing the landing rate of WT and Tri-Ala motors in the presence of ADP or AMPPNP in the motility buffer. Under ADP conditions, the landing rate of the K-loop mutant (Tri-Ala) is significantly reduced when compared to WT KIF1A ( Figure S7), consistent with previous work [22]. However, under AMPPNP conditions, there is no significant difference in landing rates between WT and Tri-Ala KIF1A motors ( Figure S7). These findings support our proposed structural mechanism of pausing in which the "downward" positioning of KIF1A loop-12 when bound to ADP enables loop-12 to interact with the microtubule C-terminal tails and facilitate pausing. Furthermore, the significant reduction of Tri-Ala KIF1A pausing and continuous run length in motility assays ( Figure 5C, 5E) highlight the importance of the K-loop for KIF1A motility. Further testing how KIF1A catalytic activity and structural states influence pausing is an enticing future direction beyond the scope of this current study.
Our results demonstrate that KIF1A responds not only to posttranslational modification of the microtubule surface but also to the state of the microtubule. Specifically, we demonstrate that KIF1A has a reduced landing rate ( Figure 7) and pause duration ( Figure 2D, Table 1) on GMPCPP microtubules as compared to taxol-stabilized microtubules. This is potentially due to a nucleotide-specific difference in positioning of the C-terminal tails on the microtubule surface. Whether the motor is influenced by protofilament number and/or nucleotide state of the GMPCPP lattice will require further study. Recent compelling work has demonstrated that microtubule associated proteins (MAPs) and septins can also influence KIF1A motility and function [36, [41][42][43]. Pausing may allow KIF1A to navigate around MAPs and other obstacles on the microtubule. The necessity to regulate kinesin cargo transport through pausing is a pre-established concept on a cellular level, as many neuronal cargo have been observed to halt processive movement in order to navigate around obstacles in the crowded cellular environment [44][45][46][47]. On the other hand, microtubule C-terminal tail polyglutamylation can regulate the microtubule affinity of MAPs such as MAP1A, MAP1B, MAP2, and Tau [48,49]. For example, Tau interacts with tubulin C-terminal tails in a unique diffusive behavior that is important for Tau function on a cellular and systemic level [50]. Tau could thus compete with KIF1A for microtubule C-terminal tails and thereby regulate KIF1A pausing and motility. This concept is a compelling topic for further study.
Understanding the mechanisms responsible for KIF1A motility is critical when one considers the extensive range of axonal cargo that KIF1A transports along neuronal microtubules [3,9,11,[51][52][53][54][55][56][57]. Furthermore, until we advance our understanding of KIF1A regulation, we are limited in our ability to address the disease state manifestations of altered KIF1A function. The regulatory capabilities of Tau's binding behavior on KIF1A cargo transport are critical to understanding KIF1A's role in in diseases known for impaired axonal transport, such as frontotemporal dementia (FTD), where changes in Tau isoform expression have been correlated to disease progression [58]. For example, pathological overexpression [59][60][61] of the more diffusive Tau isoforms [62] and reduced transport of cargo known to be transported by KIF1A are observed in FTD [11,12,63]. As we speculate that the diffusive binding state of Tau may compete with KIF1A for the microtubule Cterminal tail, this is an enticing concept for further investigation on a molecular and cellular levels. In summary, our discovery of a microtubule Cterminal tail polyglutamylation-mediated mechanism for regulation of KIF1A motility provides crucial insight as to how KIF1A cargo delivery is regulated during axonal transport and how this process may become altered in the disease state.

Tubulin isolation, microtubule preparation and labelling
Neuronal tubulin was isolated from bovine brains donated from Vermont Livestock Slaughter & Processing (Ferrisburgh, VT) using a high molarity PIPES buffer (1 M PIPES, pH 6.9 at room temperature, 10 mM MgCl 2 , and 20 mM EGTA) as previously described [64]. Purified tubulin was clarified using ultracentrifugation for 20 minutes at 95,000 rpm at 4°C in an Optima TLX Ultracentrifuge (Beckman, Pasadena, CA). After clarification, tubulin concentration was calculated using the tubulin extinction coefficient of 115,000 cm -1 M -1 and read at 280nm in the spectrophotometer.
Tubulin was purified from HeLa Kyoto cells with TOG affinity column chromatography using gravity-flow setup [65,66]. Cells were resuspended in BRB80 (80 mM PIPES, 1 mM EGTA, 1 mM MgCl 2 pH 6.8) supplemented with 1 mM DTT and protease inhibitors (cOmpleteÔ, Mini, EDTA-free [Sigma-Aldrich, St. Louis, MO] and 0.2 mM PMSF) and sonicated. Cleared lysate was loaded onto a TOG column, in which approximately 15 mg of bacterially purified GST-TOG1/2 protein was conjugated with 1 ml of NHSactivated Sepharose 4 Fast Flow resin (GE Healthcare; Marlborough, MA). Wash, elution, desalting and concentration was carried out as described in Hotta et al, 2016 [66]. Glycerol was not added to the purified tubulin. Aliquoted tubulin was snap frozen in liquid nitrogen and stored at -80ºC.

Subtilisin treatment
To remove C-terminal tails, 5 µM paclitaxel stabilized microtubules were treated with 0.05µM Subtilisin A (Sigma Aldrich), resuspended in P12 Buffer, for 45 minutes at 25°C. The reaction was stopped by the addition of 5 mM phenylmethanesulfonyl fluoride. Subtilisin treatment was confirmed by Coomassie staining of SDS-PAGE denaturing gel. Tubulin concentration was determined as previously described.

In vitro single-molecule TIRF
Flow chambers used in in vitro TIRF experiments were constructed as previously described [68]. Flow chambers were incubated with monoclonal anti-b III (neuronal) antibodies at 33 µg/ml for 5 minutes, then washed twice with 0.5 mg/ml bovine serum albumin (BSA; Sigma Aldrich) and incubated for 2 minutes. 1 µM of microtubules (any experimental condition) were administered and incubated for 8 minutes. Nonadherent microtubules were removed with a P12 wash supplemented with 20 µM paclitaxel. Kinesin motors in Motility Buffer consistent with past literature (MB; 12 mM PIPES, 1 mM MgCl 2 , 1 mM EGTA, supplemented with 20 µM paclitaxel, 10 mM DTT, 10 mg/ml BSA, 2 mM ATP and an oxygen scavenger system [5.8 mg/ml glucose, 0.045 mg/ml catalase, and 0.067 mg/ml glucose oxidase; Sigma Aldrich]) [22], or a High Salt Motility Buffer (same as Motility Buffer, with the only change being PIPES concentration raised to 80 mM), supplemented with 2 mM ATP, were added to the flow cell just before image acquisition. For landing rate assays, in vitro KIF1A motility assays were prepared as described above, with the only change being that MB was supplemented with 2 mM ADP. Control Drosophila melanogaster biotin-tagged kinesin-1 motors were labeled with streptavidin-conjugated Qdot 655 (Life Technologies, Carlsbad, CA) at a 1:4 motor:Qdot ratio as previously described [68,69].
Total internal reflection fluorescence (TIRF) microscopy was performed at room temperature using an inverted Eclipse Ti-E microscope (Nikon, Melville, NY) with a 100x Apo TIRF objective lens (1.49 N.A.) and dual iXon Ultra Electron Multiplying CCD cameras, running NIS Elements version 4.51.01. Rhodamine-labeled microtubules were excited with a 561 and a 590/50 filter. KIF1A-LZ-3xmCitrine (WT or Tri-Ala) motors were excited with a 488 laser and a 525/50 filter. Qdot 655 conjugated kinesin-1 motors were excited with a 640 laser and 655 filter. All movies were recorded with an acquisition time of 200 msec for 500 frames (100 sec observation).

Photobleaching assay
As an additional control, a photobleaching assay was performed to confirm that KIF1A run lengths were not underestimated due to photobleaching of the C-terminal 3xmCitrine tag ( Figure S6). Motors were adhered to microtubules in the presense of adenylylimidodiphosphate (AMPPNP; Sigma-Aldrich) and washed once to remove unattached motors. Using Image J , a region of interest (ROI) was drawn around each fluorescent spot and the average intensity of each pixel within the ROI was measured over time. Intensity was background corrected and a motor was considered photobleached when the ROI average intensity was 0.

Data analysis
Motility events were analyzed as previously reported [69,70]). In brief, overall run length motility data was measured using the ImageJ MTrackJ plug-in, for a frame-by-frame quantification of KIF1A motility. Pauses are defined as segments in where the average velocity is less than 0.2 µm/s over three frames or more. Continuous events were identified at the boundaries of pausing events. Average overall/continuous speed were plotted as a histogram, with mean and SD are reported in Table 1. KIF1A pause duration events were fit to a single exponential decay, represented as cumulative frequency plots with confidence intervals (CI) of 95%. Pause duration is reported as the inverse of the decay rate, representing the lifetime of pause duration events.
Kymographs of motor motility were created using the MultipleKymograph ImageJ plugin, with a set line thickness of 3. To correct for microtubule track length effects on motor motility, overall/continuous run length data was resampled to generate cumulative frequency plots (99% CI), using previously reported methods [71]. After data resampling, statistical significance between motor run length and speed data sets was determined using a paired t-test.
To determine the landing rate of KIF1A on various microtubule conditions, the total number of motor landing events on the microtubule was divided by the length of the microtubule and further divided by the duration of the movie (events/µm/minute). To be considered a landing rate event, a motor must remain on the microtubule for three consecutive frames under ADP or AMPPNP state conditions.

21.
Ikegami, K., et al.      Figure 2D). E) KIF1A pauses per distance was significantly decreased from 0.14 pauses/µm to 0.02 pauses/µm upon subtilisin-treatment of taxol-stabilized microtubules. Run length values and standard deviations were calculated as previously reported [71]. Kinesin-1 was used as an experimental control across all conditions ( Figure S2-S5). Each condition is representative of at least four independent experiments. Pause durations were fit to a single exponential decay and are represented as cumulative frequency distributions; the time constant of fit is reported in seconds with a 95% confidence interval. All other metrics are reported as Mean ± SD. * p < 0.001  Figure 2D). Pause durations were fit to a single exponential decay and are represented as cumulative frequency distributions; the time constant of fit is reported in seconds with a 95% confidence interval. All other metrics are reported as Mean ± SD.  Figure 2D). E) Mutation of the K-loop significantly decreased number of pauses per distance from 0.14 pauses/µm (WT KIF1A) to 0.05 pauses/µm (Tri-Ala KIF1A). Run length values and standard deviations were calculated as previously reported [71]. Kinesin-1 was used as an experimental control across all conditions ( Figure S2-S5). Each condition is representative of at least four independent experiments. Pause durations were fit to a single-exponential decay and are represented as cumulative frequency distributions; the time constant of fit is reported in seconds with a 95% confidence interval. All other metrics are reported as Mean ± SD. * p < 0.001  Figure 2D). E) KIF1A pauses per distance was significantly decreased from 0.14 pauses/µm on brain microtubules to 0.07 pauses/µm on HeLa microtubules. Run length values and standard deviations were calculated as previously reported [71]. Kinesin-1 was used as an experimental control across all conditions ( Figure S2-S5). Each condition is representative of at least four independent experiments. Pause durations were fit to a single-exponential decay and are represented as cumulative frequency distributions; the time constant of fit is reported in seconds with a 95% confidence interval. All other metrics are reported as Mean ± SD. * p < 0.001