Processive Motor Research Articles

Molecular interplay between HURP and Kif18A in mitotic spindle regulation.

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determined the binding mode of HURP to microtubules using Cryo-EM. The structure reveals that one HURP motif spans laterally across β-tubulin, while a second motif binds between adjacent protofilaments. HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observed that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in spindle length control.

Control of motor landing and processivity by the CAP-Gly domain in the KIF13B tail

Microtubules are major components of the eukaryotic cytoskeleton. Posttranslational modifications (PTMs) of tubulin regulates interactions with microtubule-associated proteins (MAPs). One unique PTM is the cyclical removal and re-addition of the C-terminal tyrosine of α-tubulin and MAPs containing CAP-Gly domains specifically recognize tyrosinated microtubules. KIF13B, a long-distance transport kinesin, contains a conserved CAP-Gly domain, but the role of the CAP-Gly domain in KIF13B’s motility along microtubules remains unknown. To address this, we investigate the interaction between KIF13B’s CAP-Gly domain, and tyrosinated microtubules. We find that KIF13B’s CAP-Gly domain influences the initial motor-microtubule interaction, as well as processive motility along microtubules. The effect of the CAP-Gly domain is enhanced when the motor domain is in the ADP state, suggesting an interplay between the N-terminal motor domain and C-terminal CAP-Gly domain. These results reveal that specialized kinesin tail domains play active roles in the initiation and continuation of motor movement.

The dynein adapter BicD2 recognizes its cargo through short cargo-recognition alpha-helices.

Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Using nuclear magnetic resonance, mutagenesis and circular dichroism spectroscopy, a short Nup358 α-helix was identified, which transitioned from a random coil to an α-helical conformation upon BicD2-binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 though a “cargo recognition α-helix”, a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility. To test the hypothesis that the other BicD2 cargoes are recognized by similar α-helical motifs, we identified the BicD2-binding site on Rab6GTP, which recruits BicD2 to Golgi-derived vesicles for transport. The BicD2 binding site is located in a region of Rab6 that undergoes conformational changes in the GTP- compared to the GDP-bound state, explaining why GTP-bound Rab6 has a higher affinity. This region is also intrinsically disordered, and we propose that it transitions to an alpha-helical conformation upon binding to BicD2, similar as observed for Nup358. Our results provide insights into the molecular mechanism of cargo selection of BicD2, which facilitates transport pathways that are important for brain development and the transport of Golgi-derived vesicles. Our results also pave the way for studies that establish the function of BicD2/dynein-dependent transport pathways and their regulatory mechanisms in the developing brain and their contributions to neuromuscular diseases.

Recognition of Rab6 by the dynein adaptor BicD2.

The dynein adaptor Bicaudal D2 (BicD2) recognizes cargo for dynein-dependent transport and links them to the motor complex. In addition, BicD2 also has a key role in the activation of dynein for processive motility. BicD2-dependent transport pathways are important for brain and muscle development. BicD2 also recruits dynein to Rab6, facilitating the transport of vesicles from and towards the Golgi apparatus. We recently established a structural basis of how BicD2 recognizes Nup358 which facilitates a nuclear positioning pathway that is essential for brain development. Our data suggest that BicD2 recognizes Nup358 by a short cargo recognition α-helix. It is conceivable that the other BicD2 cargoes are recognized by similar α-helical motifs. To test this hypothesis, we identified the BicD2-binding site on Rab6GTP by a combination of mutagenesis and biophysical methods. We found that the binding site of BicD2 is located in a region of Rab6GTP, which undergoes conformational changes in the GTP-bound state compared to the GDP-bound state, explaining why GTP-bound Rab6 has a higher affinity for BicD2. Furthermore, similar as observed for Nup358, the Rab6GTP region that binds to BicD2 is intrinsically disordered, and we propose that it transitions from a random coil conformation to a cargo-recognition alpha-helix in the BicD2/Rab6GTP complex. Our results provide insights into the molecular mechanism of cargo selection of BicD2, which facilitates transport pathways that are important for brain development, transport of Golgi-derived vesicles and faithful chromosome segregation.

Tension formation of a reconstructed nonmuscle sarcomere.

The interactions between actin and nonmuscle myosin 2 enable tension dependent processes in cells such as migration and adhesion. Although cortical actomyosin networks have been described as mostly disordered, several recent super resolution light microscopy studies have shown evidence for sarcomeric arrangement of actin and myosin in various cell types. Despite their resemblance to muscle sarcomeres where bipolar myosin filaments contract antiparallel actin fibers to bring about shortening, these structures are not as easily amenable to mechanical studies due to their subcellular scale and localization. To address this gap, we reconstituted a synthetic sarcomere using laser guided micropatterning of the actin elongator formin to polymerize antiparallel actin bundles. By adding nonmuscle 2 paralogs, the tension formation and shortening of these arrangements was observed using TIRF microscopy and quantified by tracking fluorescently labeled myosin filaments. Additionally, a FRET based force-sensor was added to the myosin tail and formin, elucidating the tension formation in actin and myosin filaments. Both actin and myosin displayed features resembling isometric tension like force generation within minutes after the start of the experiment, while shortening and processive motility of myosin could be observed more than 1 hour later. This allows us to propose a mechanism of tension formation inside cells, where isometric tension of 2 pNs is mediated by sliding individual actin filaments within a network of bundles, pushing against their bundling and independently of their length. This force generation mechanism at the myosin filament level would enable cells to mediate tension and contractility over the entire cortex, while deformations occur much slower, such as during the formation of a cytokinetic ring.

Towards engineering artificial kinesin-14 motors with plus-end-directed processivity.

Kinesins are motor proteins that utilize the chemical energy of ATP hydrolysis to generate movement and force inside cells on the microtubule filaments to play important roles in a variety of essential intracellular processes. Kinesin-14s are a subset of kinesins with C-terminal motor domains and commonly exhibit minus-end-directed motility on the microtubule. Our previous work revealed that KlpA, a kinesin-14 motor from the filamentous fungus Aspergillus nidulans, uniquely exhibits processive plus-end-directed motility on single microtubules as individual homodimers. We hypothesized that KlpA achieves plus-end-directed motility on a single microtubule by adopting a strained cis-configuration whereby the N-terminal tail and the C-terminal motor domains simultaneously bind to the same microtubule. To test this hypothesis, we combined bioinformatic analysis and in vitro single-molecule fluorescence microscopy to search for additional kinesin-14s that exhibit similar plus-end-directed motility as KlpA. We found that AnKinesin-14 from Aspergillus niger has a similar coiled-coil profile as that of KlpA and exhibits processive motility towards the microtubule plus ends (unpublished results). Additionally, we engineered a chimera that replaces the C-terminal motor domains of AnKinesin-14 with those of Ncd (a nonprecessive minus-end-directed kinesin-14) and retains the coiled-coil profile of AnKinesin-14. Our single-molecule motility experiments showed that the chimera moves processively on the microtubule towards the plus ends. Collectively, our findings not only provide strong support of our previous hypothesis for the plus-end-directed motility of KlpA but also lay out a conceptual framework for how to engineer artificial kinesin motors with desired directionality using a cut-and-paste approach.

Biophysical characterization of Rab6 mutants that are important for the interaction with the dynein adaptor BicD2.

Dynein adapters such as Bicaudal D2 are key players in dynein-dependent transport, as they recognize cargo, link them to the motor complex and are also required to activate dynein for processive motility. BicD recruits dynein to Rab6, which is embedded in secretory and Golgi-derived vesicles, to facilitate their minus-end directed transport. The molecular mechanism of how BicD2 recognizes Rab6 is elusive. Here, we characterize Rab6 mutants that diminish binding to BicD2 by biophysical methods, including circular dichroism spectroscopy. Similar as observed for Nup358, the Rab6GTP region that binds to BicD2 is intrinsically disordered, and we propose that it transitions from a random coil conformation to a cargo-recognition alpha-helix in the BicD2/Rab6GTP complex. Our results provide insights into the molecular mechanism of cargo selection of BicD2, which facilitates transport pathways that are important for brain development, transport of Golgi derived vesicles and faithful chromosome segregation.

Effect of the Neck Linker on Processive Stepping of Kinesin Motor

Kinesin motor protein, which is composed of two catalytic domains connected together by a long coiled-coil stalk via two flexible neck linkers (NLs), can step processively on a microtubule towards the plus end by hydrolyzing adenosine triphosphate (ATP) molecules. To understand what the role is that the NL plays in the processive stepping, the dynamics of the kinesin motor are studied theoretically here by considering the mutation or deletion of an N-terminal cover strand that contributes to the docking of the NL in kinesin-1, the extension of the NL in kinesin-1, the mutation of the NL in kinesin-1, the swapping of the NL of kinesin-2 with that of kinesin-1, the joining of the stalk and neck of Ncd that moves towards the minus end of MT to the catalytic domain of kinesin-1, the replacement of catalytic domain of kinesin-1 with that of Ncd, and so on. The theoretical results give a consistent and quantitative explanation of various available experimental results about the effects of these mutations on motor dynamics and, moreover, provide predicted results. Additionally, the processive motility of kinesin-6 MKLP2 without NL docking is also explained. The available experimental data about the effect of NL mutations on the dynamics of the bi-directional kinesin-5 Cin8 are also explained. The studies are critically implicative to the mechanism of the stepping of the kinesin motor.

Single-motor and multi-motor motility properties of kinesin-6 family members.

ABSTRACTKinesin motor proteins are responsible for orchestrating a variety of microtubule-based processes including intracellular transport, cell division, cytoskeletal organization, and cilium function. Members of the kinesin-6 family play critical roles in anaphase and cytokinesis during cell division as well as in cargo transport and microtubule organization during interphase, however little is known about their motility properties. We find that truncated versions of MKLP1 (HsKIF23), MKLP2 (HsKIF20A), and HsKIF20B largely interact statically with microtubules as single molecules but can also undergo slow, processive motility, most prominently for MKLP2. In multi-motor assays, all kinesin-6 proteins were able to drive microtubule gliding and MKLP1 and KIF20B were also able to drive robust transport of both peroxisomes, a low-load cargo, and Golgi, a high-load cargo, in cells. In contrast, MKLP2 showed minimal transport of peroxisomes and was unable to drive Golgi dispersion. These results indicate that the three mammalian kinesin-6 motor proteins can undergo processive motility but differ in their ability to generate forces needed to drive cargo transport and microtubule organization in cells.

Force generation of KIF1C is impaired by pathogenic mutations

Intracellular transport is essential for neuronal function and survival. The most effective plus-end-directed neuronal transporter is the kinesin-3 KIF1C, which transports large secretory vesicles and endosomes.1-4 Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients.5-8 In contrast to other kinesin-3s, KIF1C is a stable dimer and a highly processive motor in its native state.9,10 Here, we establish a baseline for the single-molecule mechanics of Kif1C. We show that full-length KIF1C molecules can processively step against the load of an optical trap and reach average stall forces of 3.7 pN. Compared with kinesin-1, KIF1C has a higher propensity to slip backward under load, which results in a lower maximal single-molecule force. However, KIF1C remains attached to the microtubule while slipping backward and re-engages quickly, consistent with its super processivity. Two pathogenic mutations, P176L and R169W, that cause hereditary spastic paraplegia in humans7,8 maintain fast, processive single-molecule motility invitro but with decreased run length and slightly increased unloaded velocity compared with the wild-type motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles. Our results show how its mechanics supports KIF1C's role as an intracellular transporter and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.

Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs

BackgroundKinesin-3 family motors drive diverse cellular processes and have significant clinical importance. The ATPase cycle is integral to the processive motility of kinesin motors to drive long-distance intracellular transport. Our previous work has demonstrated that kinesin-3 motors are fast and superprocessive with high microtubule affinity. However, chemomechanics of these motors remain poorly understood.ResultsWe purified kinesin-3 motors using the Sf9-baculovirus expression system and demonstrated that their motility properties are on par with the motors expressed in mammalian cells. Using biochemical analysis, we show for the first time that kinesin-3 motors exhibited high ATP turnover rates, which is 1.3- to threefold higher compared to the well-studied kinesin-1 motor. Remarkably, these ATPase rates correlate to their stepping rate, suggesting a tight coupling between chemical and mechanical cycles. Intriguingly, kinesin-3 velocities (KIF1A > KIF13A > KIF13B > KIF16B) show an inverse correlation with their microtubule-binding affinities (KIF1A < KIF13A < KIF13B < KIF16B). We demonstrate that this differential microtubule-binding affinity is largely contributed by the positively charged residues in loop8 of the kinesin-3 motor domain. Furthermore, microtubule gliding and cellular expression studies displayed significant microtubule bending that is influenced by the positively charged insert in the motor domain, K-loop, a hallmark of kinesin-3 family.ConclusionsTogether, we propose that a fine balance between the rate of ATP hydrolysis and microtubule affinity endows kinesin-3 motors with distinct mechanical outputs. The K-loop, a positively charged insert in the loop12 of the kinesin-3 motor domain promotes microtubule bending, an interesting phenomenon often observed in cells, which requires further investigation to understand its cellular and physiological significance.

The KASH5 protein involved in meiotic chromosomal movements is a novel dynein activating adaptor.

Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.

Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment.

Nup358, a protein of the nuclear pore complex, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 interacts with the dynein adaptor Bicaudal D2 (BicD2), which in turn recruits the dynein machinery to position the nucleus. However, the molecular mechanisms of the Nup358/BicD2 interaction and the activation of transport remain poorly understood. Here for the first time, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules. Using nuclear magnetic resonance titration and chemical exchange saturation transfer, mutagenesis, and circular dichroism spectroscopy, a Nup358 α-helix encompassing residues 2162-2184 was identified, which transitioned from a random coil to an α-helical conformation upon BicD2 binding and formed the core of the Nup358-BicD2 interface. Mutations in this region of Nup358 decreased the Nup358/BicD2 interaction, resulting in decreased dynein recruitment and impaired motility. BicD2 thus recognizes Nup358 through a 'cargo recognition α-helix,' a structural feature that may stabilize BicD2 in its activated state and promote processive dynein motility.

Coil-to-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment

Nup358, a nuclear pore protein, facilitates a nuclear positioning pathway that is essential for many biological processes, including neuromuscular and brain development. Nup358 binds and activates the auto-inhibited dynein adaptor Bicaudal D2 (BicD2), which in turn recruits and activates the dynein machinery to position the nucleus. However, the molecular details of the Nup358/BicD2 interaction remain poorly understood. Here, we show that a minimal Nup358 domain activates dynein/dynactin/BicD2 for processive motility on microtubules.

Elucidating tubulin E-hook peptide structure using spectroscopic approaches

Microtubules (MT) consist of α- and β-tubulin dimers and contain disordered, C-terminal tails, known as E-hooks, which extend from the MT surface to interact with the cytoplasmic environment. While the MT core is conserved, the highly negatively charged E-hook composition differs depending on its location and proposed function, and E-hooks also engage in the binding and processive motility of MT-based motors. Despite the E-hook's clear importance in cytoskeletal regulation and function, its molecular structure is not well understood.

Coiled-Coil Registry Shifts in the F684I Mutant of Bicaudal D Result in Cargo-Independent Activation of Dynein Motility

Key questions in the dynein field are how cargo is recognized by dynein adaptors, such as Drosophila Bicaudal D (BicD) and how dynein is activated for processive motility by these adaptors. BicD is auto-inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. Here, we show that the full-length BicD/F684I mutant activates dynein processivity in a cargo-independent manner, in contrast to the wild type. To obtain mechanistic insights into activation, we determined the X-ray structure of the C-terminal domain of the BicD/F684I mutant, which reveals a coiled-coil registry shift; in the N-terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild-type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. Based on our results, we propose that a coiled-coil registry shift upon cargo binding activates BicD for dynein recruitment. Activation of dynein adapters such as BicD is a key regulatory step for dynein-dependent transport, since they are required to activate dynein for processive motility. Moreover, the human homolog of the BicD/F684I mutant (BicD2/F743I) exhibits diminished binding of cargo adaptor Nup358, implying that a coiled-coil registry shift may be a mechanism to modulate cargo selection for BicD2-dependent transport pathways. Our results provide new mechanistic insights into how the dynein adapter BicD2 recognizes its cargo Nup358. This Nup358/BicD2 pathway serves to position the nucleus with respect to the centrosome and is essential for a fundamental process in brain development that is required for radial glial progenitor cells to differentiate, which are precursors for the majority of neurons and glia cells in the vertebrate neocortex.

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus–Sf9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities (kcat = ∼ 6 s−1 at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s−1 at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM–cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1–cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.

A tunable LIC1-adaptor interaction modulates dynein activity in a cargo-specific manner

Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called “activating adaptors” that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 μM. We propose that a tunable LIC1-adaptor interaction modulates dynein’s motility in a cargo-specific manner.

The Tail of Kinesin-14a in Giardia Is a Dual Regulator of Motility

Kinesin-14s are microtubule-based motor proteins that play important roles in mitotic spindle assembly [1]. Ncd-type kinesin-14s are a subset of kinesin-14 motors that exist as homodimers with an N-terminal microtubule-binding tail, a coiled-coil central stalk (central stalk), a neck, and two identical C-terminal motor domains. To date, no Ncd-type kinesin-14 has been found to naturally exhibit long-distance minus-end-directed processive motility on single microtubules as individual homodimers. Here, we show that GiKIN14a from Giardia intestinalis [2] is an unconventional Ncd-type kinesin-14 that uses its N-terminal microtubule-binding tail to achieve minus-end-directed processivity on single microtubules over micrometer distances as a homodimer. We further find that although truncation of the N-terminal tail greatly reduces GiKIN14a processivity, the resulting tailless construct GiKIN14a-Δtail is still a minimally processive motor and moves its center of mass via discrete 8-nm steps on the microtubule. In addition, full-length GiKIN14a has significantly higher stepping and ATP hydrolysis rates than does GiKIN14a-Δtail. Inserting a flexible polypeptide linker into the central stalk of full-length GiKIN14a nearly reduces its ATP hydrolysis rate to that of GiKIN14a-Δtail. Collectively, our results reveal that the N-terminal tail of GiKIN14a is a de facto dual regulator of motility and reinforce the notion of the central stalk as a key mechanical determinant of kinesin-14 motility [3].