Single-molecule Motility Assays Research Articles

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases.

Neuronal autophagosomes form and engulf cargos at presynaptic sites in the axon and are then transported to the soma to recycle their cargo. Autophagic vacuoles (AVs) mature en route via fusion with lysosomes to become degradatively competent organelles; transport is driven by the microtubule motor protein cytoplasmic dynein, with motor activity regulated by a sequential series of adaptors. Using lysate-based single-molecule motility assays and live-cell imaging in primary neurons, we show that JNK-interacting proteins 3 (JIP3) and 4 (JIP4) are activating adaptors for dynein that are regulated on autophagosomes and lysosomes by the small GTPases ARF6 and RAB10. GTP-bound ARF6 promotes formation of the JIP3/4-dynein-dynactin complex. Either knockdown or overexpression of RAB10 stalls transport, suggesting that this GTPase is also required to coordinate the opposing activities of bound dynein and kinesin motors. These findings highlight the complex coordination of motor regulation during organelle transport in neurons.

The Antiparallel Coiled-Coil Domain Allows Multiple Forward Step Sizes of Myosin X.

Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of ∼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.

Mechanistic insights into the regulation of human myosin-7a.

Human myosin-7a (Myo7a) is an actin-based motor protein essential for vision and hearing. It plays critical roles in the development and functions of actin rich protrusions. Previous studies using Drosophila homolog showed that myosin-7a is a monomeric, high duty ratio motor and can move processively upon dimerization. However, characterization of full-length mammalian myosin-7a has been limited by the difficulty of expressing and purifying stable, intact protein. Here, we report the production of a complete human myosin-7a holoenzyme in insect cells and study its regulation by intra- and intermolecular mechanisms. We found that human myosin-7a utilizes regulatory light chain (RLC), calmodulin and calmodulin like protein 4 (CALML4) as the light chain subunits. CALML4 is recently discovered highly enriched in stereocilia and identified as a deafness candidate gene. We show that CALML4 does not bind to Ca2+ but plays a key role in regulating the dynamic binding of calmodulin to myosin-7a in response to Ca2+ signaling. The cochlea expresses two myosin-7a splicing isoforms differed by a short N-terminal extension. Using combined optical trapping, biochemistry, and in vitro motility assays, we show that the N-terminal extension greatly influences the enzymatic and mechanical behaviors of mammalian myosin-7a. We propose that the hair cell regulates its mechanosensitivity by adjusting the expression levels of the two myosin-7a isoforms. Finally, using single-molecule motility assays, we show that purified full-length myosin-7a alone does not move processively on actin in vitro, but in the presence of MyRIP, a known myosin-7a binding protein in neuroretina, it exhibits processive movements. Together, our results provide new molecular insight into how myosin-7a functions in hair cell stereocilia and neuroretina. The production of intact human myosin-7a protein enables future studies to understand the molecular details of human vision and hearing loss caused by myosin-7a defects.

Reconstitution of Dynein/Dynactin Transport Using Recombinant Dynein.

Cytoplasmic dynein-1 is activated by dynactin and a cargo adaptor for processive transport along microtubules. Dynein's motility can be visualized at the single-molecule level using total internal reflection fluorescence microscopy. Our understanding of the motile behavior of the dynein/dynactin complex has been aided by advances in recombinant expression, in particular for dynein. Here, I describe the purification of recombinant dynein and cargo adaptors, and endogenous dynactin and detail a protocol for the single-molecule motility assay. In this assay, microtubules are first immobilized on a coverslip. A fluorescently labeled dynein/dynactin/cargo adaptor complex is then added, allowing for the measurement of key motility parameters as the complex walks along the microtubule.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Delineating the role of the k-loop in the KIF1A chemomechanical cycle

The kinesin-3 KIF1A has three prominent biophysical characteristics: fast velocity, superprocessivity, and low resistance to load. Using a combination of single molecule motility assay and stopped-flow spectroscopy, we measured the discrete steps of the chemomechanical cycle. The fast rear-head detachment suggests that the fast velocity originates from a reduced waiting time in the 2-head-bound state, resulting in KIF1A predominantly residing in the vulnerable 1-head-bound state during its stepping cycle and therefore being susceptible to load; while the superprocessivity arises from the relatively high microtubule affinity in the post-hydrolysis state.

The inchworm episode: Reconstituting the phenomenon of kinesin motility

New Mechanist philosophical models of "phenomenon reconstitution" understand the process to be driven by explanatory considerations. Here I discuss an episode of phenomenon reconstitution that occurred entirely within an experimental program dedicated to characterizing (rather than explaining) the phenomenon of kinesin motility. Rather than being driven by explanatory considerations, as standard mechanist views maintain, I argue that the phenomenon of kinesin motility was reconstituted to enhance researchers’ primary experimental tool—the single molecule motility assay.

The Fast and Superprocessive KIF1A Predominately Resides in a Vulnerable One-Head-Bound State during its Chemomechanical Cycle

Kinesin-3 are the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to wild-type KIF1A in BRB80. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and-2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate limiting transition in the KIF1A stepping cycle. The fast speed, superprocessivity and load sensitivity of KIF1A can be explained by a fast rear head detachment rate, a rate-limiting step of tethered head attachment that follows ATP hydrolysis, and a relatively strong microtubule interaction in the post-hydrolysis state.

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle

The kinesin-3 family contains the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle and give rise to their unique properties are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to WT KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and -2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate-limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate-limiting transition in the KIF1A stepping cycle. Thus, KIF1A's activity can be explained by a fast rear-head detachment rate, a rate-limiting step of tethered-head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly bound post-hydrolysis state.

The temperature dependence of kinesin motor-protein mechanochemistry

Biophysical studies of the mechanochemical cycle of kinesin motors are essential for understanding the mechanism of energy conversion. Here, we report a systematic study of the impact of temperature on velocity and run length of homodimeric Drosophila kinesin-1, homodimeric C. elegans OSM-3 and heterodimeric C. elegans kinesin-II motor proteins using in vitro single-molecule motility assays. Under saturated ATP conditions, kinesin-1 and OSM-3 are fast and processive motors compared to kinesin-II. From in vitro motility assays employing single-molecule fluorescence microscopy, we extracted single-motor velocities and run lengths in a temperature range from 15 °C to 35 °C. Both parameters showed a non-Arrhenius temperature dependence for all three motors, which could be quantitatively modeled using a simplified, two-state kinetic model of the mechanochemistry of the three motors, providing new insights in the temperature dependence of their mechanochemistry.

The mechanochemistry of the kinesin-2 KIF3AC heterodimer is related to strain-dependent kinetic properties of KIF3A and KIF3C

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo- and heterodimeric constructs and determine their transport potential under load, we assayed motor activity using interferometric scattering microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1 = 11 s-1) without apparent kinetic asymmetry. Asymmetry was also not apparent under hindering or assisting mechanical loads in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA yet is more capable of stepping against mechanical load than KIF3CC. Interestingly, the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, suggesting the homodimers mechanically impact each other's motility to reproduce the behavior of the heterodimer. Our observations are consistent with a mechanism in which the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner, similar to application of an assisting load. These results suggest that the mechanochemical properties of KIF3AC can be explained by the strain-dependent kinetics of KIF3A and KIF3C.

Strain-Dependent Properties of KIF3A and KIF3C Tune the Mechanochemistry of the KIF3AC Heterodimer

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo- and heterodimeric constructs, and to determine their transport potential under mechanical load, we assayed motor activity using interferometric scattering (iSCAT) microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1= 11 s−1) without apparent kinetic asymmetry in stepping. Asymmetry was also not apparent under hindering or assisting mechanical loads of 1 pN in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA, yet is more capable of stepping against mechanical load than KIF3CC. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, indicating the homodimers mechanically impact each other's motility to reproduce the behavior of the heterodimer. We conclude that the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner which is similar to application of an assisting load, and the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. These results suggest that KIF3AC-based cargo transport likely requires multiple motors, and its mechanochemical properties arise due to the strain-dependences of KIF3A and KIF3C.

A protocol for single molecule imaging and tracking of processive myosin motors

Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell’s life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins.

Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin

Neuronal function requires axon-dendrite membrane polarity, which depends on sorting of membrane traffic during entry into axons. Due to a microtubule network of mixed polarity, dendrites receive vesicles from the cell body without apparent capacity for directional sorting. We found that, during entry into dendrites, axonally destined cargos move with a retrograde bias toward the cell body, while dendritically destined cargos are biased in the anterograde direction. A microtubule-associated septin (SEPT9), which localizes specifically in dendrites, impedes axonal cargo of kinesin-1/KIF5 and boosts kinesin-3/KIF1 motor cargo further into dendrites. In neurons and invitro single-molecule motility assays, SEPT9 suppresses kinesin-1/KIF5 and enhances kinesin-3/KIF1 in a manner that depends on a lysine-rich loop of the kinesin motor domain. This differential regulation impacts partitioning of neuronal membrane proteins into axons-dendrites. Thus, polarized membrane traffic requires sorting during entry into dendrites by a septin-mediated mechanism that bestows directional bias on microtubules of mixed orientation.

An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules

Microtubule-based molecular motors mediate transport of intracellular cargo to subdomains in neurons. Previous evidence has suggested that the anesthetic propofol decreases the average run-length potential of the major anterograde transporters kinesin-1 and kinesin-2 without altering their velocity. This effect on kinesin has not been observed with other inhibitors, stimulating considerable interest in the underlying mechanism. Here, we used a photoactive derivative of propofol, meta-azipropofol (AziPm), to search for potential propofol-binding sites in kinesin. Single-molecule motility assays confirmed that AziPm and propofol similarly inhibit kinesin-1 and kinesin-2. We then applied AziPm in semiquantitative radiolabeling and MS microsequencing assays to identify propofol-binding sites within microtubule–kinesin complexes. The radiolabeling experiments suggested preferential AziPm binding to the ATP-bound microtubule–kinesin complex. The photolabeled residues were contained within the kinesin motor domain rather than at the motor domain–β-tubulin interface. No residues within the P-loop of kinesin were photolabeled, indicating an inhibitory mechanism that does not directly affect ATPase activity and has an effect on run length without changing velocity. Our results also indicated that when the kinesin motor interacts with the microtubule during its processive run, a site forms in kinesin to which propofol can then bind and allosterically disrupt the kinesin–microtubule interaction, resulting in kinesin detachment and run termination. The discovery of the propofol-binding allosteric site in kinesin may improve our understanding of the strict coordination of the motor heads during the processive run. We hypothesize that propofol's potent effect on intracellular transport contributes to various components of its anesthetic action.

Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motorcargo During Entry into Dendrites by a Microtubule-Associated Septin

Neuronal function requires axon-dendrite membrane polarity, which depends on sorting of membrane traffic during entry into axons. Due to a microtubule network of mixed polarity, dendrites receive vesicles from the cell body without apparent capacity for directional sorting. We found that in the initial segments of dendrites, axonally-destined cargos move with a retrograde bias toward the cell body, while dendritically-destined cargos are biased in the anterograde direction. A microtubule-associated septin (SEPT9), which localizes specifically in dendrites, impedes axonal cargo of kinesin-1/KIF5 and boosts kinesin-3/KIF1 motor-cargo further into dendrites. In neurons and in vitro single-molecule motility assays, SEPT9 suppresses kinesin-1/KIF5 and enhances kinesin-3/KIF1 in a manner that depends on a lysinerich loop of the kinesin motor domain. This differential regulation impacts partitioning of neuronal membrane proteins into axonsdendrites. Thus, polarized membrane traffic requires sorting during entry into dendrites by a septin-mediated mechanism that bestows directional bias on microtubules of mixed orientation.

Kinesin-1 Cargo Transport through Dense Microtubule Networks

Essential motor transport processes occurring in living cells rely heavily on trafficking of cargoes by kinesin motors through dense microtubule networks. Kinesin-1 motors in particular have been shown to walk unidirectionally along single filaments, and to fall off their tracks upon encountering obstacles, lattice defects, and network intersections. Knowing that both microtubule network density and arrangement are highly controlled in vivo, it is likely that microtubule network organization is a key component in the regulation of cargo transport. To better understand how network geometry controls multi-motor cargo transport, we have tracked quantum-dot molecular cargoes decorated with different numbers of kinesin-1 motors through microtubule networks of different densities and intersection geometries. Using single molecule motility assays and STORM imaging of the network, we have found that cargoes with greater numbers of motors travel via highly non-linear pathways, while single motor transport is confined to single filaments. These experiments help to illuminate the mechanisms by which motor number and network geometry are used in combination to carefully direct transport of essential cargoes throughout the cell.

Single Molecule Characterization of Mitotic Kif15 Reveals Capability to Generate Force in Anti-Parallel Microtubules

Proper formation of the mitotic spindle is essential for chromosome separation and cell division. Dimeric Kinesin-12, or Kif15, has been shown to serve as a backup mechanism for cell division, promoting spindle assembly in the absence of tetrameric Kinesin-5, or Eg5. The mechanism by which Kif15 aids in the assembly of the spindle is unknown. Here, single molecule motility assays were performed using optical tweezers to evaluate the mechanistic behavior of Kif15 on microtubules (MTs). Constructs containing various sub-components of the motor were utilized to determine the role of each domain, including the motor heads, stalk, MT binding domain (N700) and the full length motor (FL). Step size, dwell times, and stall forces were measured. Rupture assays were also performed on the MT binding domain (Coil-1) to determine the binding strength and the nature of the Coil-1/MT interaction. Motility and rupture assays were repeated on subtilisin-digested MTs (dMT) in which the negatively-charged carboxyl-terminal tail of MTs, called the E-hook, is removed. We found that the Coil-1 was stronger than the stall force of the motor due to its interaction with the E-hook, revealing that Kif15 has the ability to build up mechanical strain and slide MTs apart. In order to determine the importance of MT orientation, an in vitro optical trapping and fluorescence assay was developed to determine the force generating capabilities of Kif15 in different bundle environments. We determined that Kif15 slides anti-parallel MTs apart while parallel bundles remain stationary with a small amount of antagonizing force generation. Our studies provide insight regarding how Kif15 is able to rescue bipolar spindle assembly in the absence of Eg5.

Hook Adaptors Induce Unidirectional Processive Motility by Enhancing the Dynein-Dynactin Interaction

Cytoplasmic dynein drives the majority of minus end-directed vesicular and organelle motility in the cell. However, it remains unclear how dynein is spatially and temporally regulated given the variety of cargo that must be properly localized to maintain cellular function. Recent work has suggested that adaptor proteins provide a mechanism for cargo-specific regulation of motors. Of particular interest, studies in fungal systems have implicated Hook proteins in the regulation of microtubule motors. Here we investigate the role of mammalian Hook proteins, Hook1 and Hook3, as potential motor adaptors. We used optogenetic approaches to specifically recruit Hook proteins to organelles and observed rapid transport of peroxisomes to the perinuclear region of the cell. This rapid and efficient translocation of peroxisomes to microtubule minus ends indicates that mammalian Hook proteins activate dynein rather than kinesin motors. Biochemical studies indicate that Hook proteins interact with both dynein and dynactin, stabilizing the formation of a supramolecular complex. Complex formation requires the N-terminal domain of Hook proteins, which resembles the calponin-homology domain of end-binding (EB) proteins but cannot bind directly to microtubules. Single-molecule motility assays using total internal reflection fluorescence microscopy indicate that both Hook1 and Hook3 effectively activate cytoplasmic dynein, inducing longer run lengths and higher velocities than the previously characterized dynein activator bicaudal D2 (BICD2). Together, these results suggest that dynein adaptors can differentially regulate dynein to allow for organelle-specific tuning of the motor for precise intracellular trafficking.

The natural diterpene tonantzitlolone A and its synthetic enantiomer inhibit cell proliferation and kinesin-5 function

Tonantzitlolone A, a diterpene isolated from the Mexican plant Stillingia sanguinolenta, shows cytostatic activity. Both the natural product tonantzitlolone A and its synthetic enantiomer induce monoastral spindle formation in cell experiments which indicates inhibitory activity on kinesin-5 mitotic motor molecules. These inhibitory effects on kinesin-5 could be verified in in vitro single-molecule motility assays, where both tonantzitlolones interfered with kinesin-5 binding to its cellular interaction partner microtubules in a concentration-dependent manner, yet with a larger effect of the synthetic enantiomer. In contrast to kinesin-5 inhibition, both tonantzitlolone A enantiomers did not affect conventional kinesin-1 function; hence tonantzitlolones are not unspecific kinesin inhibitors. The observed stronger inhibitory effect of the synthetic enantiomer demonstrates the possibility to enhance the overall moderate anti-proliferative effect of the lead compound tonantzitlolon A by chemical modification.