Differential regulation of single microtubules and bundles by a three-protein module.

A remarkable feature of the microtubule cytoskeleton is the coexistence of subpopulations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are the dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1 and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find that their collective activity promotes elongation of single microtubules while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (1) strong rescue activity of CLASP1, which overcomes the weaker effects of Kif4A on single microtubules, and (2) lower microtubule- and PRC1-binding affinity of CLASP1, which permits the dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule length, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.

Mitochondria-adaptor TRAK1 promotes kinesin-1 driven transport in crowded environments.

Intracellular trafficking of organelles, driven by kinesin-1 stepping along microtubules, underpins essential cellular processes. In absence of other proteins on the microtubule surface, kinesin-1 performs micron-long runs. Under crowding conditions, however, kinesin-1 motility is drastically impeded. It is thus unclear how kinesin-1 acts as an efficient transporter in intracellular environments. Here, we demonstrate that TRAK1 (Milton), an adaptor protein essential for mitochondrial trafficking, activates kinesin-1 and increases robustness of kinesin-1 stepping on crowded microtubule surfaces. Interaction with TRAK1 i) facilitates kinesin-1 navigation around obstacles, ii) increases the probability of kinesin-1 passing through cohesive islands of tau and iii) increases the run length of kinesin-1 in cell lysate. We explain the enhanced motility by the observed direct interaction of TRAK1 with microtubules, providing an additional anchor for the kinesin-1-TRAK1 complex. Furthermore, TRAK1 enables mitochondrial transport in vitro. We propose adaptor-mediated tethering as a mechanism regulating kinesin-1 motility in various cellular environments.

PTPN21 and Hook3 relieve KIF1C autoinhibition and activate intracellular transport.

The kinesin-3 KIF1C is a fast organelle transporter implicated in the transport of dense core vesicles in neurons and the delivery of integrins to cell adhesions. Here we report the mechanisms of autoinhibition and release that control the activity of KIF1C. We show that the microtubule binding surface of KIF1C motor domain interacts with its stalk and that these autoinhibitory interactions are released upon binding of protein tyrosine phosphatase PTPN21. The FERM domain of PTPN21 stimulates dense core vesicle transport in primary hippocampal neurons and rescues integrin trafficking in KIF1C-depleted cells. In vitro, human full-length KIF1C is a processive, plus-end directed motor. Its landing rate onto microtubules increases in the presence of either PTPN21 FERM domain or the cargo adapter Hook3 that binds the same region of KIF1C tail. This autoinhibition release mechanism allows cargo-activated transport and might enable motors to participate in bidirectional cargo transport without undertaking a tug-of-war.

Diffusive tail anchorage determines velocity and force produced by kinesin-14 between crosslinked microtubules.

Form and function of the mitotic spindle depend on motor proteins that crosslink microtubules and move them relative to each other. Among these are kinesin-14s, such as Ncd, which interact with one microtubule via their non-processive motor domains and with another via their diffusive tail domains, the latter allowing the protein to slip along the microtubule surface. Little is known about the influence of the tail domains on the protein's performance. Here, we show that diffusive anchorage of Ncd's tail domains impacts velocity and force considerably. Tail domain slippage reduced velocities from 270 nm s-1 to 60 nm s-1 and forces from several piconewtons to the sub-piconewton range. These findings challenge the notion that kinesin-14 may act as an antagonizer of other crosslinking motors, such as kinesin-5, during mitosis. It rather suggests a role of kinesin-14 as a flexible element, pliantly sliding and crosslinking microtubules to facilitate remodeling of the mitotic spindle.

Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s.

Kinesin-13s constitute a distinct group within the kinesin superfamily of motor proteins that promote microtubule depolymerization and lack motile activity. The molecular mechanism by which kinesin-13s depolymerize microtubules and are adapted to perform a seemingly very different activity from other kinesins is still unclear. To address this issue, here we report the near atomic resolution cryo-electron microscopy (cryo-EM) structures of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to curved or straight tubulin in different nucleotide states. These structures show how nucleotide induced conformational changes near the catalytic site are coupled with movement of the kinesin-13-specific loop-2 to induce tubulin curvature leading to microtubule depolymerization. The data highlight a modular structure that allows similar kinesin core motor-domains to be used for different functions, such as motility or microtubule depolymerization.

Biochemical and Biophysical characterization of curcumin binding to human mitotic kinesin Eg5: Insights into the inhibitory mechanism of curcumin on Eg5.

In this study we have characterized the biochemical and biophysical interactions of curcumin with the mitotic kinesin Eg5 which plays a pivotal role in the separation of centrosomes during cell division. Curcumin bound to the purified Eg5 (Eg5-437H) with a Kd value of 7.8µM. The temperature dependent binding analysis and evaluation of thermodynamic parameters indicated the involvement of static quenching mechanism in the binding process. Evidences from competition experiment with monastrol indicated that curcumin bound to Eg5 at a novel druggable site. Using Förster resonance energy transfer the distance between curcumin and monastrol binding site from TRP127 on Eg5-437H was found to be 33Å and 17Å respectively. Curcumin inhibited the ATPase activity of Eg5 motor and perturbed the dynamic interactions between Eg5 and microtubules. Results from circular dichroism studies and molecular dynamics simulations suggest that curcumin binding might perturb the Eg5-437H secondary structure which could be the reason behind its inhibitory effects on Eg5. Cell culture studies performed in HeLa cells indicated that curcumin potentiated the mitotic arrest and monopolar spindle formation in synergism with monastrol, indicating that both ligands could bind simultaneously to the same target.

Preparation of centralspindlin as an active heterotetramer of kinesin and GTPase activating protein subunits for in vitro structural and functional assays.

Centralspindlin is a crucial regulator of animal cytokinesis, consisting of MKLP1 kinesin-6 and CYK4 Rho-family GTPase activating protein (RhoGAP). As a microtubule-bundling protein, it plays a crucial role in the formation of the central spindle. Through distinct accumulation to the antiparallel microtubule overlaps at the central spindle and the midbody, it recruits various downstream factors to the site of cell division as well as anchors the plasma membrane to maintain the narrow intercellular channels between the daughter cells until their final separation (abscission). A unique and functionally important feature of centralspindlin as a kinesin-containing protein complex is that the nonmotor component, CYK4, is not a passive cargo of the MKLP1 motor, but an integrated component of a microtubule-organizing machinery. Thus, for in vitro structural and functional assays, it is pivotal to prepare active stoichiometric complexes of the two components. Discussed here are two complimentary approaches, (1) reconstitution of the complex in bacterial extracts (in extract reconstitution) and (2) purification of a native complex from a mammalian cell line using a localization and affinity purification (LAP) tag.

Interferometric Scattering Microscopy for the Study of Molecular Motors.

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

[Expression, purification and crystallization of rat brain kinesin].

Kinesin is a motor protein that uses the energy from ATP hydrolysis to move along the microtubule system. To investigate how the chemical energy stored in ATP is converted to mechanical movement, the corresponding N-terminal region of rat brain kinesin was expressed in BL21-Codon Plus (DE3)-RP competent cells. After SP-cation exchange chromatography and size exclusion chromatography, the protein yield reached 10 mg/L culture with the purity above 95%. The purified protein had ATPase activity and specifically reacted with the kinesin antibody in the Western blotting analysis. The purified kinesin was crystallized under the following condition: 1.7 mol/L (NH4)2SO4, 500 mmol/L NaCl, 20% glycerol. The kinesin crystal can diffract up to 2.0 angstroms resolution.

Biomolecular motor modulates mechanical property of microtubule.

The microtubule (MT) is the stiffest cytoskeletal filamentous protein that takes part in a wide range of cellular activities where its mechanical property plays a crucially significant role. How a single biological entity plays multiple roles in cell has been a mystery for long time. Over the recent years, it has been known that modulation of the mechanical property of MT by different cellular agents is the key to performing manifold in vivo activities by MT. Studying the mechanical property of MT thus has been a prerequisite in understanding how MT plays such diversified in vivo roles. However, the anisotropic structure of MT has been an impediment in obtaining a precise description of the mechanical property of MT along its longitudinal and lateral directions that requires employment of distinct experimental approach and has not been demonstrated yet. In this work, we have developed an experimental system that enabled us to investigate the effect of tensile stress on MT. By using our newly developed system, (1) we have determined the Young's modulus of MT considering its deformation under applied tensile stress and (2) a new role of MT associated motor protein kinesin in modulating the mechanical property of MT was revealed for the first time. Decrease in Young's modulus of MT with the increase in interaction with kinesin suggests that kinesin has a softening effect on MT and thereby can modulate the rigidity of MT. This work will be an aid in understanding the modulation of mechanical property of MTs by MT associated proteins and might also help obtain a clear insight of the endurance and mechanical instability of MTs under applied stress.

Isolation and purification of kinesin from Drosophila embryos.

Motor proteins move cargoes along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Crystal structure of the Kar3-like kinesin motor domain from the filamentous fungus Ashbya gossypii.

Kar3 kinesins are microtubule (MT) minus-end-directed motors with pleiotropic functions in mitotic spindle formation and nuclear movement in budding and fission yeasts. A Kar3-like kinesin is also expressed by the filamentous fungus Ashbya gossypi, which exhibits different nuclear movement challenges from its yeast relatives. Presented here is a 2.35 Å crystal structure and enzymatic analysis of the AgKar3 motor domain (AgKar3MD). Compared to the previously published Saccharomyces cerevisiae Kar3MD structure (ScKar3MD), AgKar3MD displays differences in the conformation of some of its nucleotide-binding motifs and peripheral elements. Unlike ScKar3MD, the salt bridge between Switch I and Switch II in AgKar3MD is broken. Most of the Switch I, and the adjoining region of helix α3, are also disordered instead of bending into the active site cleft as is observed in ScKar3MD. These aspects of AgKar3MD are highly reminiscent of the ScKar3 R598A mutant that disrupts the Switch I-Switch II salt bridge and impairs MT-stimulated ATPase activity of the motor. Subtle differences in the disposition of secondary structure elements in the small lobe (ß1a, ß1b, and ß1c) at the edge of the MD are also apparent even though it contains approximately the same number of residues as ScKar3. These differences may reflect the unique enzymatic properties we measured for this motor, which include a lower MT-stimulated ATPase rate relative to ScKar3, or they could relate to its interactions with different regulatory companion proteins than its budding yeast counterpart.

A novel actin-microtubule cross-linking kinesin, NtKCH, functions in cell expansion and division.

• Kinesins with a calponin homology domain (KCHs) have been identified recently as a plant-specific subgroup of the kinesin-14 family and are suspected to act as microtubule-actin filament cross-linkers. The cellular function, however, has remained elusive. • In order to address the function of KCHs, we isolated NtKCH, a novel KCH homologue from tobacco BY-2 cells. Following synchronization, NtKCH transcripts were shown to be abundant during mitosis, whereas, during interphase, expression was low. • Using fluorescent-tagged cell lines and immunolabelling techniques, the localization of tobacco KCH was found to differ depending on the cell cycle. During interphase, NtKCH mainly associated with cortical microtubules, whereas a subfraction also co-localized with perinuclear actin cables. In dividing cells, NtKCH accumulated at the pre-prophase band and at the phragmoplast. However, it remained absent from spindle microtubules, but, instead, concentrated at two agglomerations in proximity to the two cell poles. • This work develops a detailed model for the dual localization and function of NtKCH during cell division vs cell expansion. This model implies two dynamic states of KCHs that differ with regard to actin interaction. This allows the modulation of force generation by KCH in a cell cycle-dependent capture mechanism.

PH-domain-dependent selective transport of p75 by kinesin-3 family motors in non-polarized MDCK cells.

A key process during epithelial polarization involves establishment of polarized transport routes from the Golgi to distinct apical and basolateral membrane domains. To do this, the machinery involved in selective trafficking must be regulated during differentiation. Our previous studies showed that KIF5B selectively transports vesicles containing p75-neurotrophin receptors to the apical membrane of polarized, but not non-polarized MDCK cells. To identify the kinesin(s) responsible for p75 trafficking in non-polarized MDCK cells we expressed KIF-specific dominant-negative constructs and assayed for changes in post-Golgi transport of p75 by time-lapse fluorescence microscopy. Overexpression of the tail domains of kinesin-3 family members that contain a C-terminal pleckstrin homology (PH) domain, KIF1A or KIF1Bbeta, attenuated the rate of p75 exit from the Golgi in non-polarized MDCK cells but not in polarized cells. Analysis of p75 post-Golgi transport in cells expressing KIF1A or KIF1Bbeta with their PH domains deleted revealed that vesicle transport by these motors depends on the PH domains. Furthermore, purified KIF1A and KIF1Bbeta tails interact with p75 vesicles and these interactions require the PH domain. Knockdown of canine KIF1A also inhibited exit of p75 from the Golgi, and this was rescued by expression of human KIF1A. Together these data demonstrate that post-Golgi transport of p75 in non-polarized epithelial cells is mediated by kinesin-3 family motors in a PH-domain-dependent process.

Size sorting of kinesin-driven microtubules with topographical grooves on a chip.

Gliding microtubules (MTs) on a surface coated with kinesin biomolecular motors have been suggested for the development of nanoscale transport systems. In order to establish a sorting function for gliding MTs, events for MTs approaching micro-scale grooves were investigated. MTs longer than the width of grooves fabricated on a Si substrate bridged the grooves (bridging) and many MTs shorter than the groove width almost began to bridge, but returned to the surface that they approached from (guiding). Occurrence probabilities for the events were analyzed with focus on the geometric conditions, such as length of the MTs, width of the grooves, and the incident angle (alpha) of the MTs approaching the grooves. The occurrence probability for bridging increased with an increase in the incident angle (16%, alpha = 0-30 degrees; 51%, alpha = 30-60 degrees; 75%, alpha = 60-90 degrees), and the probability for guiding decreased with an increase in the incident angle (79%, alpha = 0-30 degrees; 55%, alpha = 30-60 degrees; 5%, alpha = 60-90 degrees). The results indicate that an incident angle of 30-60 degrees is an effective condition for MT sorting, because the bridging and guiding events can sort MTs that are longer and shorter than the groove widths, respectively. Furthermore, the occurrence probabilities of both bridging and guiding in a higher concentration of methylcellulose (0.5%) increased up to approximately 70% at incident angles of 30-60 degrees, indicating good feasibility for the development of devices for the sorting of MTs on surfaces with topographical grooves.

Analysis of intraflagellar transport in C. elegans sensory cilia.

Cilia are assembled and maintained by intraflagellar transport (IFT), the motor-dependent, bidirectional movement of multiprotein complexes, called IFT particles, along the axoneme. The sensory cilia of Caenorhabditis elegans represent very useful objects for studying IFT because of the availability of in vivo time-lapse fluorescence microscopy assays of IFT and multiple ciliary mutants. In this system there are 60 sensory neurons, each having sensory cilia on the endings of their dendrites, and most components of the IFT machinery operating in these structures have been identified using forward and reverse genetic approaches. By analyzing the rate of IFT along cilia within living wild-type and mutant animals, two anterograde and one retrograde IFT motors were identified, the functional coordination of the two anterograde kinesin-2 motors was established and the transport properties of all the known IFT particle components have been characterized. The anterograde kinesin motors have been heterologously expressed and purified, and their biochemical properties have been characterized using MT gliding and single molecule motility assays. In this chapter, we summarize how the tools of genetics, cell biology, electron microscopy, and biochemistry are being used to dissect the composition and mechanism of action of IFT motors and IFT particles in C. elegans.

Ring-shaped assembly of microtubules shows preferential counterclockwise motion.

In this paper, we reveal that microtubules (MTs), reconstructed from tubulin in vitro in the presence of guanosine-5'-triphosphate (GTP), have a ring or spiral shape on a motor protein-fixed surface, and these MTs show biased motion in the counterclockwise direction. By cross-linking these MTs during the sliding motion, we obtained large ring-shaped MT assemblies, 1 approximately 12.6 microm in diameter. The ratio of the rings rotating in the counterclockwise direction to those rotating in the clockwise direction was approximately 3/1. Under optimized conditions, the ratio was as high as 14/1. Thus, we successfully obtained aggregated MTs with a large hierarchic structure that shows a preferential motion, through a dynamic process in vitro.

Molecular characterization and expression of a novel kinesin which localizes with the kinetoplast in the human pathogen, Leishmania donovani.

Using a variety of molecular and cell biological approaches, we identified, characterized and expressed a novel kinesin, LdK39B in the protozoan pathogen Leishmania donovani. Results of RT-PCR revealed two distinct LdK39B products with different splice leader mini-exon sites, indicative of two potentially mature mRNA transcripts. Analyses indicated that LdK39B had a calculated molecular mass of >261, 327 Da and contained multiple amino acid repeat units. Several GFP-LdK39B fusion constructs were generated and used for episomal-expression in these parasites. Results of confocal and immunoelectron microscopy indicated that the GFP-LdK39B-fusion proteins localized to a region adjacent to the flagellar pocket and the kinetoplast i.e. the mitochondrial-DNA containing organelle that is physically tethered to the flagellar basal bodies. Sub-cellular fractionation results showed that GFP-LdK39B proteins were insoluble in nature and remained tightly associated with purified flagella/kinetoplasts following their extraction with detergent and high salts. Our cumulative results suggest that the LdK39B may play a scaffold-like role in facilitating and maintaining the unique spatial/structural association between the flagellum-basal body-kinetoplast complex in these parasites.

Effect of spastic paraplegia mutations in KIF5A kinesin on transport activity.

Hereditary spastic paraplegia (HSP) is a neurodegenerative disease caused by motoneuron degeneration. It is linked to at least 30 loci, among them SPG10, which causes dominant forms and originates in point mutations in the neuronal Kinesin-1 gene (KIF5A). Here, we investigate the motility of KIF5A and four HSP mutants. All mutations are single amino-acid exchanges and located in kinesin's motor or neck domain. The mutation in the neck (A361V) did not change the gliding properties in vitro, the others either reduced microtubule affinity or gliding velocity or both. In laser-trapping assays, none of the mutants moved more than a few steps along microtubules. Motility assays with mixtures of homodimeric wild-type, homodimeric mutant and heterodimeric wild-type/mutant motors revealed that only one mutant (N256S) reduces the gliding velocity at ratios present in heterozygous patients, whereas the others (K253N, R280C) do not. Attached to quantum dots as artificial cargo, mixtures involving N256S mutants produced slower cargo populations lagging behind in transport, whereas mixtures with the other mutants led to populations of quantum dots that rarely bound to microtubules. These differences indicate that the dominant inheritance of SPG10 is caused by two different mechanisms that both reduce the gross cargo flux, leading to deficient supply of the synapse.