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1.  Reconstitution of dynein transport to the microtubule plus end by kinesin 
eLife  2014;3:e02641.
Cytoplasmic dynein powers intracellular movement of cargo toward the microtubule minus end. The first step in a variety of dynein transport events is the targeting of dynein to the dynamic microtubule plus end, but the molecular mechanism underlying this spatial regulation is not understood. Here, we reconstitute dynein plus-end transport using purified proteins from S. cerevisiae and dissect the mechanism using single-molecule microscopy. We find that two proteins–homologs of Lis1 and Clip170–are sufficient to couple dynein to Kip2, a plus-end-directed kinesin. Dynein is transported to the plus end by Kip2, but is not a passive passenger, resisting its own plus-end-directed motion. Two microtubule-associated proteins, homologs of Clip170 and EB1, act as processivity factors for Kip2, helping it overcome dynein's intrinsic minus-end-directed motility. This reveals how a minimal system of proteins transports a molecular motor to the start of its track.
eLife digest
Eukaryotic cells use transport systems to efficiently move materials from one location to another. Much transport in the cell interior is achieved using molecular motors, which carry cargoes along tracks called microtubules.
Unlike roads of human construction, microtubules are very dynamic. One of their ends (the ‘plus’ end) explores the outskirts of the cell, growing and shrinking through the addition and loss of protein building blocks. The other microtubule end (the ‘minus’ end) typically lies in a hub near the center of the cell.
There are two types of molecular motor that move on microtubules. Kinesin motors move toward the plus end of the microtubule, and dynein motors move in the opposite direction, toward the minus end. But if dynein only moves to the minus end of the microtubule, a problem arises: how would dynein initially reach the plus end of the microtubule and the outskirts of the cell, where it collects cargoes?
Using purified yeast proteins, Roberts et al. reveal that a group of three proteins can solve this problem by transporting dynein to the plus end of the microtubule. The proteins comprise a kinesin motor, and two additional proteins that connect the dynein motor to the kinesin. Imaging the transport process shows that the dynein motor is not a passive passenger: it is able to resist against the kinesin. However, an additional microtubule-associated protein can help the kinesin motor to win this ‘tug of war’, and so the protein complex—including the dynein motor—moves toward the plus end of the microtubule.
PMCID: PMC4046564  PMID: 24916158
dynein; kinesin; microtubule; cytoskeleton; DNA origami; S. cerevisiae
2.  Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains 
eLife  2013;2:e00943.
Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins.
eLife digest
When cells divide, they must also divide their contents. In particular, both ‘mother’ and ‘daughter’ cells require full sets of chromosomes, which must first be duplicated, and then evenly distributed between the cells. Protein filaments called microtubules form a network that helps to accurately segregate the chromosomes. Microtubules emanate from structures at each end of the dividing cell known as spindle poles; after the chromosomes have duplicated, the microtubules latch onto them and align the pairs in the middle of the cell. As the two cells separate, microtubules at opposite spindle poles reel in one chromosome from each pair.
Microtubules are composed of alternating copies of two different types of a protein called tubulin, and have ends with distinct properties. The ‘minus’ ends are directed outwards, away from the chromosomes; the ‘plus’ ends—which can actively add tubulin—grow toward the middle of the cell, and can also bind to chromosomes. Microtubules can be manipulated by motor proteins that ‘walk’ along them carrying cargoes, which can include other microtubules. The combined actions of many motor proteins rearrange the microtubule network into a configuration that enables the chromosomes, and other cellular structures, to partition equally between the mother and daughter cells.
Motor proteins such as dynein and kinesin transport cargoes along microtubules; each motor is composed of two identical copies of the protein bound to each other. Kinesin walks toward the plus end of a microtubule, propelling itself using ‘feet’ that are called motor domains; it binds cargoes (including other microtubules) through additional regions located at the opposite end of the protein. In contrast, dynein walks toward the minus end of a microtubule. Although dynein is known to carry certain cargoes through regions outside its motor domain, how it transports other microtubules is not well understood.
Tanenbaum et al. now show that regions outside the motor domain of dynein are unnecessary to transport microtubule cargoes. When two dynein motor domains are isolated and linked to each other in vitro, each can bind to a separate microtubule. By walking toward the minus ends of their respective microtubules, the motor domains drive the microtubules in opposite directions, sliding them apart. These studies thus provide insight into the mechanism through which dynein works with additional motor proteins (such as kinesin) to rearrange microtubules during cell division—and also to ensure that chromosomes segregate evenly between mother and daughter cells.
PMCID: PMC3762337  PMID: 24015359
dynein; spindle; microtubule; Eg5; cytoskeleton; mitosis; Human; S. cerevisiae
3.  The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors 
PLoS Computational Biology  2012;8(11):e1002783.
Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (φ-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain.
Author Summary
Proteins belonging to the kinesin superfamily are responsible for vesicle or organelle transport, spindle morphogenesis, and chromosome sorting during cell division. Interestingly, while most proteins in kinesin superfamily that share the common catalytic motor head domain have plus-end directionality along microtubules, kinesin-14 (Ncd) exhibits minus-end directionality. Despite the several circumstantial evidences on the determining factors for the motor directionality in the last decade, a comprehensive understanding of the mechanism governing the Ncd minus-end directionality is still missing. Our studies provide a clear explanation for this minus-end directionality and the associated mechanochemical cycle. Here, we modeled an Ncd motor by employing structural details available in the literature to simulate its conformational dynamics. Simulations using our structure-based model of Ncd assert that the dynamics due to a simple rearrangement of structural elements, peripheral to the catalytic motor domain, is the key player in determining both the directionality and mechanochemistry unique to Ncd motors. Although Ncd has a different directionality, it uses a similar strategy to kinesin-1 of structural adaptation of the catalytic motor domain. Therefore using the same physical principle of protein folding and very similar structural elements, motors in the kinesin superfamily are able to achieve a variety of biological function.
PMCID: PMC3499263  PMID: 23166486
4.  Electrostatically Biased Binding of Kinesin to Microtubules 
PLoS Biology  2011;9(11):e1001207.
An electrostatic field rotates, slides, and guides the kinesin head to bind the microtubule at a site a short distance ahead, thus determining the direction of movement of the motor.
The minimum motor domain of kinesin-1 is a single head. Recent evidence suggests that such minimal motor domains generate force by a biased binding mechanism, in which they preferentially select binding sites on the microtubule that lie ahead in the progress direction of the motor. A specific molecular mechanism for biased binding has, however, so far been lacking. Here we use atomistic Brownian dynamics simulations combined with experimental mutagenesis to show that incoming kinesin heads undergo electrostatically guided diffusion-to-capture by microtubules, and that this produces directionally biased binding. Kinesin-1 heads are initially rotated by the electrostatic field so that their tubulin-binding sites face inwards, and then steered towards a plus-endwards binding site. In tethered kinesin dimers, this bias is amplified. A 3-residue sequence (RAK) in kinesin helix alpha-6 is predicted to be important for electrostatic guidance. Real-world mutagenesis of this sequence powerfully influences kinesin-driven microtubule sliding, with one mutant producing a 5-fold acceleration over wild type. We conclude that electrostatic interactions play an important role in the kinesin stepping mechanism, by biasing the diffusional association of kinesin with microtubules.
Author Summary
Animal and plant cells contain a molecular-scale “railway” network, in which the tracks, called microtubules, radiate out from the cell centre and locomotive proteins, called kinesins, haul their molecular cargoes along the microtubule tracks. This railway system transports many different cargoes to where they are needed, so it is crucial for the cell's organization and function. Breakdowns in this transport system can cause diseases like Alzheimer's, and drugs that temporarily halt transport make powerful anti-cancer agents. Precisely how kinesin motor proteins move along their microtubule tracks is an important question in biology. We know that some kinesins have twin “heads” that alternately bind to and step along microtubules in a coordinated walking action. But more usually, kinesins have only one head. How single-headed kinesins produce force and movement is poorly understood. In this study, we address this question and show that electrical attraction between single kinesin heads and microtubules is a critical factor deciding the direction of movement: each time the head approaches a microtubule, it slides forwards by the electrical attraction between the engine and the track.
PMCID: PMC3226556  PMID: 22140358
5.  Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2 
Molecular Biology of the Cell  2012;23(7):1254-1266.
The kinesin Eg5 moves toward minus ends of astral microtubules in early mitosis, switching to plus-end motion in anaphase. Dynein is required for minus-end motion; depletion of TPX2 results in a switch to plus-end motion. On midzone microtubules, Eg5 moves in both directions. Our results explain the redistribution of Eg5 throughout mitosis.
Kinesin-5 is an essential mitotic motor. However, how its spatial–temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification–tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end–directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end–directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.
PMCID: PMC3315814  PMID: 22337772
6.  Kinesin-1 regulates dendrite microtubule polarity in Caenorhabditis elegans 
eLife  2013;2:e00133.
In neurons, microtubules (MTs) span the length of both axons and dendrites, and the molecular motors use these intracellular ‘highways' to transport diverse cargo to the appropriate subcellular locations. Whereas axonal MTs are organized such that the plus-end is oriented out from the cell body, dendrites exhibit a mixed MTs polarity containing both minus-end-out and plus-end-out MTs. The molecular mechanisms underlying this differential organization, as well as its functional significance, are unknown. Here, we show that kinesin-1 is critical in establishing the characteristic minus-end-out MT organization of the dendrite in vivo. In unc-116 (kinesin-1/kinesin heavy chain) mutants, the dendritic MTs adopt an axonal-like plus-end-out organization. Kinesin-1 protein is able to cross-link anti-paralleled MTs in vitro. We propose that kinesin-1 regulates the dendrite MT polarity through directly gliding the plus-end-out MTs out of the dendrite using both the motor domain and the C-terminal MT-binding domain.
eLife digest
Neurons, or nerve cells, are excitable cells that transmit information using electrical and chemical signals. Nerve cells are generally composed of a cell body, multiple dendrites, and a single axon. The dendrites are responsible for receiving inputs and for transferring these signals to the cell body, whereas the axon carries signals away from the cell body and relays them to other cells.
Like all cells, nerve cells have a cytoskeleton made up of microtubules, which help to determine cellular shape and which act as ‘highways' for intracellular transport. Microtubules are long hollow fibers composed of alternating α- and β-tubulin proteins: each microtubule has a ‘plus'-end, where the β subunits are exposed, and a ‘minus'-end, where the α subunits are exposed. Nerve cells are highly polarized: within the axon, the microtubules are uniformly oriented with their plus-ends pointing outward, whereas in dendrites, there are many microtubules with their minus-ends pointing outward. This arrangement is conserved across the animal kingdom, but the mechanisms that establish it are largely unknown.
Yan et al. use the model organism Caenorhabditis elegans (the nematode worm) to conduct a detailed in vivo analysis of dendritic microtubule organization. They find that a motor protein called kinesin-1 is critical for generating the characteristic minus-end-out pattern in dendrites: when the gene that codes for this protein is knocked out, the dendrites in microtubules undergo a dramatic polarity shift and adopt the plus-end-out organization that is typical of axons. The mutant dendrites also show other axon-like features: for example, they lack many of the proteins that are usually found in dendrites. Based on these and other data, Yan et al. propose that kinesin-1 determines microtubule polarity in dendrites by moving plus-end-out microtubules out of dendrites.
These first attempts to explain, at the molecular level, how dendritic microtubule polarity is achieved in vivo could lead to new insights into the structure and function of the neuronal cytoskeleton.
PMCID: PMC3591006  PMID: 23482306
polarity; cytoskeleton; kinesin-1; C. elegans
7.  Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei 
PLoS Pathogens  2010;6(8):e1001050.
Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target.
Author Summary
Kinesins represent a class of mechanochemical enzymes that are able to move along microtubule filaments and transport cargo in a directional manner within the cell. Of particular importance are mitotic kinesins, as they ensure the accurate segregation of chromosomes and therefore cell survival. Such kinesins are involved in building and maintaining the mitotic microtubule-based spindle and in chromosome translocation during mitosis. Mitotic kinesins are potentially excellent drug targets because of their roles in an essential process of cell multiplication. Unregulated cell proliferation is associated with diseases such as cancer, but also many infectious diseases. Therefore, the identification of kinesins essential for the proliferation of parasites in the human host offers an attractive prospect for intervention. In our study we present a comprehensive biochemical and cell biological analysis of a mitotic kinesin in the protozoan parasite Trypanosoma brucei, causative agent of sleeping sickness in Africa. We show that this kinesin is essential for parasite survival not only in cultured cells but also in mice infected with this parasite and therefore establish this kinesin as a potential drug target in parasitic infections.
PMCID: PMC2924347  PMID: 20808899
8.  Chromosome congression by kinesin-5 motor-mediated disassembly of longer kinetochore microtubules 
Cell  2008;135(5):894-906.
During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.
PMCID: PMC2683758  PMID: 19041752
9.  Molecular Requirements for Bi-directional Movement of Phagosomes Along Microtubules 
The Journal of Cell Biology  1997;137(1):113-129.
Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with “late” phagosomes moving threefold more frequently than “early” ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.
PMCID: PMC2139871  PMID: 9105041
10.  Microtubule cross-linking triggers the directional motility of kinesin-5 
The Journal of Cell Biology  2008;182(3):421-428.
Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)–dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.
PMCID: PMC2500128  PMID: 18678707
11.  B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast 
PLoS Genetics  2010;6(5):e1000935.
Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.
Author Summary
The assembly of a bipolar mitotic spindle is essential for the accurate segregation of sister chromatids during mitosis and, hence, for successful cell division. Spindle assembly depends on the successful duplication of the spindle poles, followed by their separation to opposing ends of the cell. Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle, the relevant CDK targets have not been identified. Motor proteins of the kinesin-5 family generate movement on the microtubules that make up the spindle and are believed to power spindle pole separation. By employing the budding yeast Saccharomyces cerevisiae as a model, we have found evidence that cyclin/CDKs control spindle assembly by phosphorylating the kinesins-5 Kip1 and Cin8. When phosphorylation at a conserved CDK site in the motor domain of Kip1 is blocked, spindle pole separation is greatly diminished but neither protein abundance nor localization is affected. We have also obtained direct evidence by mass spectrometry that Kip1 and Cin8 are phosphorylated in vivo at consensus CDK sites in their tail domains. Our findings suggest that B-cyclin/CDKs regulate spindle assembly by regulating kinesin-5 motor activity.
PMCID: PMC2865516  PMID: 20463882
12.  Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors 
PLoS Biology  2009;7(10):e1000216.
Molecular motors differentially recognize and move cargo along discrete microtubule subpopulations in cells, resulting in preferential transport and targeting of subcellular cargoes.
Cells generate diverse microtubule populations by polymerization of a common α/β-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.
Author Summary
Eukaryotic cells assemble a variety of cytoskeletal structures from a set of highly conserved building blocks. For example, all microtubules are generated by the polymerization of a common α/β-tubulin subunit, yet cells can contain diverse, discrete populations of microtubule structures such as axonemes, spindles, and radial arrays. This diversity must be read and translated by cellular components in order to carry out population-specific functions. We use single-molecule imaging to study how molecular motors navigate the heterogeneous microtubule populations present in interphase cells. We show that different kinesin motors select different subpopulations of microtubules for transport. This selectivity, based solely on the motor-microtubule interface, may enable kinesin motors to segregate transport events to distinct microtubule populations and thus to target cargoes to specific subcellular destinations.
PMCID: PMC2749942  PMID: 19823565
13.  Role of a Class Dhc1b Dynein in Retrograde Transport of Ift Motors and Ift Raft Particles along Cilia, but Not Dendrites, in Chemosensory Neurons of Living Caenorhabditis elegans 
The Journal of Cell Biology  1999;147(3):519-530.
The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 μm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope–based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at ∼1.1 μm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end–directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.
PMCID: PMC2151193  PMID: 10545497
dynein; kinesin; intraflagellar transport; Caenorhabditis elegans; neuron transport
14.  Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry 
PLoS Biology  2010;8(4):e1000350.
Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis.
BICD2 is one of the two mammalian homologues of the Drosophila Bicaudal D, an evolutionarily conserved adaptor between microtubule motors and their cargo that was previously shown to link vesicles and mRNP complexes to the dynein motor. Here, we identified a G2-specific role for BICD2 in the relative positioning of the nucleus and centrosomes in dividing cells. By combining mass spectrometry, biochemical and cell biological approaches, we show that the nuclear pore complex (NPC) component RanBP2 directly binds to BICD2 and recruits it to NPCs specifically in G2 phase of the cell cycle. BICD2, in turn, recruits dynein-dynactin to NPCs and as such is needed to keep centrosomes closely tethered to the nucleus prior to mitotic entry. When dynein function is suppressed by RNA interference-mediated depletion or antibody microinjection, centrosomes and nuclei are actively pushed apart in late G2 and we show that this is due to the action of kinesin-1. Surprisingly, depletion of BICD2 inhibits both dynein and kinesin-1-dependent movements of the nucleus and cytoplasmic NPCs, demonstrating that BICD2 is needed not only for the dynein function at the nuclear pores but also for the antagonistic activity of kinesin-1. Our study demonstrates that the nucleus is subject to opposing activities of dynein and kinesin-1 motors and that BICD2 contributes to nuclear and centrosomal positioning prior to mitotic entry through regulation of both dynein and kinesin-1.
Author Summary
Bidirectional microtubule-based transport is responsible for the positioning of a large variety of cellular organelles, but the molecular mechanisms underlying the recruitment of microtubule-based motors to their cargoes and their activation remain poorly understood. In particular, the molecular players involved in the important processes of nuclear and centrosomal positioning prior to the onset of cell division are not known. In this study we focus on the function of one of the mammalian homologues of Drosophila Bicaudal D, an adaptor for the microtubule minus-end-directed dynein-dynactin motor complex. Previously, Drosophila Bicaudal D and its mammalian homologues were shown to act as linkers between the dynein motor and mRNP complexes or secretory vesicles. Here, we identify a new cargo for mammalian Bicaudal D2 (BICD2)–the nucleus. We show that BICD2 specifically binds to nuclear pore complexes in cells in G2 phase of the cell division cycle. We also show that this interaction is required for G2-specific recruitment of dynein to the nuclear envelope and thus for proper positioning of the nucleus relative to centrosomes prior to the onset of mitosis. Further, our findings demonstrate that the motor protein kinesin-1 opposes dynein's activity during this process and requires BICD2 for its activity. Our study therefore reveals BICD2 as the critical molecular adaptor that allows molecular motors to regulate nuclear and centrosomal positioning before cell division.
PMCID: PMC2850381  PMID: 20386726
15.  Structure-based Molecular Simulations Reveal the Enhancement of Biased Brownian Motions in Single-headed Kinesin 
PLoS Computational Biology  2013;9(2):e1002907.
Kinesin is a family of molecular motors that move unidirectionally along microtubules (MT) using ATP hydrolysis free energy. In the family, the conventional two-headed kinesin was experimentally characterized to move unidirectionally through “walking” in a hand-over-hand fashion by coordinated motions of the two heads. Interestingly a single-headed kinesin, a truncated KIF1A, still can generate a biased Brownian movement along MT, as observed by in vitro single molecule experiments. Thus, KIF1A must use a different mechanism from the conventional kinesin to achieve the unidirectional motions. Based on the energy landscape view of proteins, for the first time, we conducted a set of molecular simulations of the truncated KIF1A movements over an ATP hydrolysis cycle and found a mechanism exhibiting and enhancing stochastic forward-biased movements in a similar way to those in experiments. First, simulating stand-alone KIF1A, we did not find any biased movements, while we found that KIF1A with a large friction cargo-analog attached to the C-terminus can generate clearly biased Brownian movements upon an ATP hydrolysis cycle. The linked cargo-analog enhanced the detachment of the KIF1A from MT. Once detached, diffusion of the KIF1A head was restricted around the large cargo which was located in front of the head at the time of detachment, thus generating a forward bias of the diffusion. The cargo plays the role of a diffusional anchor, or cane, in KIF1A “walking.”
Author Summary
It is one of the major issues in biophysics how molecular motors such as conventional two-headed kinesin convert the chemical energy released at ATP hydrolysis into mechanical work. While most molecular motors move with more than one catalytic domain working in coordinated fashions, there are some motors that can move with only a single catalytic domain, which provides us a possibly simpler case to understand. A single-headed kinesin, KIF1A, with only one catalytic domain, has been characterized by in vitro single-molecule assay to generate a biased Brownian movement along the microtubule. Here, we conducted a set of structure-based coarse-grained molecular simulations for KIF1A system over an ATP hydrolysis cycle for the first time to our knowledge. Without cargo the simulated stand-alone KIF1A could not generate any directional movement, while a large-friction cargo-analog linked to the C-terminus of KIF1A clearly enhanced the forward-biased Brownian movement of KIF1A significantly. Interestingly, the cargo-analog here is not merely load but an important promoter to enable efficient movements of KIF1A.
PMCID: PMC3572960  PMID: 23459019
16.  Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins 
The Journal of Cell Biology  1992;119(6):1589-1596.
Kinesin and dynein are motor proteins that move in opposite directions along microtubules. In this study, we examine the consequences of having kinesin and dynein (ciliary outer arm or cytoplasmic) bound to glass surfaces interacting with the same microtubule in vitro. Although one might expect a balance of opposing forces to produce little or no net movement, we find instead that microtubules move unidirectionally for several microns (corresponding to hundreds of ATPase cycles by a motor) but continually switch between kinesin-directed and dynein- directed transport. The velocities in the plus-end (0.2-0.3 microns/s) and minus-end (3.5-4 microns/s) directions were approximately half those produced by kinesin (0.5 microns/s) and ciliary dynein (6.7 microns/s) alone, indicating that the motors not contributing to movement can interact with and impose a drag upon the microtubule. By comparing two dyneins with different duty ratios (percentage of time spent in a strongly bound state during the ATPase cycle) and varying the nucleotide conditions, we show that the microtubule attachment times of the two opposing motors as well as their relative numbers determine which motor predominates in this assay. Together, these findings are consistent with a model in which kinesin-induced movement of a microtubule induces a negative strain in attached dyneins which causes them to dissociate before entering a force-generating state (and vice versa); reversals in the direction of transport may require the temporary dissociation of the transporting motor from the microtubule. The bidirectional movements described here are also remarkably similar to the back-and-forth movements of chromosomes during mitosis and membrane vesicles in fibroblasts. These results suggest that the underlying mechanical properties of motor proteins, at least in part, may be responsible for reversals in microtubule-based transport observed in cells.
PMCID: PMC2289742  PMID: 1469050
17.  Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules 
The Journal of Cell Biology  1996;135(2):383-397.
Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)- induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.
PMCID: PMC2121042  PMID: 8896596
18.  Posterior Localization of Dynein and Dorsal-Ventral Axis Formation Depend on Kinesin in Drosophila Oocytes 
Current biology : CB  2002;12(17):1541-1545.
To establish the major body axes, late Drosophila oocytes localize determinants to discrete cortical positions: bicoid mRNA to the anterior cortex, oskar mRNA to the posterior cortex, and gurken mRNA to the margin of the anterior cortex adjacent to the oocyte nucleus (the “anterodorsal corner”) [1–3]. These localizations depend on microtubules [4–7] that are thought to be organized such that plus end-directed motors can move cargoes, like oskar, away from the anterior/lateral surfaces and hence toward the posterior pole [8–10]. Likewise, minus end-directed motors may move cargoes toward anterior destinations [6, 11–13]. Contradicting this, cytoplasmic dynein, a minus-end motor, accumulates at the posterior [14]. Here, we report that disruption of the plus-end motor kinesin I causes a shift of dynein from posterior to anterior. This provides an explanation for the dynein paradox, suggesting that dynein is moved as a cargo toward the posterior pole by kinesin-generated forces. However, other results present a new transport polarity puzzle. Disruption of kinesin I causes partial defects in anterior positioning of the nucleus and severe defects in anterodorsal localization of gurken mRNA. Kinesin may generate anterodorsal forces directly, despite the apparent preponderance of minus ends at the anterior cortex. Alternatively, kinesin I may facilitate cytoplasmic dynein-based anterodorsal forces by repositioning dynein toward microtubule plus ends.
PMCID: PMC3209760  PMID: 12225672
19.  A kinesin motor in a force-producing conformation 
Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states.
Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central β-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1.
Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.
PMCID: PMC2906495  PMID: 20602775
20.  Novel Dendritic Kinesin Sorting Identified by Different Process Targeting of Two Related Kinesins: KIF21A and KIF21B  
The Journal of Cell Biology  1999;145(3):469-479.
Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.
PMCID: PMC2185086  PMID: 10225949
kinesin; protein sorting; WD-40 repeats; neuron transport; dendrite
21.  Structural basis for the assembly of the mitotic motor Kinesin-5 into bipolar tetramers 
eLife  2014;3:e02217.
Chromosome segregation during mitosis depends upon Kinesin-5 motors, which display a conserved, bipolar homotetrameric organization consisting of two motor dimers at opposite ends of a central rod. Kinesin-5 motors crosslink adjacent microtubules to drive or constrain their sliding apart, but the structural basis of their organization is unknown. In this study, we report the atomic structure of the bipolar assembly (BASS) domain that directs four Kinesin-5 subunits to form a bipolar minifilament. BASS is a novel 26-nm four-helix bundle, consisting of two anti-parallel coiled-coils at its center, stabilized by alternating hydrophobic and ionic four-helical interfaces, which based on mutagenesis experiments, are critical for tetramerization. Strikingly, N-terminal BASS helices bend as they emerge from the central bundle, swapping partner helices, to form dimeric parallel coiled-coils at both ends, which are offset by 90°. We propose that BASS is a mechanically stable, plectonemically-coiled junction, transmitting forces between Kinesin-5 motor dimers during microtubule sliding.
eLife digest
Successful cell division requires copies of the chromosomes containing the genetic material of a cell to be accurately copied and then separated so that when a cell divides, each new daughter cell contains exactly one copy of each chromosome. If this does not happen, the cell may malfunction or die.
To separate the duplicated chromosomes, a biological machine called the mitotic spindle forms inside the cell. This has two poles, one at each end, with each pole being responsible for gathering together the chromosomes for delivery to each of the daughter cells. Large numbers of long, thin protein tubes called microtubules extend out of each pole. Some microtubules attach to the chromosomes, whilst others are responsible for pushing apart the two poles—and the chromosomes attached to them—to the opposite sides of the cell before it divides.
To move the poles, motor proteins slide pairs of microtubules that are attached to opposite poles over each other. The Kinesin-5 family of motor proteins is particularly important for mitosis, because it is essential for forming the mitotic spindle and for making it work correctly. These motors assemble into motile machines that can apply a force to both of the microtubules in a sliding pair at the same time because they contain motor units at each end connected by a central rod.
The structure of this central rod is crucial for the successful operation of Kinesin-5. Scholey, Nithianantham et al. have now worked out the structure of a region of this filament called the bipolar assembly, or BASS domain. This structure is more complicated than expected: it contains four helixes made of protein that are all intertwined with each other.
In addition, Scholey, Nithianantham et al. found two ‘molecular pockets’ that small molecules can access. By entering the pockets, the molecules could disrupt the structure of the BASS domain, and consequently prevent Kinesin-5 from forming the dual-ended machines required to work properly. As Kinesin-5 is required to build the mitotic spindle, this would interfere with cell division. Targeting molecules into these pockets could therefore potentially form part of an anti-cancer therapy, preventing the rapid cell divisions behind the spread of the disease.
PMCID: PMC3978770  PMID: 24714498
mitosis; Kinesin-5; microtubule; motor protein; coiled-coil; X-ray structure; D. melanogaster
22.  The influence of dynein processivity control, MAPs, and microtubule ends on directional movement of a localising mRNA 
eLife  2014;3:e01596.
Many cellular constituents travel along microtubules in association with multiple copies of motor proteins. How the activity of these motors is regulated during cargo sorting is poorly understood. In this study, we address this issue using a novel in vitro assay for the motility of localising Drosophila mRNAs bound to native dynein-dynactin complexes. High precision tracking reveals that individual RNPs within a population undergo either diffusive, or highly processive, minus end-directed movements along microtubules. RNA localisation signals stimulate the processive movements, with regulation of dynein-dynactin’s activity rather than its total copy number per RNP, responsible for this effect. Our data support a novel mechanism for multi-motor translocation based on the regulation of dynein processivity by discrete cargo-associated features. Studying the in vitro responses of RNPs to microtubule-associated proteins (MAPs) and microtubule ends provides insights into how an RNA population could navigate the cytoskeletal network and become anchored at its destination in cells.
eLife digest
For a cell to do its job, the different components inside it need to be moved to different locations. This is achieved by an elaborate cellular transport system. To move a component to where it needs to be, motor proteins bind to it, often with the assistance of other ‘accessory’ proteins. This cargo-motor complex then moves along a network of tracks within the cell. Viruses also exploit this transport system in order to be trafficked to specific parts of the cell during their life cycles.
Many cargos are moved along microtubule tracks. Multiple microtubule motor proteins often attach to the same cargo, but it is unclear how they work together during transport. Previous studies have attempted to address this issue by attaching motor proteins to artificial cargoes, such as synthetic beads. However, these experiments did not include some of the accessory proteins that are thought to play a role during transport within the living cell.
Soundararajan and Bullock have now examined how complexes containing multiple motors bound to accessory proteins move molecules of messenger RNA to specific sites within cells. By visualising fruit fly mRNA moving along microtubules attached to a glass surface, the transport process can be studied in detail. It appears that the complexes travel using one of two methods: they either diffuse along the microtubules, which they can do in either direction, or they power themselves along the microtubules, which they can only do in one direction. Although previous experiments with artificial cargos suggested that the number of motors in the complex determines the likelihood of one-way traffic, it appears that one or more accessory proteins are actually in control during mRNA transport.
Soundararajan and Bullock also documented how the mRNA-motor complexes react to roadblocks and dead-ends on the microtubule highway. Rather than letting go of the microtubule upon such an encounter, the complexes can reverse back down the track. This behaviour may help them to find a new route to their destination.
PMCID: PMC3985186  PMID: 24737859
molecular motors; mRNA localisation; in vitro transport; microtubules; D. melanogaster
23.  Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures 
PLoS Pathogens  2010;6(7):e1000991.
Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly.
Author Summary
Many viruses, particularly neurotropic alphaherpesviruses such as herpes simplex virus (HSV), require an intact microtubule network for efficient replication and pathogenesis. In living cells, host and viral cargo show rapid reversals in transport direction, suggesting that they can recruit motors of opposing directionality simultaneously. To elucidate the molecular mechanisms for specific motor-cargo recognition, it is necessary to characterize the surface of such cargos. We established a cell-free system that reconstitutes the binding of native, mammalian microtubule motors to intact tegumented HSV capsids. Our data suggest that the inbound motor dynein and the outbound motor kinesin-1 bind directly and independently of other host factors to the inner tegument that coats the capsids during cytosolic transport. Identifying viral receptors for the hosts' transport machinery will provide us on the one hand with new potential targets for antiviral therapy. On the other hand, such viral protein domains could be added to viral vectors or even to artificial nano carriers designed to deliver therapeutic genes or molecules to the nucleus or other subcellular destinations.
PMCID: PMC2900298  PMID: 20628567
24.  Kinesin is bound with high affinity to squid axon organelles that move to the plus-end of microtubules 
The Journal of Cell Biology  1992;119(2):389-399.
This paper addresses the question of whether microtubule-directed transport of vesicular organelles depends on the presence of a pool of cytosolic factors, including soluble motor proteins and accessory factors. Earlier studies with squid axon organelles (Schroer et al., 1988) suggested that the presence of cytosol induces a > 20-fold increase in the number of organelles moving per unit time on microtubules in vitro. These earlier studies, however, did not consider that cytosol might nonspecifically increase the numbers of moving organelles, i.e., by blocking adsorption of organelles to the coverglass. Here we report that treatment of the coverglass with casein, in the absence of cytosol, blocks adsorption of organelles to the coverglass and results in vigorous movement of vesicular organelles in the complete absence of soluble proteins. This technical improvement makes it possible, for the first time, to perform quantitative studies of organelle movement in the absence of cytosol. These new studies show that organelle movement activity (numbers of moving organelles/min/micron microtubule) of unextracted organelles is not increased by cytosol. Unextracted organelles move in single directions, approximately two thirds toward the plus-end and one third toward the minus-end of microtubules. Extraction of organelles with 600 mM KI completely inhibits minus-end, but not plus-end directed organelle movement. Upon addition of cytosol, minus-end directed movement of KI organelles is restored, while plus--end directed movement is unaffected. Biochemical studies indicate that KI-extracted organelles attach to microtubules in the presence of AMP-PNP and copurify with tightly bound kinesin. The bound kinesin is not extracted from organelles by 1 M KI, 1 M NaCl or carbonate (pH 11.3). These results suggest that kinesin is irreversibly bound to organelles that move to the plus-end of microtubules and that the presence of soluble kinesin and accessory factors is not required for movement of plus-end organelles in squid axons.
PMCID: PMC2289649  PMID: 1400582
25.  Kinesin-13 Regulates Flagellar, Interphase, and Mitotic Microtubule Dynamics in Giardia intestinalis▿ †  
Eukaryotic Cell  2007;6(12):2354-2364.
Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.
PMCID: PMC2168246  PMID: 17766466

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