MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.     

A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.     

Subcellular motility: A correlated light and electron microscopic study using cultured cells

To assess the possible role of filaments in subcellular motility, particular cultured cells were studied by light and electron microscopy. Motile cell margins always contained meshworks of ~50 Å diam. filaments. Organelles moved within cytoplasm occupied by a meshwork of 50–100 Å filaments and microtubules. When cells were treated with cytochalasin B, movements of cell margins stopped, but organelle movements continued; electron microscopically, while subplasmalemmal filaments had disappeared, subcortical filaments and microtubules remained. When cells were treated with hypertonic medium, organelle movements ceased but marginal movements continued; electron microscopically, although cell margins contained normal filament arrays, few subcortical filaments remained. It is concluded that while cell margins are moved by a meshwork of filaments, organelle movement is mediated by a subcortical meshwork of filaments and microtubules.     

F-actin involvement in guinea pig sperm motility

Sperm motility is a must for natural fertilization to occur. During their travel through the epididymis, mammalian spermatozoa gradually acquire the ability to move. This is accomplished through a sliding movement of the outer doublet microtubules of the axoneme which is energized by the dynein ATPase. Within its complex structure, the mammalian sperm flagellum contains F-actin and thus, we decided to test in the guinea pig sperm flagellum the role of F-actin in motility. During maturation, capacitation, and the acrosome reaction, a gradual decrease of the relative concentration of F-actin was observed. Motility increased as spermatozoa became able to fertilize. Gelsolin, phalloidin, and KI inhibited sperm motility. Gelsolin canceled sperm motility within 20 min of treatment while 0.6 M KI had immediate effects. Phalloidin diminished hyperactive sperm motility slightly. All three compounds significantly increased the relative concentration of F-actin. Latrunculins are conventional drugs that destabilize the F-actin cytoskeleton. Latrunculin A (LAT A) did not affect sperm motility; but significantly increased F-actin relative concentration. The results suggested that in guinea pig spermatozoa, randomly severing F-actin filaments inhibits flagellar motility; while end filament alteration does not. Thus, specific filament regions seem to be important for sperm motility.     

Phosphorylation of Tetrahymena 22 S dynein

Studies involving 32P labeling and wet ashing of isolated dynein reveal that isolated dynein contains approximately 6 mol of phosphate predominantly distributed over four polypeptides of molecular masses of 78, 76, 47, and 23 kDa. Dynein must, therefore, be phosphorylated to at least this extent in vivo. The catalytic subunit of cAMP-dependent protein kinase and an axonemal cAMP-dependent protein kinase contaminating the dynein preparation can further phosphorylate dynein in vitro. Each kinase can place up to 0.5 mol of phosphate on native dynein polypeptides of molecular masses of 78 and 34 kDa. Removal of two of the phosphates on isolated dynein by either acid or alkaline phosphatase results in a 28% decrease in the specific activity of dynein in the presence or absence of microtubules. Selective attenuation of the microtubule-activated ATPase, but not the uncoupled free dynein ATPase, would be indicative of a regulatory function of the phosphates. The in vivo regulation of the dynein ATPase by the two phosphates accessible to acid or alkaline phosphatase is therefore subject to question. Other phosphates on dynein must be examined for their effect on the microtubule-dynein cross-bridge cycle and motility before phosphorylation can definitively be established as a mode of dynein regulation.     

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either tracking dynamic microtubule plus‐ends in an EB‐dependent manner or moving processively towards minus ends in an adaptor protein‐dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal‐D2 (BicD2) or the multifunctional regulator Lissencephaly‐1 (Lis1). BicD2 and Lis1 together control the overall efficiency of motility initiation. Remarkably, dynactin can bias motility initiation locally from microtubule plus ends by autonomous plus‐end recognition. This bias is further enhanced by EBs and Lis1. Our study provides insight into the mechanism of dynein regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.     

Dynactin increases the processivity of the cytoplasmic dynein motor

Cytoplasmic dynein supports long-range intracellular movements of cargo in vivo but does not appear to be a processive motor protein by itself. We show here that the dynein activator, dynactin, binds microtubules and increases the average length of cytoplasmic-dynein-driven movements without affecting the velocity or microtubule-stimulated ATPase kinetics of cytoplasmic dynein. Enhancement of microtubule binding and motility by dynactin are both inhibited by an antibody to dynactin's microtubule-binding domain. These results indicate that dynactin acts as a processivity factor for cytoplasmic-dynein-based motility and provide the first evidence that cytoskeletal motor processivity can be affected by extrinsic factors.     

Evidence for cooperative interactions between the two motor domains of cytoplasmic dynein.

Cytoplasmic dynein is a force-transducing ATPase that powers the movement of cellular cargoes along microtubules. Two identical heavy chain polypeptides (> 500 kDa) of the cytoplasmic dynein complex contain motor domains that possess the ATPase and microtubule-binding activities required for force production [1]. It is of great interest to determine whether both heavy chains (DHCs) in the dynein complex are required for progression of the mechanochemical cycle and motility, as observed for other dimeric motors. We have used transgenic constructs to investigate cooperative interactions between the two motor domains of the Drosophila cytoplasmic dynein complex. We show that 138 kDa and 180 kDa amino-terminal fragments of DHC can assemble with full-length DHC to form heterodimeric complexes containing only a single motor domain. The single-headed dynein complexes can bind and hydrolyze ATP, yet do not show the ATP-induced detachment from microtubules that is characteristic of wild-type homodimeric dynein. These results suggest that cooperative interactions between the monomeric units of the dimer are required for efficient ATP-induced detachment of dynein and unidirectional movement along the microtubule.     

Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport

Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ～1.0 μm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.     

Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins

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.     

Redundant mechanisms for anaphase chromosome movements: crane-fly spermatocyte spindles normally use actin filaments but also can function without them

Summary. Actin inhibitors block or slow anaphase chromosome movements in crane-fly spermatocytes, but stopping of movement is only temporary; we assumed that cells adapt to loss of actin by switching to mechanism(s) involving only microtubules. To test this, we produced actin-filament-free spindles: we added latrunculin B during prometaphase, 9–80 min before anaphase, after which chromosomes generally moved normally during anaphase. We confirmed the absence of actin filaments by staining with fluorescent phalloidin and by showing that cytochalasin D had no effect on chromosome movement. Thus, actin filaments are involved in normal anaphase movements, but in vivo, spindles nonetheless can function normally without them. We tested whether chromosome movements in actin-filament-free spindles arise via microtubules by challenging such spindles with anti-myosin drugs. Y-27632 and BDM (2,3-butanedione monoxime), inhibitors that affect myosin at different regulatory levels, blocked chromosome movement in normal spindles and in actin-filament-free spindles. We tested whether BDM has side effects on microtubule motors. BDM had no effect on ciliary and sperm motility or on ATPase activity of isolated ciliary axonemes, and thus it does not directly block dynein. Nor does it block kinesin, assayed by a microtubule sliding assay. BDM could conceivably indirectly affect these microtubule motors, though it is unlikely that it would have the same side effect on the motors as Y-27632. Since BDM and Y-27632 both affect chromosome movement in the same way, it would seem that both affect spindle myosin; this suggests that spindle myosin interacts with kinetochore microtubules, either directly or via an intermediate component. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s00709-005-0094-6 Correspondence and reprints: Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase

Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge- membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.     

A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules

Microtubule-associated proteins (MAPs) use particular microtubule-binding domains that allow them to interact with microtubules in a manner specific to their individual cellular functions. Here, we have identified a highly basic microtubule-binding domain in the p150 subunit of dynactin that is only present in the dynactin members of the CAP-Gly family of proteins. Using single-particle microtubule-binding assays, we found that the basic domain of dynactin moves progressively along microtubules in the absence of molecular motors - a process we term 'skating'. In contrast, the previously described CAP-Gly domain of dynactin remains firmly attached to a single point on microtubules. Further analyses showed that microtubule skating is a form of one-dimensional diffusion along the microtubule. To determine the cellular function of the skating phenomenon, dynein and the dynactin microtubule-binding domains were examined in single-molecule motility assays. We found that the basic domain increased dynein processivity fourfold whereas the CAP-Gly domain inhibited dynein motility. Our data show that the ability of the basic domain of dynactin to skate along microtubules is used by dynein to maintain longer interactions for each encounter with microtubules.     

Cellular organelle transport and positioning by plasma streaming

Our analysis of known data reveals that translocations of passively movable cellular organelles from tiny granules up to large cell nuclei can be ascribed to transport by streaming cytoplasm. The various behaviours, such as velocity changes during more or less interrupted movements, forth and back shuttling and particle rotation result from different types of plasma circulation. Fast movements over long distances, as observed in the large characean internodial cells occur in strong streams generated by myosin in bundles of actin filaments in the direction of the barbed filament ends. Slow movements with frequent reversions of the direction are typical for neuronal axons, in which an anterograde plasma flow, produced in a thin layer of membrane-attached actin filaments, is compensated by a retrograde stream, produced by dynein activity in the central bundle of microtubules. Here particle rotation is due to steep flow velocity gradients, and frequent changes of particle movements result from minor particle displacements in radial directions. Similar shuttling of pigment granules in the lobes of epidermal chromatophores results from the same mechanism, whereby the centrifugal movement along astral microtubules is due to flow generated by excess of kinesin activity and the centripetal movement to the plasma recycling through the intermicrotubular space. If the streaming pattern is reversed by switching to excess dynein activity, the moving granules are trapped in the high microtubule density at the aster center. The presence of larger bodies in asters disturbs the regular, kinesin-dependent microtubule distribution in such a way that a superimposed centrifugal plasma flow develops in the microtubule-dense layer along them, which is recycled in the microtubule-free space, created by their presence. Consequently, at excess kinesin activity, nuclei, mitochondria as well as chromosome fragments move towards the aster center until they reach a dynamically stabilized position that depends on the local microtubule density. These various behaviours are not rationally explainable by models based on a mechanical stepping along microtubules or actin filaments.     

Early endosomes motility in filamentous fungi: How and why they move

Elongate hyphae of filamentous fungi grow predominantly at their tips, whereas organelles are positioned in the subapical parts of the cell. Organelle positioning and long-distance intracellular communication involves active, energy-dependent transport along microtubules (MTs). This is mediated by specialized molecular motors, named kinesins and dynein, which utilize ATP hydrolysis to “walk” along the tubulin polymers. Work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans has shown that early endosomes (EEs) are one of the major cargos of MT-dependent motors in fungi. EEs are part of the early endocytic pathway, and their motility behavior and the underlying transport machinery is well understood. However, the physiological role of constant bi-directional EE motility remains elusive. Recent reports, conducted in the corn smut fungus U. maydis, have provided novel insights into the cellular function of EE motility. They show that EE motility is crucial for the distribution of the protein synthesis machinery, and also that EEs transmit signals during plant infection that trigger the production of fungal effector proteins, required for successful invasion into host plants.     

Molecular motors: from one motor many tails to one motor many tales

Kinesin and dynein molecular motor proteins generate the movement of a wide variety of materials in cells. Such movements are crucial for many different cellular and developmental functions, including organelle movement, localization of developmental determinants, mitosis, meiosis and possibly long-range signaling in neurons. Kinesins that control the dynamics of microtubules have also been discovered. Recent work has begun to identify processes in which defective molecular motor function can cause human disease.     

The mechanism of dynein motility: insight from crystal structures of the motor domain

Dynein is a large cytoskeletal motor protein that belongs to the AAA+ (ATPases associated with diverse cellular activities) superfamily. While dynein has had a rich history of cellular research, its molecular mechanism of motility remains poorly understood. Here we describe recent X-ray crystallographic studies that reveal the architecture of dynein's catalytic ring, mechanical linker element, and microtubule binding domain. This structural information has given rise to new hypotheses on how the dynein motor domain might change its conformation in order to produce motility along microtubules.     

Mechanochemical coupling in eukaryotic flagella

Quantitative analyses of ATP hydrolysis coupled to movement of eukaryotic flagella is important for understanding the relationship between ATP hydrolysis and movement. The difference in ATPase activity between intact motile axonemes (that is the cytoskeletal core of flagella) and homogenized or immotile axonemes has been assumed to be coupled to movement. However, recent findings on rates of steps in the dynein ATPase cycle and the effect of interaction with microtubules on those steps call for reassessment of movement-coupled ATPase. From these studies, it is clear that dynein ATPase activity is not as tightly coupled to interaction with microtubules as myosin ATPase activity is coupled to interaction with actin. The method by which axonemal movement is inhibited will critically affect the interpretation of difference in ATPase activity. If the homogenization or similar methods uncouple dynein, the difference in ATPase activity is not a useful measurement. Greater understanding of the relationship between dynein kinetics and axonemal movement may be obtained by use of conditions and substrates with known effects at specific steps in the dynein mechanochemical cycle and quantitating their effects on movement.     

Kinesin-1 (uKHC/KIF5B) is required for bidirectional motility of ER exit sites and efficient ER-to-Golgi transport

Transport of proteins and lipids between intracellular compartments is fundamental to the organization and function of eukaryotic cells. The efficiency of this process is greatly enhanced through coupling of membranes to microtubules. This serves two functions, organelle positioning and vesicular transport. In this study, we show that in addition to the well-known role for the minus-end motor dynein in endoplasmic reticulum (ER)-to-Golgi transport, the plus-end-directed motor kinesin-1 is involved in positioning coat protein II-coated ER exit sites (ERES) in cells as well as the formation of transport carriers and their movement to the Golgi. Using two-dimensional Gaussian fitting to determine their location at high spatial resolution, we show that ERES undergo short-range bidirectional movements. Bidirectionality depends on both kinesin-1 and dynein. Suppression of kinesin-1 (KIF5B) also inhibits ER-to-Golgi transport and affects the morphology of ER-to-Golgi transport carriers. Furthermore, we show that suppression of dynein heavy chain expression increases the range of movement of ERES, suggesting that dynein might anchor ERES, or the ER itself, to microtubules. These data implicate kinesin-1 in the spatial organization of the ER/Golgi interface as well as in traffic outside the ER.     

Living markers for actin block myosin-dependent motility of plant organelles and auxin

Expression-based techniques using recombinant actin-binding proteins (ABPs) have been developed as advantageous means of visualising actin filaments. As actin function is linked to the movement of cellular cargoes, and overexpression of ABPs may compete with endogenous cytoskeletal proteins, such as myosins, secondary effects on cellular motility might be observed during actin visualisation. Cytoplasmic streaming and auxin transport were chosen as examples of cargo movement and investigated in two Arabidopsis thaliana lines stably transformed with fluorescently labelled talin (GFP-mTn) or fimbrin (GFP-FABD2). In both lines, the maximal streaming velocity of organelles was reduced to 80% in hypocotyl epidermal cells, where actin was broadly equally labelled by both ABPs. In contrast, observations of streaming and actin organisation during treatments with cytochalasin D (CD) suggested GFP-mTn-labelled actin to remain more stable. Furthermore, basipetal auxin transport was undisturbed in the GFP-FABD2 line but reduced by GFP-mTn. Remarkably, treatments with CD and 2,3-butanedione monoxime, which immobilizes myosin by impairing its ATPase, produced not only failures in organelle movement but also in basipetal auxin transport in the wild-type. These observations suggest that myosin is involved in processes of auxin translocation. In parallel, reduced motility in transgenic plants may be explained by a disturbed acto-myosin interplay, if overexpressed ABPs block the processive movement of myosin along actin filaments. This report shows that the use of live markers for actin visualisation may affect motility of cellular compounds and underlines the general need for critical investigation of actin-related processes in wild-type as well as transgenic plants prior to further interpretation.