In vitro assays demonstrate that pollen tube organelles use kinesin-related motor proteins to move along microtubules

The movement of pollen tube organelles relies on cytoskeletal elements. Although the movement of organelles along actin filaments in the pollen tube has been studied widely and is becoming progressively clear, it remains unclear what role microtubules play. Many uncertainties about the role of microtubules in the active transport of pollen tube organelles and/or in the control of this process remain to be resolved. In an effort to determine if organelles are capable of moving along microtubules in the absence of actin, we extracted organelles from tobacco pollen tubes and analyzed their ability to move along in vitro-polymerized microtubules under different experimental conditions. Regardless of their size, the organelles moved at different rates along microtubules in the presence of ATP. Cytochalasin D did not inhibit organelle movement, indicating that actin filaments are not required for organelle transport in our assay. The movement of organelles was cytosol independent, which suggests that soluble factors are not necessary for the organelle movement to occur and that microtubule-based motor proteins are present on the organelle surface. By washing organelles with KI, it was possible to release proteins capable of gliding carboxylated beads along microtubules. Several membrane fractions, which were separated by Suc density gradient centrifugation, showed microtubule-based movement. Proteins were extracted by KI treatment from the most active organelle fraction and then analyzed with an ATP-sensitive microtubule binding assay. Proteins isolated by the selective binding to microtubules were tested for the ability to glide microtubules in the in vitro motility assay, for the presence of microtubule-stimulated ATPase activity, and for cross-reactivity with anti-kinesin antibodies. We identified and characterized a 105-kD organelle-associated motor protein that is functionally, biochemically, and immunologically related to kinesin. This work provides clear evidence that the movement of pollen tube organelles is not just actin based; rather, they show a microtubule-based motion as well. This unexpected finding suggests new insights into the use of pollen tube microtubules, which could be used for short-range transport, as actin filaments are in animal cells.     

Microtubule motors and pollen tube growth—still an open question

The growth of pollen tubes is supported by the continuous supply of secretory vesicles in the tip area. Movement and accumulation of vesicles is driven by the dynamic interplay between the actin cytoskeleton and motor proteins of the myosin family. A combination of the two protein systems is also responsible for the bidirectional movement of larger organelle classes. In contrast, the role of microtubules and microtubule-based motors is less clear and often ambiguous. Nevertheless, there is evidence which shows that the pollen tube contains a number of microtubule-based motors of the kinesin family. These motor proteins are likely to be associated with pollen tube organelles and, consequently, they have been hypothesized to participate in the distribution of organelles during pollen tube growth. Whether microtubule motor proteins take part in either the transport or positioning of organelles is not known for sure, but there is evidence for this second possibility. This review will discuss the current knowledge of microtubule-based motor proteins (including kinesins and hypothetical dyneins) and will make some hypothesis about their role in the pollen tube.     

Identification and characterization of a novel microtubule-based motor associated with membranous organelles in tobacco pollen tubes

Pollen tube growth depends on the differential distribution of organelles and vesicles along the tube. The role of microtubules in organelle movement is uncertain, mainly because information at the molecular level is limited. In an effort to understand the molecular basis of microtubule-based movement, we isolated from tobacco pollen tubes polypeptides that cosediment with microtubules in an ATP-dependent manner. Major polypeptides released from microtubules by ATP (ATP-MAPs) had molecular masses of 90, 80, and 41 kD. Several findings indicate that the 90-kD ATP-MAP is a kinesin-related motor: binding of the polypeptide to microtubules was enhanced by the nonhydrolyzable ATP analog AMP-PNP; the 90-kD polypeptide reacted specifically with a peptide antibody directed against a highly conserved region in the motor domain of the kinesin superfamily; purified 90-kD ATP-MAP induced microtubules to glide in motility assays in vitro; and the 90-kD ATP-MAP cofractionated with microtubule-activated ATPase activity. Immunolocalization studies indicated that the 90-kD ATP-MAP binds to organelles associated with microtubules in the cortical region of the pollen tube. These findings suggest that the 90-kD ATP-MAP is a kinesin-related microtubule motor that moves organelles in the cortex of growing pollen tubes.     

Microtubule motor proteins and the organization of the pollen tube cytoplasm

Molecular motors are molecules that drive a wide range of activities (for example, organelle movement, chromosome segregation, and flagellar movement) in cells. Thus, they play essential roles in diverse cellular functions. Understanding their structures, mechanisms of action and different roles is therefore of great practical importance. The role of microtubules during pollen tube growth is presently not identified, nor are basic properties. We do not know, for example, where microtubules are organized, the extent of microtubule dynamics, and the polarity of microtubules in the pollen tube. Roles of microtubules and related motors in organelle trafficking are not clear. Regardless of scarce information, microtubule-based motors of both the kinesin and dynein families have been identified in the pollen tube. Most of these microtubule motors have also been found in association with membrane-bounded organelles, which suggest that these proteins could translocate organelles or vesicles along microtubules. The biochemical features of these proteins are typical of the motor protein class. Immunofluorescence microscopy of pollen tubes probed with antibodies that cross-react with microtubule motors indicate that these proteins are localized in different regions of the pollen tube; therefore, they could have different roles. Although a number of microtubule motors have been identified in the pollen tube, the role of these proteins during pollen tube germination and growth or organelle movement is not yet recognized, as tube elongation and organelle movement in the pollen tube depend mostly on actin filaments. In the effort to understand the specific role that microtubules and related motors have in the pollen tube, it is therefore necessary to identify the molecular machinery that interacts with microtubules. Furthermore, it is crucial to clearly establish the types of interaction between organelles and microtubules. This review summarizes the current state of the art on microtubule motors in the pollen tube, mainly surrounding the putative roles of microtubule motors in organelle movement and cytoplasmic organization. Some hypotheses and speculations are also presented.     

The kinesin-immunoreactive homologue from Nicotiana tabacum pollen tubes: Biochemical properties and subcellular localization

In plant cells, microtubule-based motor proteins have not been characterized to the same degree as in animal cells; therefore, it is not yet clear whether the movement of organelles and vesicles is also dependent on the microtubular cytoskeleton. In this work the kinesinimmunoreactive homologue from pollen tubes of Nicotiana tabacum L. has been purified and biochemically characterized. The protein preparation mainly contained a polypeptide with a relative molecular weight of approx. 100 kDa. This polypeptide bound to animal microtubules in an ATP-dependent manner and it further copurified with an ATPase activity fourfold-stimulated by the presence of microtubules. In addition, the sedimentation coefficient (approx. 9S) was similar to those previously shown for other kinesins. Immunofluorescence analyses revealed a partial co-distribution of the protein with microtubules in the pollen tube. These data clearly indicate that several properties of the kinesin-immunoreactive homologue are similar to those of kinesin proteins, and suggest that molecular mechanisms analogous to those of animal cells may drive the microtubule-based motility of organelles and vesicles in plants.Abbreviations AE-LPLC anion-exchange low-pressure liquid chromatography - AMPPNP 5-adenylylimidodiphosphate - PKH pollen kinesin homologue - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Alignment, zippering and splaying of microtubules during axogenesis

Neurodegenerative diseases may result in part from defects in motor-driven vesicle transport in neuronal cells. Myosin-V, an actin-based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin-V interacts with the microtubule-based motor, kinesin, to form a 'hetero-motor' complex on vesicles. The complex of these two motors, one microtubule-based and the other actin-based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin-V to kinesin and to neuronal vesicles, we used a GST-tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin-V presumably by binding to vesicles and competing away the native myosin-V motors. The GST-MyoV-tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin-V. These data show that the headless myosin-V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin-V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

Cell cycle control of microtubule-based membrane transport and tubule formation in vitro

When higher eukaryotic cells enter mitosis, membrane organization changes dramatically and traffic between membrane compartments is inhibited. Since membrane transport along microtubules is involved in secretion, endocytosis, and the positioning of organelles during interphase, we have explored whether the mitotic reorganization of membrane could involve a change in microtubule-based membrane transport. This question was examined by reconstituting organelle transport along microtubules in Xenopus egg extracts, which can be converted between interphase and metaphase states in vitro in the absence of protein synthesis. Interphase extracts support the microtubule-dependent formation of abundant polygonal networks of membrane tubules and the transport of small vesicles. In metaphase extracts, however, the plus end- and minus end-directed movements of vesicles along microtubules as well as the formation of tubular membrane networks are all reduced substantially. By fractionating the extracts into soluble and membrane components, we have shown that the cell cycle state of the supernatant determines the extent of microtubule-based membrane movement. Interphase but not metaphase Xenopus soluble factors also stimulate movement of membranes from a rat liver Golgi fraction. In contrast to above findings with organelle transport, the minus end-directed movements of microtubules on glass surfaces and of latex beads along microtubules are similar in interphase and metaphase extracts, suggesting that cytoplasmic dynein, the predominant soluble motor in frog extracts, retains its force-generating activity throughout the cell cycle. A change in the association of motors with membranes may therefore explain the differing levels of organelle transport activity in interphase and mitotic extracts. We propose that the regulation of organelle transport may contribute significantly to the changes in membrane structure and function observed during mitosis in living cells.     

Kinectin-kinesin binding domains and their effects on organelle motility

Intracellular organelle motility involves motor proteins that move along microtubules or actin filaments. One of these motor proteins, kinesin, was proposed to bind to kinectin on membrane organelles during movement. Whether kinectin is the kinesin receptor on organelles with a role in organelle motility has been controversial. We have characterized the sites of interaction between human kinectin and conventional kinesin using in vivo and in vitro assays. The kinectin-binding domain on the kinesin tail partially overlaps its head-binding domain and the myosin-Va binding domain. The kinesin-binding domain on kinectin resides near the COOH terminus and enhances the microtubule-stimulated kinesin-ATPase activity, and the overexpression of the kinectin-kinesin binding domains inhibited kinesin-dependent organelle motility in vivo. These data, when combined with other studies, suggest a role for kinectin in organelle motility.     

An internal motor kinesin is associated with the Golgi apparatus and plays a role in trichome morphogenesis in Arabidopsis

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.     

Motoring down the highways of the cell

All eukaryotic cells contain large numbers of motor proteins (kinesins, dyneins and myosins), each of which appears to carry out a specialized force-generating function within the cell. They are known to have roles in muscle contraction, ciliary movement, organelle and vesicle transport, mitosis and cytokinesis. These motor proteins operate on different cytoskeletal filaments; myosins move along actin filaments, and kinesins and dyneins along microtubules. Recently published crystal structures of the motor domains of two members of the kinesin superfamily reveal that they share the same overall fold that is also found at the core of the larger myosin motor. This suggests that they may share a common mechanism as well as a common ancestry.     

Association of kinesin with characterized membrane-bounded organelles.

The family of molecular motors known as kinesin has been implicated in the translocation of membrane-bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane-bounded organelles in the cell. In addition, immunoelectron microscopy of mitochondria revealed a patch-like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility.     

RNA processing bodies, peroxisomes, Golgi bodies, mitochondria, and endoplasmic reticulum tubule junctions frequently pause at cortical microtubules

Organelle motility, essential for cellular function, is driven by the cytoskeleton. In plants, actin filaments sustain the long-distance transport of many types of organelles, and microtubules typically fine-tune the motile behavior. In shoot epidermal cells of Arabidopsis thaliana seedlings, we show here that a type of RNA granule, the RNA processing body (P-body), is transported by actin filaments and pauses at cortical microtubules. Interestingly, removal of microtubules does not change the frequency of P-body pausing. Similarly, we show that Golgi bodies, peroxisomes, and mitochondria all pause at microtubules, and again the frequency of pauses is not appreciably changed after microtubules are depolymerized. To understand the basis for pausing, we examined the endoplasmic reticulum (ER), whose overall architecture depends on actin filaments. By the dual observation of ER and microtubules, we find that stable junctions of tubular ER occur mainly at microtubules. Removal of microtubules reduces the number of stable ER tubule junctions, but those remaining are maintained without microtubules. The results indicate that pausing on microtubules is a common attribute of motile organelles but that microtubules are not required for pausing. We suggest that pausing on microtubules facilitates interactions between the ER and otherwise translocating organelles in the cell cortex.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

Molecular motors and their role in pigmentation.

Skin pigmentation is orchestrated through a series of complementary processes. After migration of melanoblasts out of the neural crest to epidermis and hair follicle, these cells mature into melanocytes. Differentiated melanocytes produce melanin in specialized organelles, the melanosomes. Moreover, the cytoplasm of melanocytes branches into extensions, the dendrites. Via the tips of these dendrites they donate their mature melanosomes to the keratinocytes resulting in skin pigmentation. Thus, one essential part of the process of pigmentation is the translocation of melanosomes from their site of origin in the perinuclear cytoplasm towards the dendrite tips. Motor proteins are molecules which use the energy derived from ATP hydrolysis to move along cytoskeletal elements, either actin filaments or microtubules, to transport their cargo, which can be organelles, vesicles or chromosomes. This review describes the different classes of microtubule-based and actin-based motor proteins with their characteristics and functional importance in cell biology and organelle transport. Some of them will be highlighted and several recent studies in mammalian pigment cells indicating their role in pigment granule transport will be discussed. As a result of these data and previous suggestions, a model will be proposed for the possible cooperation of both systems in melanosome movement.     

Cytoplasmic motors and pollen tube growth

The growth of pollen tubes is characterized by an intense cytoplasmic streaming, during which the movements of smaller organelles (like secretory vesicles) and larger ones (including the generative cell and vegetative nucleus) are precisely coordinated. A well-characterized cytoskeletal apparatus is likely responsible for these intracellular movements. In recent years both microfilament and microtubule-based motor proteins have been identified and assumed to be the translocators of the several organelle categories. Their precise function during pollen tube growth is not yet clear, but apparently an actomyosin-based system is mainly responsible for pollen tube elongation. On the other hand, microtubules and microtubule-based motors have been thought to play a role in the maintenance of cell polarity. Both cytoskeletal systems (and their respective motor activities) could cooperate to ensure a precise regulation of pollen tube growth.     

Actin dynamics is essential for myosin-based transport of membrane organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.     

An immunoreactive homolog of mammalian kinesin in Nicotiana tabacum pollen tubes.

A cytoskeletal apparatus is involved in the movement of vesicles, organelles, and gametes in the pollen tube. The function of microfilaments has been defined quite precisely, but the role of microtubules needs to be further clarified. On the basis of immunological and biochemical investigations, we have identified a polypeptide showing common properties with kinesin, a microtubule-based motor mainly described in nonplant tissues, in the pollen tube of Nicotiana tabacum. Like mammalian kinesin, the kinesin-immunoreactive homolog from Nicotiana tabacum pollen tubes binds to mammalian microtubules in an AMP-PNP dependent manner. The kinesin-like component is likely to be involved in the movement of vesicular material in the growing pollen tube.     

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.     

Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.     

A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.