Single fungal kinesin motor molecules move processively along microtubules

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.     

Kinesin is bound with high affinity to squid axon organelles that move to the plus-end of microtubules

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.     

In vitro measurement of pollen tube growth inhibition

A method for estimating inhibition of pollen tube growth was developed. Pollen is placed in straight lines on an agar surface where it responds uniformly and predictably to aqueous solutions of germination-inhibiting substances located in wells at the ends of the lines. A scale of ratings, roughly corresponding to serial, doubled concentrations of inhibiting substances, was devised. Water-soluble organic solvents are relatively noninhibitory, salts are variable, and metabolic inhibitors have strong inhibitory effects. Pollens differ in their susceptibility to inhibition and in their response to particular substances.     

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.     

Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.     

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.     

In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 μm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 μM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 μM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 μm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 μM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.     

In vitro germination and pollen tube growth of maize (Zea mays L.) pollen

Summary Pollen grains containing either theWx,wx,Su ₁,Su ₁,Sh ₂orsh ₂alleles were stored for 0, 1, 2, 3, 4 or 5 days at 2 °C. After each storage period, a portion of each genotype was cultured on a 15% sucrose, 0.6% bacto-agar, 0.03% calcium nitrate and 0.01% boric acid medium, while another portion was placed on receptive silks, the number of kernels produced being a measure of fertilization ability. Regardless of the allele present in the pollen grain, 1 day of storage greatly increased the germination percentage and significantly increased pollen tube length. After 4 days of storage, there was noin vitro germination but some fertilization ability was found. The experiment was designed so that comparisons free from genetic background effects could be made between alleles at each locus. Significant differences at each storage period and a differential response to storage were obtained at some loci for germination percentage, ruptured percentage, pollen tube length and fertilization ability. A relationship between dominance of the allele and response to storage was detected only for fertilization ability. Since alleles at these loci affect the biochemical composition of pollen grains containing them, the results suggest that differences inin vitro germination characteristics and fertilization ability may be associated with biochemical composition.Journal Series Paper No. 3950, Florida Agricultural Experiment Station.     

In vitro germination and pollen tube growth of maize (Zea mays L.) pollen

Summary Pollen grains from two hybrids, WF9xH55 (W) and K64xK55 (K) were collected and a sample from each was cultured immediately (0 h). The remainder was subdivided and stored at 2, 20, and 35° C. At 3, 6, 12, 24, 36, 48, 72, 96, and 120 h, a sample was cultured. The culture medium contained 15% sucrose, 0.6% bacto-agar, 0.03% calcium nitrate and 0.01% boric acid. Storage at 2° C resulted in a large increase in germination percentage in both W and K reaching a maximum at 24 h and then slowly decreasing with additional storage. No germination was observed at 96 h with W and at 120 h with K. The complete loss of germination occurred during a 24 h period and was very abrupt. At 20° C, a similar but less pronounced pattern was observed. However, after 24 h, aggregates of 50–1 000 pollen grains developed during storage in both W and K. Storage of W at 35° C slightly decreased the germination percentage at 3 h and eliminated it at 6 h. Storage of K at 35° C substantially increased the germination percentage at 3 h with further increases in storage periods resulting in the aggregation of grains. This general pattern of an increase at shorter storage periods followed by a gradual decrease as the storage period was extended, was found for pollen tube length and growth rate. In vitro germination characteristics can be substantially altered by the temperature and length of storage and the response to storage is associated with pollen source.Journal Series Paper No. 4566, Florida Agricultural Experiment Station.     

Identification and characterization of plasma membrane proteins that bind to microtubules in pollen tubes and generative cells of tobacco

The organization and function of microtubules in plant cells are important in many developmental stages. Connections between microtubules and the endomembrane system of plant cells have been discovered by microscopy, but the molecular characteristics of these relationships are mostly unknown except for a few cases. Using two antibodies raised against microtubule-associated proteins (MAPs) from maize, we have identified two polypeptides that share properties of the MAP family in the pollen tube of Nicotiana tabacum. The two polypeptides (with an apparent Mr of 161 and 90 kDa) bind efficiently to animal and plant microtubules and are found in association with the cellular membranes of the pollen tube, from which they can be solubilized with a zwitterionic detergent. One of these proteins has been purified and shown to promote the assembly of tubulin and, to a lesser extent, the bundling of microtubules. Subcellular fractionation indicated that the two proteins are associated with the plasma membrane compartment. The two proteins are found to co-localize in situ with cortical microtubules in the vegetative cytoplasm of tobacco pollen tubes; co-localization is also evident in the generative cell. According to these data, both the 161 and 90 kDa polypeptides are likely to mediate the interactions between the plasma membrane and microtubules in pollen tubes. In addition, functional data indicate that these MAP-like proteins take part in the process of microtubule assembly and reorganization occurring during cell growth. The evidence that both proteins associate with different cellular compartments also suggests a broad-spectrum role in mediating the dynamic relationships between microtubules and plant cell membranes.     

In vitro pollen germination and pollen tube growth differences among Quercus robur L. clones in response to meteorological conditions

The impact of meteorological conditions on in vitro pollen germination and pollen tube growth during the initial phases of the development of male flowers in the Pedunculate Oak, Quercus robur, is studied. Phenological observations of male flowers and pollen sampling were performed on the field trial established with grafted Pedunculate Oak clones. During the investigation, weather conditions (absolute minimum and maximum daily air temperature, minimum absolute relative humidity of air and amount of precipitation) were recorded by an automatic meteorological station installed at the field trial. Influence of meteorological conditions on pollen germination and pollen tube growth was studied in the following stages of male flower: (I) during the last ten days of flower bud dormancy, (II) during swelling of the buds, (III) during bud burst and beginning of male catkins elongation, (IV) during the final stage of male flower catkins elongation. High temperatures and low relative air humidity during the bud burst and beginning of the male catkins elongation reduced pollen germination and pollen tube growth. Weather conditions did not significantly affect pollen germination and pollen tube growth during the swelling of flower buds, or in the final stage of male catkins elongation.     

Kinesin-73 is a processive motor that localizes to Rab5-containing organelles

Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its "tail" domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.     

Active motor proteins can couple cargo to the ends of growing microtubules

In living cells, dynamic microtubule ends interact with specialized protein complexes located on microtubule targets such as chromosomes and the cell cortex. A significant role in coupling microtubule ends to these complexes has been attributed to motor proteins, which are thought to provide a physical link while at the same time allowing for microtubule growth or shrinkage. In the past, motor-coated beads have been shown to be able to follow the ends of depolymerizing microtubules, in a direction opposite to their natural walking direction. Here we show that beads coated with plus-end-directed motors can also stay attached for several seconds to the ends of growing microtubules. Upon arrival at the microtubule end, fast-moving beads reduce their velocity to the microtubule growth velocity. We show that the tendency to stay attached depends on the initial bead velocity and that the microtubule growth velocity is unaffected by the presence of the bead.     

Accumulation of adrenocorticotropin secretory granules in the midbody of telophase AtT20 cells: evidence that secretory granules move anterogradely along microtubules

During the cell cycle the distribution of the ACTH-containing secretory granules in AtT20 cells, as revealed by immunofluorescence labeling and electron microscopy of thin sections, undergoes a cycle of changes. In interphase cells the granules are concentrated in the Golgi region, where they form, and also at the tips of projections from the cells, where they accumulate. These projections contain many microtubules extending to their tips. During metaphase and anaphase the granules are randomly distributed in the cytoplasm of the rounded-up mitotic cells. On entry into telophase there is a rapid and striking redistribution of the granules, which accumulate in large numbers in the midbody as it develops during cytokinesis. This accumulation of secretory granules in the midbody is dependent upon the presence of microtubules. The changing pattern of distribution of the secretory granules during the cell cycle fulfills the predictions of a model envisaging first that secretory granules associate with and move along interphase microtubules in a net anterograde direction away from the centrioles, and secondly that they do not associate with microtubules of the mitotic spindle during metaphase and anaphase.     

Cytoplasmic illuminations: In planta targeting of fluorescent proteins to cellular organelles

Summary Use of the jellyfish green-fluorescent protein as an in vivo reporter is in the process of revolutionising plant cell biology. By fusing the protein to specific targeting peptides or to sequences of complete proteins, it is now possible to observe the location, structure, and dynamics of a number of intracellular organelles over extended periods of time. In this review we discuss the most recent developments and unexpected results originating from the targeting of this unique protein and its derivatives to elements of the cytoskeleton and to membrane-bounded organelles in a range of plant cell types.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

In vitro soil receptivity assays to egg-parasitic nematophagous fungi

Soil application of nematophagous fungi for the biological control of plant-parasitic nematodes often fails, and in many cases it has been difficult to reisolate the agent delivered to the soil. A reason for these results could be the inability of the fungi to proliferate in soil. We used a soil–membrane technique to study the capacity of several isolates of the nematophagous fungi Pochonia chlamydosporia and Paecilomyces lilacinus to grow and establish in sterilized and nonsterilized sandy soils from SE Spain and Western Australia. Growth of all fungi tested was inhibited in nonsterilized soil, although there was intraspecific variability in sensitivity among isolates of the same species. With respect to hyphal density, P. chlamydosporia isolate 5 (from Italy) was the least inhibited in nonsterilized soil from both sites. Relative growth analyses confirmed this result for soil from SE Spain, while with this method, P. chlamydosporia isolate 4624 (from Australia) appeared to be least inhibited in the Australian soil. The results indicate that a soil can be more receptive to its indigenous isolates than to nonindigenous isolates. Apparently, soil microbiota can determine the ability of nematophagous fungi to proliferate in soil.