The analysis was performed using in vitro motility assays in which organelles move along both microtubules and actin filaments. The results indicated that the movement of mitochondria and Golgi vesicles is slow and continuous along microtubules but fast and irregular along actin filaments. In addition, the presence of microtubules in the motility assays forces organelles to use lower velocities.
Actin- and tubulin-binding tests, immunoblotting and immunogold labeling indicated that different organelles bind to identical myosins but associate with specific kinesins. In vitro and in vivo motility assays indicate that microtubules and kinesins decrease the speed of mitochondria, thus contributing to their positioning in the pollen tube.
The pollen tube is a highly polarized and rapidly tip-growing cell used to deliver the male genetic material to the ovule Wilhelmi and Preuss In angiosperms, pollen tubes show a strong cytoplasmic streaming of organelles, which move along actin filaments, as shown by studies with cytoskeleton inhibitors Mascarenhas and Lafountain , Lancelle and Hepler , Gibbon et al.
Furthermore, organelles isolated from Lilium pollen tubes moved along actin filament bundles of Characeae Kohno and Shimmen Immunocytochemical analysis has shown that myosins, the actin filament motors, are associated with pollen tube organelles Tang et al. This myosin is also present in tobacco plants Yokota et al.
Although less well supported, it is nevertheless evident that microtubules also participate in the regulation of motility in pollen tubes. Microtubules apparently correlate with the movement of both the generative cell and the vegetative nucleus Astrom et al.
In addition, kinesin-like and dynein-like proteins have been biochemically identified and characterized Tiezzi et al. These motors show different distributions inside the pollen tube Cai et al. Furthermore, organelles isolated from tobacco pollen tubes have been shown to move in vitro along microtubules, and kinesin-related motor proteins are probably involved in this activity Romagnoli et al.
The in vitro velocity of organelles along microtubules is far slower than that of the streaming induced by actin filament—myosin in pollen tubes, suggesting that the microtubule-dependent transport of organelles is overwhelmed in vivo by the rapid transport generated by the actin filament—myosin system. To sum up, current data indicate that both actin filament- and microtubule-based motors contribute, although differently, to organelle movement in the pollen tube.
However, some issues are unresolved; for example, it is unclear if the two motor systems cooperate with each other and how different motors distribute between the several organelle types. In other eukaryotic cells mainly in specialized animal cells such as neurons and melanophores , the functional cooperation between microtubules, actin filaments and molecular motors in organelle trafficking has already been described Rogers and Gelfand Some models propose that microtubule- and actin filament-dependent motors perform spatially distinct activities in animal cells, with the long-range transport of organelles mediated by microtubules and the short-range local transport provided by actin filaments Goode et al.
Alternatively, it has been proposed that the movement of a single organelle is the result of the forces exerted simultaneously on the organelle itself by different motors Tabb et al. Although the above-mentioned models derive essentially from animal cell systems, plants also exhibit examples of functional cooperation between actin filaments and microtubules. In non-vascular plants, such as the alga Chara , both microtubules and actin filaments are involved in the transport and immobilization of mitochondria Foissner Cooperation between actin filament- and microtubule-dependent motility also extends to the transport of chloroplasts in Bryopsis Menzel and Schliwa and in Physcomitrella patens Sato et al.
In vascular plants, actin filaments are the main tracks along which organelle transport occurs Shimmen and Yokota Nevertheless, analysis of organelle movement and positioning in some plant species has revealed that both microtubules and actin filaments mediate the movement and the anchoring of organelles.
In cultured tobacco cells, for example, the fast directional movement of mitochondria is dependent on the actin filament—myosin system, while the positioning of immobile mitochondria in the cortical cytoplasm is based on both actin filaments and microtubules Van Gestel et al.
In Arabidopsis thaliana leaves, one kinesin is associated with the Golgi apparatus and is critical for the dispersal of the Golgi along microtubules, while the movement of Golgi from the center to the cell cortex depends on myosin Lu et al.
Because current literature suggests that microtubule- and actin filament-dependent motors may cooperate for organelle movement in plant cells, we analyzed the motility of individual pollen tube organelles along microtubules and actin filaments, and assayed these organelles for the presence of specific motor proteins.
Mitochondria have been chosen as a model organelle, because information on their movement already exists in the literature and because they can be easily isolated from plant cells.
Mitochondria are distributed all along the pollen tube length and move backward and forward along the pollen tube according to the reverse-fountain streaming pathway Parton et al. Results obtained for mitochondria were compared with those from Golgi vesicles because the two organelle classes distribute differently in the tube. Vesicles are generated by the Golgi bodies, transported along actin filaments and accumulated in the apical region Wang et al.
We used in vitro motility assays coupled to microtubule- and actin filament-binding analysis, immunochemical and immunocytological techniques for the characterization of organelle movement and motor proteins. Our aim was to define how mitochondria move along microtubules and actin filaments, to determine the relative contribution of each motor system to their movement and to understand whether mitochondria possess specific motor proteins kinesin and myosin.
We used published protocols to purify either mitochondria or Golgi vesicles from tobacco pollen tubes. Although the original method for the isolation of membrane fractions from pollen tubes claims to yield relatively uncontaminated fractions, we assayed marker enzymes for mitochondria, Golgi apparatus, endoplasmic reticulum and plasma membrane to assess the purity of the isolated organelles Fig.
As expected, the activities of these enzymes were equivalent in the post-nuclear supernatant PNS. The activity of the marker enzymes indicated that the mitochondrial and vesicular fractions were largely free of contamination from other organelles. To confirm this assessment, mitochondria Fig. Analysis of the organelle fractions used in the motility assays. Error bars indicate the standard deviation. In this work, we examined mitochondria and Golgi vesicles purified from pollen tubes moving along microtubules and actin filaments.
Both cytoskeletal filaments were derived from animal sources tubulin from bovine brain and actin from rabbit skeletal muscle but they were scarcely contaminated by animal organelles. In addition, exogenous motor proteins were not detected in the tubulin or actin samples as revealed by Western blotting with antibodies to animal myosin and kinesin; data not shown. On microtubules, mitochondria, initially free in solution, attached and, after a lag phase, moved slowly while retaining a stable contact with the microtubule surface Fig.
After movement, mitochondria usually detached from the filaments and became free in solution. Exchange between different microtubules was uncommon. Only some microtubule-associated mitochondria exhibited active movement Table 1. In vitro motility assay of mitochondria along microtubules or actin filaments. A Time-lapse video sequences of mitochondria arrowheads moving along in vitro polymerized microtubules MT or B in vitro polymerized fluorescent actin filaments AF.
Numbers on the top right indicate the time in seconds between each video frame. The movement of pollen tube mitochondria along actin filaments Fig. On actin, mitochondria moved rapidly and irregularly, detaching and then attaching to the same or different actin filaments see the mitochondrion in Fig.
Long-lived contact with the same actin filament was not rated Table 1. After movement, organelles again detached from the filaments. Unlike along microtubule substrate, most mitochondria moved actively along actin filaments Table 1. When both cytoskeletal elements were present in the assays, we observed three different conditions. The velocity of mitochondria during each step was equivalent to the velocity observed under separate conditions.
In this case, mitochondria alternated between rapid movements presumably along actin filaments and slow ones most probably along microtubules ; the average speed was lower than the velocity observed along actin filaments but higher compared with that along microtubules Table 1.
The three different conditions were observed in separate samples; however, their relative incidence was difficult to evaluate because it depended on the relative organization of actin filaments and microtubules.
In vitro motility assay of mitochondria along microtubules and actin filaments. A Time-lapse video sequences of one mitochondrion arrow that moves in vitro along a single microtubule MT. One actin filament AF is aligned with the microtubule but is relatively distant. Numbers on the top right indicate the time in seconds. B Time-lapse video sequences of the sequential movement of one mitochondrion arrow that firstly moves along actin filaments AF , then switches to and moves on one microtubule MT.
The black line in the last video frame of B shows the course covered by the mitochondrion. C Time-lapse video sequences showing one microtubule MT and one actin filament AF that align closely; one single mitochondrion arrow moves while simultaneously interacting with both cytoskeletal filaments. The black line in the last video frame of C shows the route covered by the mitochondrion. Numbers in all frames indicate the time in seconds.
Like mitochondria, Golgi vesicles from tobacco pollen tubes moved in vitro along microtubules and actin filaments in an ATP-dependent and cytosol-independent manner. The movement of Golgi vesicles along microtubules was slow and continuous; the vesicles did not detach from microtubules.
Unlike mitochondria, moving Golgi vesicles frequently switched to different microtubules and resumed their movement Fig. The run length of Golgi vesicles along microtubules was shorter than that of mitochondria, in agreement with the short time that Golgi vesicles spent along microtubules Table 1.
On the other hand, the movement of Golgi vesicles along actin filaments was similar to the movement of mitochondria on actin Fig. When Golgi vesicles were tested on both cytoskeletal filaments together, they interacted with both Fig. In the most frequent condition, Golgi vesicles bound to and moved rapidly along actin filaments, with many saltations; when Golgi vesicles contacted microtubules, they often stopped or resumed a slow movement along them.
The mean velocity of Golgi vesicles along microtubule—actin filament matrices was lower that the speed of vesicles along actin filaments but higher than their velocity along microtubules Table 1. A One vesicle arrow moves along and switches between two different microtubules MT. B One vesicle arrow moves in vitro along fluorescent actin filaments AF. The long black line in the last video frame of B indicates the route covered by the vesicle. One vesicle arrow firstly travels along one single actin filament, then switches to and moves slowly along microtubules.
A second vesicle arrowhead initially moves along microtubules and then switches to actin filaments. The black line in the last video frame of C shows the route covered by the first vesicle. Numbers on the top right of each frame indicate the time in seconds. The dotted line in the first video frame indicates the microtubule.
The movement of mitochondria and Golgi vesicles was evaluated statistically. The velocity distribution of mitochondria and vesicles along microtubules was indistinguishable from a normal distribution Fig. However, the distribution of organelle velocity on actin differed significantly from a normal distribution, being spread out rather evenly among represented velocities Fig. The statistical analysis of mitochondria and Golgi vesicles on the combined actin and microtubule system was performed taking into account the conditions of consecutive and simultaneous contact which are supposed to occur in the pollen tube.
The velocity distribution of Golgi vesicles Fig. Likewise for mitochondria Fig. The velocity distributions of both organelles moving in the combined system could be distinguished statistically from distributions for movement on actin alone compare Fig. Velocity distribution of mitochondria and Golgi vesicles along microtubules. Velocity distribution of mitochondria and Golgi vesicles along microtubule—actin filaments and in living pollen tubes.
C Golgi vesicles analyzed in the presence of microtubules and actin filaments lack the highest velocities while the frequency of the lowest velocities increases compared with A. E Velocity distribution of mitochondria in living pollen tubes. The lowest velocity ranges are recognizable. F Velocity distribution of mitochondria in living pollen tubes treated with oryzalin. The lowest ranges disappear and the frequency of the highest velocities increases.
The velocity distribution of organelles was determined using Retrac software. To extend these results, we obtained velocity distributions for organelles moving in living pollen tubes. The results observed in vitro compared closely with those observed in in vivo conditions Fig. Given the absence of Golgi vesicle-specific dyes, we focused our attention on mitochondria. In control conditions when both microtubules and actin filaments are present, Fig.
In the case of oryzalin-treated pollen tubes when only actin filaments are supposed to be present, Fig. Treatment with cytochalasin D inhibited the movement of mitochondria within a few minutes following drug application data not shown. As mitochondria and Golgi vesicles from tobacco pollen tubes moved along actin filaments in an ATP-dependent manner, we investigated the presence of myosins associated with pollen tube organelles using actin filament binding assays coupled to immunoblotting.
As shown by the binding assay, the PNS fraction contained membrane proteins that bind to actin filaments in the absence of ATP gel, pellet in lane 5. Assay of binding to actin filaments and characterization of pollen tube myosin.
A, gel Binding assay of PNS proteins to actin filaments. Microtubules are cylindrical tubes, nm in diameter. They are composed of subunits of the protein tubulin--these subunits are termed alpha and beta. Microtubules act as a scaffold to determine cell shape, and provide a set of "tracks" for cell organelles and vesicles to move on. Microtubules also form the spindle fibers for separating chromosomes during mitosis.
When arranged in geometric patterns inside flagella and cilia, they are used for locomotion. In the epithelial skin cells of the intestine, all three types of fibers are present. Aa Aa Aa. Microtubules and Filaments. What Is the Cytoskeleton Made Of? The cytoskeleton of eukaryotic cells is made of filamentous proteins, and it provides mechanical support to the cell and its cytoplasmic constituents. All cytoskeletons consist of three major classes of elements that differ in size and in protein composition.
Microtubules are the largest type of filament, with a diameter of about 25 nanometers nm , and they are composed of a protein called tubulin. Actin filaments are the smallest type, with a diameter of only about 6 nm, and they are made of a protein called actin. Intermediate filaments, as their name suggests, are mid-sized, with a diameter of about 10 nm.
Unlike actin filaments and microtubules, intermediate filaments are constructed from a number of different subunit proteins. What Do Microtubules Do?
Figure 1. What Do Actin Filaments Do? Figure 2. What Do Intermediate Filaments Do? Figure 4: The structure of intermediate filaments. Intermediate filaments are composed of smaller strands in the shape of rods. How Do Cells Move? The cytoskeleton of a cell is made up of microtubules, actin filaments, and intermediate filaments. These structures give the cell its shape and help organize the cell's parts.
In addition, they provide a basis for movement and cell division. Cell Biology for Seminars, Unit 3. Topic rooms within Cell Biology Close. No topic rooms are there. Or Browse Visually. Student Voices. Creature Cast. Simply Science. Green Screen. Green Science. Bio 2. The Success Code. Why Science Matters. The Beyond. Plant ChemCast. Postcards from the Universe.
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