Stepwise movements in vesicle transport of HER2 by motor proteins in living cells

The stepwise movements generated by myosin, dynein, and kinesin were observed in living cells in an attempt to understand the molecular mechanisms of movement within cells. First, the sequential process of the transport of vesicles, including human epidermal factor 2 receptor, after endocytosis was observed for long periods in three dimensions using quantum dots (QDs) and a three-dimensional confocal microscope. QD vesicles, after being endocytosed into the cells, moved along the membrane by transferring actin filaments and were then rapidly transported toward the nucleus along microtubules. Second, the position of vesicles was detected with a precision up to 1.9 nm and 330 micros using a new two-dimensional tracking method. The movement of the QDs transported by myosin VI lying just beneath the cell membrane consisted of 29- and 15-nm steps with a transition phase between these two steps. QD vesicles were then transported toward the nucleus or away from the nucleus toward the cell membrane with successive 8-nm steps. The stepwise movements of these motor proteins in cells were observed using new imaging methods that allowed the molecular mechanisms underlying traffic to and from the membrane to be determined.     

Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules

Kinesin is a molecular motor that moves processively by regular 8-nm steps along microtubules. The processivity of this movement is explained by a hand-over-hand model in which the two heads of kinesin work in a coordinated manner. One head remains bound to the microtubule while the other steps from the alphabeta-tubulin dimer behind the attached head to the dimer in front. The overall movement is 8 nm per ATPase cycle. To investigate elementary processes within the 8-nm step, we have developed a new assay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved into fast and slow substeps, each corresponding to a displacement of approximately 4 nm. The substeps are most probably generated by structural changes in one head of kinesin, leading to rectified forward thermal motions of the partner head. It is also possible that the kinesin steps along the 4-nm repeat of tubulin monomers.     

Cargo-binding makes a wild-type single-headed myosin-VI move processively

Class VI myosin is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. Recently, however, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting a doubt on its processivity. By using single molecule techniques, we show that green-fluorescent-protein-tagged single-headed, wild-type myosin-VI does not move processively. However, when coupled to 200-nm polystyrene beads (comparable to intracellular vesicles in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40-nm) steps. The characteristics of this monomer-driven movement were different to that of artificial dimer-driven movement: Compared to the artificial dimer, the monomer-bead complex had a reduced stall force (1 pN compared to 2 pN), an average run length 2.5-fold shorter (91 nm compared to 220 nm) and load-dependent step size. Furthermore, we found that a monomer-bead complex moved more processively in a high viscous solution (40-fold higher than water) similar to cellular environment. Because the diffusion constant of the bead is 60-fold lower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing its rebinding following detachment and supporting processive movement of the bead-monomer complexes. Although a single-headed myosin-VI was able to move processively with a large cargo, the travel distance was rather short. Multiple molecules may be involved in the cargo transport for a long travel distance in cells.     

Differential behavior of photoactivated microtubules in growing axons of mouse and frog neurons

To characterize the behavior of axonal microtubules in vivo, we analyzed the movement of tubulin labeled with caged fluorescein after activation to be fluorescent by irradiation of 365-nm light. When mouse sensory neurons were microinjected with caged fluorescein-labeled tubulin and then a narrow region of the axon was illuminated with a 365-nm microbeam, photoactivated tubulin was stationary regardless of the position of photoactivation. We next introduced caged fluorescein-labeled tubulin into Xenopus embryos and nerve cells isolated from injected embryos were analyzed by photoactivation. In this case, movement of the photoactivated zone toward the axon tip was frequently observed. The photoactivated microtubule segments in the Xenopus axon moved out from their initial position without significant spreading, suggesting that fluorescent microtubules are not sliding as individual filaments, but rather translocating en bloc. Since these observations raised the possibility that the mechanism of nerve growth might differ between two types of neurons, we further characterized the movement of another component of the axon structure, the plasma membrane. Analysis of the position of polystyrene beads adhering to the neurites of Xenopus neurons revealed anterograde movement of the beads at the rate similar to the rate of microtubule movement. In contrast, no movement of the beads relative to the cell body was observed in mouse sensory neurons. These results suggest that the mode of translocation of cytoskeletal polymers and some components of the axon surface differ between two neuron types and that most microtubules are stationary within the axon of mammalian neurons where the surface-related motility of the axon is not observed.     

Nucleotide-Induced Flexibility Change in Neck Linkers of Dimeric Kinesin as Detected by Distance Measurements Using Spin-Labeling EPR

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3-2.5 nm. In the absence of nucleotide but in the presence of microtubules, ∼ 40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5′-(β,γ-imido)triphosphate and adenosine 5′-(γ-thio)triphosphate, 40-50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4-0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered.We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.     

Kinesin steps do not alternate in size

Kinesin is a two-headed motor protein that transports cargo inside cells by moving stepwise on microtubules. Its exact trajectory along the microtubule is unknown: alternative pathway models predict either uniform 8-nm steps or alternating 7- and 9-nm steps. By analyzing single-molecule stepping traces from “limping” kinesin molecules, we were able to distinguish alternate fast- and slow-phase steps and thereby to calculate the step sizes associated with the motions of each of the two heads. We also compiled step distances from nonlimping kinesin molecules and compared these distributions against models predicting uniform or alternating step sizes. In both cases, we find that kinesin takes uniform 8-nm steps, a result that strongly constrains the allowed models.     

Force-induced bidirectional stepping of cytoplasmic dynein

Cytoplasmic dynein is a minus-end-directed microtubule motor whose mechanism of movement remains poorly understood. Here, we use optical tweezers to examine the force-dependent stepping behavior of yeast cytoplasmic dynein. We find that dynein primarily advances in 8 nm increments but takes other sized steps (4-24 nm) as well. An opposing force induces more frequent backward stepping by dynein, and the motor walks backward toward the microtubule plus end at loads above its stall force of 7 pN. Remarkably, in the absence of ATP, dynein steps processively along microtubules under an external load, with less force required for minus-end- than for plus-end-directed movement. This nucleotide-independent walking reveals that force alone can drive repetitive microtubule detachment-attachment cycles of dynein's motor domains. These results suggest a model for how dynein's two motor domains coordinate their activities during normal processive motility and provide new clues for understanding dynein-based motility in living cells.     

Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.     

Processivity of the Motor Protein Kinesin Requires Two Heads

A single kinesin molecule can move for hundreds of steps along a microtubule without dissociating. One hypothesis to account for this processive movement is that the binding of kinesin's two heads is coordinated so that at least one head is always bound to the microtubule. To test this hypothesis, the motility of a full-length single-headed kinesin heterodimer was examined in the in vitro microtubule gliding assay. As the surface density of single-headed kinesin was lowered, there was a steep fall both in the rate at which microtubules landed and moved over the surface, and in the distance that microtubules moved, indicating that individual single-headed kinesin motors are not processive and that some four to six single-headed kinesin molecules are necessary and sufficient to move a microtubule continuously. At high ATP concentration, individual single-headed kinesin molecules detached from microtubules very slowly (at a rate less than one per second), 100-fold slower than the detachment during two-headed motility. This slow detachment directly supports a coordinated, hand-over-hand model in which the rapid detachment of one head in the dimer is contingent on the binding of the second head.     

Kinesin passing permanent blockages along its protofilament track

During movement along microtubules, kinesin usually follows a track parallel to the axis of a single protofilament. The question arises what happens when kinesin encounters blockages. The present study describes the movement of kinesin labeled by 20-nm gold beads along immobilized microtubules artificially decorated with blocking proteins. To guarantee that exactly the kinesin-binding sites were occupied and to avoid steric effects exerted by large molecules, the KIF5A motor domain was used for blocking. After binding, the blockages were irreversibly cross-linked to the microtubules to make them non-exchangeable. Under such conditions, kinesin movement became a non-continuous one. As a rule, after temporary stopping the kinesin moved on without being released from the microtubule. The results strongly suggest a bypassing mechanism based on the postulation that kinesin changes to and continues movement along a neighbouring protofilament. Bypassing is considered to ensure an efficient long-distance transport of cellular cargoes by kinesins.     

The Role of Microtubules and Their Dynamics in Cell Migration

Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.     

Biased binding of single molecules and continuous movement of multiple molecules of truncated single-headed kinesin

Conventional kinesin has a double-headed structure consisting of two motor domains and moves processively along a microtubule using the two heads cooperatively. The movement of single and multiple truncated heads of Drosophila kinesin was measured using a laser trap and nanometer detecting apparatus. Single molecules of single-headed kinesin bound to the microtubules with a 3.5 nm biased displacement toward the plus end of the microtubule. The position of these single-headed kinesin molecules bound to a microtubule did not change until they had dissociated, indicating that single kinesin heads utilize nonprocessive movement processes. Two molecules of single-headed kinesin moved continuously along a microtubule with a lower velocity and force than that of single molecules of double-headed kinesin. The biased binding of the heads determines the directionality of movement, whereas two molecules of single-headed kinesin move continuously without dissociation from a microtubule.     

Differential labeling of myosin V heads with quantum dots allows direct visualization of hand-over-hand processivity

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.     

Plus-end motors override minus-end motors during transport of squid axon vesicles on microtubules

Plus- and minus-end vesicle populations from squid axoplasm were isolated from each other by selective extraction of the minus-end vesicle motor followed by 5'-adenylyl imidodiphosphate (AMP-PNP)- induced microtubule affinity purification of the plus-end vesicles. In the presence of cytosol containing both plus- and minus-end motors, the isolated populations moved strictly in opposite directions along microtubules in vitro. Remarkably, when treated with trypsin before incubation with cytosol, purified plus-end vesicles moved exclusively to microtubule minus ends instead of moving in the normal plus-end direction. This reversal in the direction of movement of trypsinized plus-end vesicles, in light of further observation that cytosol promotes primarily minus-end movement of liposomes, suggests that the machinery for cytoplasmic dynein-driven, minus-end vesicle movement can establish a functional interaction with the lipid bilayers of both vesicle populations. The additional finding that kinesin overrides cytoplasmic dynein when both are bound to bead surfaces indicates that the direction of vesicle movement could be regulated simply by the presence or absence of a tightly bound, plus-end kinesin motor; being processive and tightly bound, the kinesin motor would override the activity of cytoplasmic dynein because the latter is weakly bound to vesicles and less processive. In support of this model, it was found that (a) only plus-end vesicles copurified with tightly bound kinesin motors; and (b) both plus- and minus-end vesicles bound cytoplasmic dynein from cytosol.     

The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1

We have examined the cytoskeletal architecture and its relationship with synaptic vesicles in synapses by quick-freeze deep-etch electron microscopy (QF.DE). The main cytoskeletal elements in the presynaptic terminals (neuromuscular junction, electric organ, and cerebellar cortex) were actin filaments and microtubules. The actin filaments formed a network and frequently were associated closely with the presynaptic plasma membranes and active zones. Short, linking strands approximately 30 nm long were found between actin and synaptic vesicles, between microtubules and synaptic vesicles. Fine strands (30-60 nm) were also found between synaptic vesicles. Frequently spherical structures existed in the middle of the strands between synaptic vesicles. Another kind of strand (approximately 100 nm long, thinner than the actin filaments) between synaptic vesicles and plasma membranes was also observed. We have examined the molecular structure of synapsin 1 and its relationship with actin filaments, microtubules, and synaptic vesicles in vitro using the low angle rotary shadowing technique and QF.DE. The synapsin 1, approximately 47 nm long, was composed of a head (approximately 14 nm diam) and a tail (approximately 33 nm long), having a tadpole-like appearance. The high resolution provided by QF.DE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles, forming approximately 30-nm short strands. The head was on the actin and the tail was attached to the synaptic vesicle or actin filament. Microtubules were also cross-linked by a single synapsin 1, which also connected a microtubule to synaptic vesicles, forming approximately 30 nm strands. The spherical head was on the microtubules and the tail was attached to the synaptic vesicles or to microtubules. Synaptic vesicles incubated with synapsin 1 were linked with each other via fine short fibrils and frequently we identified spherical structures from which two or three fibril radiated and cross-linked synaptic vesicles. We have examined the localization of synapsin 1 using ultracryomicrotomy and colloidal gold-immunocytochemistry of anti-synapsin 1 IgG. Synapsin 1 was exclusively localized in the regions occupied by synaptic vesicles. Statistical analyses indicated that synapsin 1 is located mostly at least approximately 30 nm away from the presynaptic membrane. These data derived via three different approaches suggest that synapsin 1 could be a main element of short linkages between actin filaments and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles in the nerve terminals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Firm structural associations between migratory pigment granules and microtubules in crayfish retinula cells

The morphology of associations between mobile pigment granules and microtubules of the crayfish retinula cells was examined with transmission electron microscopy. Many pigment granules were found associated with microtubules through linkages of fuzzy appearance in thin sections. The linkages were revealed as discrete strands of variable shape in rotary-shadowed replicas of freeze-fractured and deep- etched specimens. The only feature of constant morphology among these connections consisted of 2-4-nm filaments projecting laterally from the microtubules. The firmness of the pigment granule-microtubule associations was judged by their ability to hold up during cell disruption procedures of increasing disaggregation effects in a low- Ca++ stabilization buffer. The results of these tests were inspected with scanning electron microscopy and with transmission electron microscopy of negatively stained preparations. Numerous pigment granules remained associated with a stable microtubule framework after the plasma membrane had been stripped away. Moreover, granule- microtubule attachments survived breakdown of this framework into free fascicles of microtubules. The pigment granules were associated with the free microtubules either individually or as clusters entangled in a fibrous material interwoven with 10-nm filaments. These findings attest that many pigment granules are bound to microtubules through linkages that constitute effective attachments. Further, it is demonstrated that a highly cohesive substance associates the pigment granules with one another. These conclusions are discussed in terms of a pigment transport mechanism in which a network of interconnected granules would establish firm transient interactions with a supporting skeleton of microtubules.     

Motility of single one-headed kinesin molecules along microtubules

The motility of single one-headed kinesin molecules (K351 and K340), which were truncated fragments of Drosophila two-headed kinesin, has been tested using total internal reflection fluorescence microscopy. One-headed kinesin fragments moved continuously along the microtubules. The maximum distance traveled until the fragments dissociated from the microtubules for both K351 and K340 was approximately 600 nm. This value is considerably larger than the space resolution of the measurement system (SD approximately 30 nm). Although the movements of the fragments fluctuated in forward and backward directions, statistical analysis showed that the average movements for both K340 and K351 were toward the plus end of the microtubules, i.e., forward direction. When BDTC (a 1.3-S subunit of Propionibacterium shermanii transcarboxylase, which binds weakly to a microtubule), was fused to the tail (C-terminus) of K351, its movement was enhanced, smooth, and unidirectional, similar to that of the two-headed kinesin fragment, K411. However, the travel distance and velocity of K351BDTC molecules were approximately 3-fold smaller than that of K411. These observations suggest that a single kinesin head has basal motility, but coordination between the two heads is necessary for stabilizing the basal motility for the normal level of kinesin processivity.     

Directional loading of the kinesin motor molecule as it buckles a microtubule.

Single kinesin motor molecules were observed to buckle the microtubules along which they moved in a modified in vitro gliding assay. In this assay a central portion of the microtubule was clamped to the glass substrate via biotin-streptavidin bonds, while the plus end of the microtubule was free to interact with motors adsorbed at low density to the substrate. A statistical analysis of the length of microtubules buckled by single motors showed a decreasing probability of buckling for loads greater than 4-6 pN parallel to the filament. This is consistent with kinesin stalling forces found in other experiments. A detailed analysis of some buckling events allowed us to estimate both the magnitude and direction of the loading force as it developed a perpendicular component tending to pull the motor away from the microtubule. We also estimated the motor speed as a function of this changing vector force. The kinesin motors consistently reached unexpectedly high speeds as the force became nonparallel to the direction of motor movement. Our results suggest that a perpendicular component of load does not hinder the kinesin motor, but on the contrary causes the motor to move faster against a given parallel load. Because the perpendicular force component speeds up the motor but does no net work, perpendicular force acts as a mechanical catalyst for the reaction. A simple explanation is that there is a spatial motion of the kinesin molecule during its cycle that is rate-limiting under load; mechanical catalysis results if this motion is oriented away from the surface of the microtubule.     

Novel Ways to Determine Kinesin-1's Run Length and Randomness Using Fluorescence Microscopy

The molecular motor protein Kinesin-1 drives intracellular transport of vesicles, by binding to microtubules and making hundreds of consecutive 8-nm steps along them. Three important parameters define the motility of such a linear motor: velocity, run length (the average distance traveled), and the randomness (a measure of the stochasticity of stepping). We used total internal reflection fluorescence microscopy to measure these parameters under conditions without external load acting on the motor. First, we tracked the motility of single motor proteins at different adenosine triphosphate (ATP) concentrations and determined both velocity and (for the first time, to our knowledge, by using single-molecule fluorescence assays) randomness. We show that the rate of Kinesin-1 at zero load is limited by two or more exponentially distributed processes at high ATP concentrations, but that an additional, ATP-dependent process becomes the sole rate-limiting process at low ATP concentrations. Next, we measured the density profile of moving Kinesin-1 along a microtubule. This allowed us to determine the average run length in a new way, without the need to resolve single-molecules and to correct for photobleaching. At saturating ATP concentration, we measured a run length of 1070 ± 30 nm. This value did not significantly change for different ATP concentrations.     

Evidence for firm linkages between microtubules and membrane-bounded vesicles

Direct evidence is presented in support of the widely held idea that membrane-bounded vesicles can bind firmly to microtubules. This is shown in P. caudatum which contains ribbons of straight microtubules located in open cytoplasm and uniquely associated with the disk-shaped vesicles. These vesicles frequently lie flat against the face of the ribbons at a constant distance of 30-40 nm. Under certain conditions the ribbons are compressed into zigzag pattern, but the vesicles continue to maintain their 30-40 nm spacing with the tubules and The author's interpretation of this phenomena is that the vesicles and the microtubules are strongly bound together. This interaction appears to be via a filamentous material rather than bridges.