Requirement for microtubule integrity in the SOCS1‐mediated intracellular dynamics of HIV‐1 Gag
Mayuko NishiAkihide RyoNaomi TsurutaniKenji OhbaTatsuya SawasakiRyo MorishitaKilian PerremIchiro AokiYuko MorikawaNaoki Yamamoto
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MINT‐7014221: Cullin 2 (uniprotkb:Q13617) physically interacts (MI:0218) with Gag (uniprotkb:P05888), elongin C (uniprotkb:Q15370), elongin B (uniprotkb:Q15369), SOCS1 (uniprotkb:O15524) and RBX1 (uniprotkb:P62877) by pull‐down (MI:0096)Keywords:
Nocodazole
Axonal growth is believed to depend on microtubule transport and microtubule dynamic instability. We now report that the growth of axon collateral branches can occur independent of microtubule dynamic instability and can rely mostly on the transport of preassembled polymer. Raising embryonic sensory neurons in concentrations of either taxol or nocodazole (NOC) that largely inhibit microtubule dynamics significantly inhibited growth of main axonal shafts but had only minor effects on collateral branch growth. The collaterals of axons raised in taxol or nocodazole often contained single microtubules with both ends clearly visible within the collateral branch (“floating” microtubules), which we interpret as microtubules undergoing transport. Furthermore, in these collaterals there was a distoproximal gradient in microtubule mass, indicating the distal accumulation of transported polymer. Treatment of cultures with a high dose of nocodazole to deplete microtubules from collaterals, followed by treatment with 4–20 n m vinblastine to inhibit microtubule repolymerization, resulted in the time-dependent reappearance and subsequent distal accumulation of floating microtubules in collaterals, providing further evidence for microtubule transport into collateral branches. Our data show that, surprisingly, the contribution of microtubule dynamics to collateral branch growth is minor compared with the important role of microtubule dynamics in growth cone migration, and they indicate that the transport of microtubules may provide sufficient cytoskeletal material for the initial growth of collateral branches.
Nocodazole
Growth cone
Axoplasmic transport
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In the interphase PK cells, more than 85% of microtubules radiating from the centrosome were not longer than 1.5 microns. A half of microtubules had their proximal ends free. After nocodazole treatment (20 microM), the number of microtubules attached to the centrosome decreased by 20% after 10 min of treatment, remained the same after 20 min of treatment, and increased after 60 min of nocodazole treatment slightly above the control level. After 5 and 60 min of treatment, the number of attached microtubules with the length over 0.7 micron increased twice as compared to the control level. During the first 20 min of nocodazole treatment, the immunofluorescent staining of cells with antibodies to gamma-tubulin was the same as in the control cells. The number of free microtubules decreased fourfold during the first 5 min, then it decreased slowly (for 20 min) and remained at the same level after 60 min. After 10 min of taxol (12 microM) treatment, the number of attached and free microtubules increased more than two times, whereas the number of attached microtubules with the length over 0.7 micron increased more than tenfold. After 15 min of treatment, the number of attached microtubules was slightly higher, and the number of free microtubules was half of the control level. After 20-60 min of treatment, the number of microtubules of all types decreased. Thus, upon the nocodazole treatment, the microtubules attached to the centrosome were more resistant to depolymerization: however, these microtubules were the most reactive to taxol treatment. The data obtained suggest that (a) in PK cells, the centrosome-attached microtubules occupy not all of the existing templates; (b) during prolonged treatment with inhibitors, the centrosome performed the compensatory reaction-the inhibition of microtubule assembly results in the decrease in the number of active templates on the centrosome; the inhibition of microtubule depolymerization results in the inactivation of hitherto active reserve templates. The microtubules formed on the centrosome within the first minutes disengage from it and then leave the chromosomal region.
Nocodazole
Astral microtubules
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Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.
Nocodazole
Microtubule-associated protein
Microtubule polymerization
Fragmentation
Microtubule organizing center
Treadmilling
Microtubule nucleation
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Nocodazole
Microtubule-associated protein
Depolymerization
Microtubule nucleation
Microtubule organizing center
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Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.
Nocodazole
Depolymerization
Video microscopy
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We previously defined two classes of microtubule polymer in the axons of cultured sympathetic neurons that differ in their sensitivity to nocodazole by roughly 35-fold (Baas and Black (1990) J. Cell Biol. 111, 495-509). Here we demonstrate that virtually all of the microtubule polymer in these axons, including the drug-labile polymer, is stable to cold. What factors account for the unique stability properties of axonal microtubules? In the present study, we have focused on the role of tau, a microtubule-associated protein that is highly enriched in the axon, in determining the stability of microtubules to nocodazole and/or cold in living cells. We used a baculovirus vector to express very high levels of tau in insect ovarian Sf9 cells. The cells respond by extending processes that contain dense bundles of microtubules (Knops et al. (1991) J. Cell Biol. 114, 725-734). Cells induced to express tau were treated with either cold or 2 micrograms/ml nocodazole for times ranging from 5 minutes to 6 hours. The results with each treatment were very different from one another. Virtually all of the polymer was depolymerized within the first 30 minutes in cold, while little or no microtubule depolymerization was detected even after 6 hours in nocodazole. Based on these results, we conclude that tau is almost certainly a factor in conferring drug stability to axonal microtubules, but that factors other than or in addition to tau are required to confer cold stability.
Nocodazole
Microtubule-associated protein
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By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a “cross talk” between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.
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Nocodazole
Stathmin
Immunofluorescence
Platelet lysate
Microtubule-associated protein
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Nocodazole
Kinesin
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Microtubules are cylindrical protein polymers that play important roles in a number of cellular functions. The properties of microtubules are dynamically changed by interacting with many microtubule-related proteins and drugs. In this study, we used atomic force microscopy to evaluate the changes in microtubule mechanical properties induced by treatment with nocodazole, which is a microtubule-destabilizing drug. The average spring constant of the microtubules, which was used as a measure of microtubule lateral stiffness, was drastically decreased by treatment with nocodazole within 30 min from 0.052 +/- 0.014 N/m to 0.029 +/- 0.015 N/m. Our findings will aid in the understanding of microtubule dynamics, protein interactions in response to drug treatment, microtubule-related diseases, and drug development.
Nocodazole
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