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1.
Microtubules control cell architecture by serving as a scaffold for intracellular transport, signaling, and organelle positioning. Microtubules are intrinsically polarized, and their orientation, density, and post-translational modifications both respond and contribute to cell polarity. Animal cells that can rapidly reorient their polarity axis, such as fibroblasts, immune cells, and cancer cells, contain radially organized microtubule arrays anchored at the centrosome and the Golgi apparatus, whereas stably polarized cells often acquire non-centrosomal microtubule networks attached to the cell cortex, nucleus, or other structures. Microtubule density, longevity, and post-translational modifications strongly depend on the dynamics of their plus ends. Factors controlling microtubule plus-end dynamics are often part of cortical assemblies that integrate cytoskeletal organization, cell adhesion, and secretion and are subject to microtubule-dependent feedback regulation. Finally, microtubules can mechanically contribute to cell asymmetry by promoting cell elongation, a property that might be important for cells with dense microtubule arrays growing in soft environments. 相似文献
2.
Epithelial polarization and neuronal outgrowth require the assembly of microtubule arrays that are not associated with centrosomes. As these processes generally involve contact interactions mediated by cadherins, we investigated the potential role of cadherin signalling in the stabilization of non-centrosomal microtubules. Here we show that expression of cadherins in centrosome-free cytoplasts increases levels of microtubule polymer and changes the behaviour of microtubules from treadmilling to dynamic instability. This effect is not a result of cadherin expression per se but depends on the formation of cell-cell contacts. The effect of cell-cell contacts is mimicked by application of beads coated with stimulatory anti-cadherin antibody and is suppressed by overexpression of the cytoplasmic cadherin tail. We therefore propose that cadherins initiate a signalling pathway that alters microtubule organization by stabilizing microtubule ends. 相似文献
3.
M M Mogensen 《Biology of the cell / under the auspices of the European Cell Biology Organization》1999,91(4-5):331-341
Many differentiated cells including polarised epithelial cells display a non-radial, apico-basal microtubule array. In some cells the centrosome disassembles and new nucleating sites are created at more appropriate locations. In others the centrosome remains, but relatively few microtubules radiate from it's immediate environs. Instead, the majority of the microtubule minus-ends are associated with apical cell surface sites. Centrosomal microtubule release and capture is evidently a mechanism exploited by some polarised epithelial cells as a means of producing non-centrosomal, apico-basal microtubule arrays. This involves microtubule nucleation at the centrosome, release and subsequent translocation and capture at the apical sites. Two functionally distinct centrosomal complexes dedicated to the control of microtubule nucleation and anchorage have been suggested to be essential and universal features of all centrosomes. The centrosomal proteins ninein and R2 are potential microtubule anchoring proteins and their discovery has exciting implications for centrosomal organisation and microtubule positioning in cells. 相似文献
4.
The spatial organization of microtubules is crucial for different cellular processes. It is traditionally supposed that fibroblasts have radial microtubule arrays consisting of long microtubules that run from the centrosome. However, a detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study, we used laser photobleaching to analyze the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, after which the growth of microtubules in the centrosome region and peripheral parts of cytoplasm was assayed in the bleached zone. In most cases, microtubule growth in the bleached zone occurred rectilinearly; at distances of up to 5 μm, microtubules seldom bend more than 10°–15°. We considered a growing fragment of the microtubule as a vector with the beginning at the point of occurrence and the end at the point where the growth terminated (or the end point after 30 s if microtubule persistent growth proceeded for longer). We defined the direction of microtubule growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome, we directed a comparison vector inside the bleached zone from the centrosome to the beginning of the growing microtubule segment; in the lamella and trailing part of the fibroblast, we used the vector of comparison directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing straight away from the centrosome grew along the cell radius. However, at a distance of 10 μm from the centrosome, radially growing microtubules comprised 40% of the overall number, while at a distance of 20 μm, they made up only 25%. The rest of the microtubules grew in different directions, with the preferred angle between their growth direction and cell radius equaling around 90 °. In the lamella and trailing part of the fibroblast, 80% of all microtubules grew along the long axis of the cell or at an angle of no more than 20 °; 10–15% of microtubules grew along axis of the cell but towards the centrosome. Thus, in 3T3 fibroblasts, the radial system of microtubules is perturbed starting at a distance of several microns from the centrosome. In the internal cytoplasm, the microtubule system is completely disordered and, in the stretched parts of the polarized cell (lamella, trailing edge), the microtubule system again becomes well organized; microtubules are preferentially oriented along the long axis of the cell. From the results obtained, we conclude that the orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome; rather, their orientation is preset by local factors. 相似文献
5.
In response to locomotory cues, many motile cells have been shown to reposition their centrosome to a location in front of the nucleus, towards the direction of cell migration. We examined centrosome position in PtK(2) epithelial cells treated with hepatocyte growth factor (HGF), which stimulates motility but, unlike chemotactic agents or wounding of a monolayer, provides no directional cues. To observe centrosome movement directly, a plasmid encoding human gamma tubulin fused to the green fluorescent protein was expressed in HGF-treated cells. In cells whose movements were unconstrained by neighboring cells, we found that the position of the centrosome was not correlated with the direction of cell locomotion. Further, in cells where the direction of locomotion changed during the observation period, the centrosome did not reorient toward the new direction of locomotion. Analysis of centrosome and nuclear movement showed that motion of the centrosome often lagged behind that of the nucleus. Analysis of 249 fixed cells stained with an antibody to gamma tubulin confirmed our observations in live cells: 69% of the cells had centrosomes behind the nucleus, away from the direction of locomotion. Of these, 41% had their centrosome in the retraction tail. Confocal microscopy showed that the microtubule array in HGF treated PtK(2) cells was predominantly non-centrosomal. Because microtubules are required for efficient cellular locomotion, we propose that non-centrosomal microtubules stabilize the direction of locomotion without a requirement for reorientation of the centrosome. 相似文献
6.
T J Keating G G Borisy 《Biology of the cell / under the auspices of the European Cell Biology Organization》1999,91(4-5):321-329
While microtubule (MT) arrays in cells are often focused at the centrosome, a variety of cell types contain a substantial number of non-centrosomal MTs. Epithelial cells, neurons, and muscle cells all contain arrays of non-centrosomal MTs that are critical for these cells' specialized functions. There are several routes by which non-centrosomal MTs can arise, including release from the centrosome, cytoplasmic assembly, breakage or severing, and stabilization from non-centrosomal sites. Once formed, MTs that are not tethered to the centrosome must be organized, which can be accomplished by means of self-organization or by capture and nucleation of MTs where they are needed. The presence of free MTs requires stabilization of minus ends, either by MT-associated proteins or by an end-capping complex. Although some of the basic elements of free MT formation and organization are beginning to be understood, a great deal of work is still necessary before we have a complete picture of how non-centrosomal MT arrays are assembled in specific cell types. 相似文献
7.
Microtubules in interphase mammalian cells usually form a radial array with minus-ends concentrated in the central region and plus-ends placed at the periphery. This is accepted as correct, that two factors determinate the radial organization of microtubules - the centrosome, which nucleate and anchor the microtubules minus-ends, and the interaction of microtubules with cortical dynein, which positions centrosome in the cell center. However, it looks as if there are additional factors, affecting the radial structure of microtubule system. We show here that in aged Vero cytoplasts (17 h after enucleation) microtubule system lost radial organization and became chaotic. To clear up the reasons of that, we studied centrosome activity, its position in the cytoplasts and microtubule dynamics. We found that centrosome in aged cytoplasts was still active and placed in the central region of the cytoplasm, while after total disruption of the microtubules it was displaced from the center. Microtubules in aged cytoplasts were not stabilized, but they lost their ability to stop to grow near cell cortex and continued to grow reaching it. Aged cytoplast lamellae was partially depleted with dynactin though Golgi remained compact indicating dynein activity. We conclude that microtubule stoppage at cell cortex is mediated by some (protein) factors, and these factors influence radial structure of microtubule system. It seems that the key role in centrosome positioning is played by dynein complexes anchored everywhere in the cytoplasm rather than anchored in cell cortex. 相似文献
8.
I. B. Brodsky E. S. Nadezhdina 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2010,4(3):249-256
Microtubule array in eukaryotic cells supports directed transport of various cargoes driven by motor proteins. The arrangement
of microtubules in cytoplasm is not stochastic; they are organized in a certain way setting a system of coordinates for intracellular
transport. Most cultured fibroblast-like cells possess a radial microtubule array with the minus ends of microtubules gathered
on the centrosome and plus ends directed towards the periphery of the cell. Mechanisms that regulate the formation of radial
microtubule system remain unclear. Usually centrosome works as a microtubule-organizing center; however, the radial system
of microtubules can be formed without centrosome participation. At least in some cases microtubule network can be organized
by dynein-dynactin complexes associated with membrane vesicles. Membrane vesicles can nucleate microtubules, anchor them and
move along them. However, the role of membrane organelles in microtubule organization began to attract attention of researches
only recently. It this review we summarize the data indicating that membrane organelles can organize microtubules, providing
“tracks” for their subsequent transport. 相似文献
9.
Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network 总被引:6,自引:0,他引:6
Efimov A Kharitonov A Efimova N Loncarek J Miller PM Andreyeva N Gleeson P Galjart N Maia AR McLeod IX Yates JR Maiato H Khodjakov A Akhmanova A Kaverina I 《Developmental cell》2007,12(6):917-930
Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front. 相似文献
10.
《Cell cycle (Georgetown, Tex.)》2013,12(7):921-928
Centrosome is the major microtubule organizing center in mammalian cells that plays a critical role in a variety of cellular events by the microtubule arrays emanating from it. Despite its significance, the molecular mechanisms underlying the structure and function of the centrosome are still not clear. Herein we describe the identification of three isotypes of human ninein by expression library screening with autoimmune sera from CREST patients. All three ninein isotypes exhibit centrosomal localization throughout the cell cycle when GFP-tagged fusion proteins are expressed transiently in mammalian cells. Construction of serial deletions of GFP-tagged ninein reveals that a stretch of three leucine zippers with a flanking sequence is required and sufficient for centrosomal targeting. Overexpression of ninein results in mislocalization of ?-tubulin, recruiting it to ectopic (non-centrosomal) ninein-containing sites which are not active in nucleating microtubules. In these cells, nucleation of microtubules from the centrosome is also inhibited. These results thus suggest a regulatory role for ninein in microtubule nucleation. 相似文献
11.
《Cell cycle (Georgetown, Tex.)》2013,12(15)
Although the centrosome is traditionally viewed as cell’s principle microtubule organizing center (MTOC), regulation of microtubule dynamics at the cell cortex plays an equally important role in the formation of the steady-state microtubule network. Several recent studies, including one published in this issue, reveal that complex signaling mechanisms associated with adherence junctions influence both microtubule nucleation at the centrosome, and the stability of non-centrosomal microtubules. In the mid 1980s Marc Kirschner and Timothy Mitchison proposed an elegant “search-and-capture” hypothesis that seemed to explain how cells manage to convert a simple radial array of microtubules produced by the centrosome into the complex and precisely regulated asymmetric network found in a typical polarized cell. The key to this mechanism was the selective stabilization of inherently dynamic microtubule plus ends at the certain parts of cell cortex.4 Subsequently, it was shown that microtubule plus ends can in fact be captured and stabilized at diverse cortical loci including focal adhesions and adherence junctions. These observations provided direct support to the search-and-capture hypothesis. However, in recent years it became clear that role of cell cortex in the regulation of microtubule dynamics goes beyond simple stabilization of the plus ends. For example, there is evidence that integrin β1 is involved in the regulation of microtubule nucleation at the centrosome.6 Further, in polarized epithelia, cell cortex serves as the dominant MTOC, effectively replacing the centrosome.5 Thus, cell-cortex mechanisms affect microtubule dynamics both at their plus- and minus ends. The challenge now is to identify molecular pathways underlying this regulation. A study in this issue of Cell Cycle (Shtutman et al.) suggests that α-catenin, a major component of adherence junctions is responsible for promoting microtubule nucleation and/or stability in a centrosome-independent fashion. Shtutman and coworkers used centrosome-free cytoplasts. The number of microtubules in these cytoplasts is low in the absence of cell-cell contacts but increases to near-normal levels in confluent cultures3 or upon overexpression of cadherins1 suggesting that adherence junctions somehow regulate microtubule dynamics. Shtutman and coworkers now demonstrate a similar increase in microtubule density can be induced by overexpression of a membrane-targeted α catenin. This is an exciting finding because α-catenin is also directly involved in the regulation of actin dynamics2 and thus this molecule emerges as a central player in the global regulation of the cytoskeleton in response to extracellular interactions. Interestingly, expression of non-membrane-targeted α-catenin only mildly increased the density of microtubule network in centrosome-free cytoplasts suggesting that α-catenin needs to be engaged in an activation event at the cell cortex, perhaps within the adherence junction. Although formation of cell-cell junctions clearly increases the density of microtubule network, microtubule nucleation appears to occur throughout the cytoplasm and not preferentially at adherence junctions in these cells.1 Thus, local interactions at adherence junctions ultimately result in the propagation of a certain factor(s) that influences global microtubule dynamics. The exact nature of this factor or even the general layout of the pathway that alters microtubule dynamics in response to cortical interactions remain unknown. However, the demonstration that α-catenin is one of the molecular players required for this pathway is an important towards the understanding the link between extracellular interactions and microtubule dynamics. Further ReadingChausovsky A, Bershadsky AD, Borisy GG. Cadherin-mediated regulation of microtubule dynamics. Nat Cell Biol 2000; 2:797- 804. Gates J, Peifer M. Can 1000 reviews be wrong? Actin, alpha-Catenin, and adherens junctions. Cell 2005; 123:769-72. Karsenti E, Kobayashi S, Mitchison T, Kirschner M. Role of the centrosome in organizing the interphase microtubule array: properties of cytoplasts containing or lacking centrosomes. J Cell Biol 1984; 98:1763-76. Kirschner M, Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell 1986; 45:329-42. Reilein A, Yamada S, Nelson WJ. Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells. J Cell Biol 2005; 171:845-55. Reverte CG, Benware A, Jones CW, LaFlamme SE. Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis. J Cell Biol 2006; 174:491-7. 相似文献
12.
Ste20-related protein kinase LOSK (SLK) controls microtubule radial array in interphase 总被引:1,自引:0,他引:1
Burakov AV Zhapparova ON Kovalenko OV Zinovkina LA Potekhina ES Shanina NA Weiss DG Kuznetsov SA Nadezhdina ES 《Molecular biology of the cell》2008,19(5):1952-1961
Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types. 相似文献
13.
Gamma-tubulin complexes and microtubule organization 总被引:6,自引:0,他引:6
Microtubule nucleation requires gamma-tubulin, which exists in two main protein complexes: the gamma-tubulin small complex, and the gamma-tubulin ring complex. During mitosis, these complexes accumulate at the centrosome to support spindle formation. Gamma-tubulin complexes are also present at non-centrosomal microtubule nucleation sites, both in interphase and in mitosis. In interphase, non-centrosomal nucleation enables the formation of microtubule bundles or networks of branched microtubules. Gamma-tubulin complexes may be involved not only in microtubule nucleation, but also in regulating microtubule dynamics. Recent findings indicate that the dynamics of microtubule plus-ends are altered, depending on the expression of gamma-tubulin complex proteins. 相似文献
14.
There is broad agreement that cells reconfigure their microtubules through rapid bouts of assembly and disassembly, as described by the mechanism known as dynamic instability. However, many cell types have complex patterns of microtubule organization that are not entirely explicable by dynamic instability. There is growing evidence that microtubules can be moved into new patterns of organization by forces generated by molecular motor proteins. Studies on several cell types support a model called 'cut and run' in which long microtubules are stationary, but relatively short microtubules are mobile. In this model, cells mobilize their microtubules by severing them into short pieces, using enzymes such as katanin and spastin that break the lattice of the microtubule polymer. After being reorganized, the short microtubules can once again elongate and lose their mobility. Microtubule severing is also crucial for a variation of 'cut and run' in which the severed microtubules are reorganized by means of treadmilling. 相似文献
15.
EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and fusion 总被引:1,自引:0,他引:1
During muscle differentiation, myoblasts elongate and fuse into syncytial myotubes [1]. An early event during this process is the remodeling of the microtubule cytoskeleton, involving disassembly of the centrosome and, crucially, the alignment of microtubules into a parallel array along the long axis of the cell [2-5]. To further our understanding on how microtubules support myogenic differentiation, we analyzed the role of EB1-related microtubule-plus-end-binding proteins. We demonstrate that EB3 [6] is specifically upregulated upon myogenic differentiation and that knockdown of EB3, but not that of EB1, prevents myoblast elongation and fusion into myotubes. EB3-depleted cells show disorganized microtubules and fail to stabilize polarized membrane protrusions. Using live-cell imaging, we show that EB3 is necessary for the regulation of microtubule dynamics and microtubule capture at the cell cortex. Expression of EB1/EB3 chimeras on an EB3-depletion background revealed that myoblast fusion depends on two specific amino acids in the calponin-like domain of EB3, whereas the interaction sites with Clip-170 and CLASPs are dispensable. Our results suggest that EB3-mediated microtubule regulation at the cell cortex is a crucial step during myogenic differentiation and might be a general mechanism in polarized cell elongation. 相似文献
16.
Plant cell cortical microtubule arrays attain a high degree of order without the benefit of an organizing center such as a centrosome. New assays for molecular behaviors in living cells and gene discovery are yielding insight into the mechanisms by which acentrosomal microtubule arrays are created and organized, and how microtubule organization functions to modify cell form by regulating cellulose deposition. Surprising and potentially important behaviors of cortical microtubules include nucleation from the walls of established microtubules, and treadmilling-driven motility leading to polymer interaction, reorientation, and microtubule bundling. These behaviors suggest activities that can act to increase or decrease the local level of order in the array. The SPIRAL1 (SPR1) and SPR2 microtubule-localized proteins and the radial swollen 6 (rsw-6) locus are examples of new molecules and genes that affect both microtubule array organization and cell growth pattern. Functional tagging of cellulose synthase has now allowed the dynamic relationship between cortical microtubules and the cell-wall-synthesizing machinery to be visualized, providing direct evidence that cortical microtubules can organize cellulose synthase complexes and guide their movement through the plasma membrane as they create the cell wall. 相似文献
17.
A microtubule network on the basal cortex of polarized epithelial cells consists of non-centrosomal microtubules of mixed polarity. Here, we investigate the proteins that are involved in organizing this network, and we show that end-binding protein 1 (EB1), adenomatous polyposis coli protein (APC) and p150Glued - although considered to be microtubule plus-end-binding proteins - are localized along the entire length of microtubules within the network, and at T-junctions between microtubules. The network shows microtubule behaviours that arise from physical interactions between microtubules, including microtubule plus-end stabilization on the sides of other microtubules, and sliding of microtubule ends along other microtubules. APC also localizes to the basal cortex. Microtubules grew over and paused at APC puncta; an in vitro reconstituted microtubule network overlaid APC puncta; and microtubule network reconstitution was inhibited by function-blocking APC antibodies. Thus, APC is a component of a cortical template that guides microtubule network formation. 相似文献
18.
Microtubules take part in various cell processes, including cell polarization, migration, intercellular transport, and some
others. Therefore, the spatial organization of microtubules is crucial for normal cell behavior. Fibroblasts have radial microtubule
arrays that consist of microtubules that run from the centrosome. Two components compose this microtubule array, i.e., (1)
minus ends attached to the centrosome microtubules with their plus ends radiating to the cell periphery and (2) free microtubules
with ends not attached to the centrosome. Distinctions in the dynamic properties, intercellular organization, and structure
of centrosome-attached and free microtubules allow us to assume that their cellular functions are also different. To study
centrosome-attached and free microtubules functions, we used cytoplasts, i.e., nucleus-lacking cellular fragments that, under
certain conditions, also lose their centrosomes. In these cytoplasts, there are only free microtubules. The shape, general
morphology, and size of cytoplasts that retain their centrosomes differ only slightly from whole cells. Cytoplasts who have
lost their centrosomes have an extremely thin network of microtubules located in their central region; furthermore, they lose
the shape that is typical for fibroblast and become rough lamellae with protrusions. The internal architecture of the cytoplasm
and organoid arrangement are also broken. Saltatory movements in cytoplasts with centrosomes are similar to those in whole
cells; in cytoplasts without centrosomes, saltatory movements occur with velocities that are twofold less and by shorter distances.
Saltatory movements of granules in centrosome-lacking cytoplasts took place basically in the central region of cytoplast and
were less ordered than in whole cells and in cytoplasts with centrosomes. We believe that radial organized microtubules ensure
the effective transport and dynamical interaction of microtubule plus ends with cellular cortical structures, which is sufficient
to support the common fibroblast-like shape, whereas the disorganized free microtubules are not able to maintain the external
fibroblast shape and its intercellular organization. 相似文献
19.
In the internal cytoplasm of interphase cells the density of microtubules is the highest in the centrosome area and decreases to the cell periphery. As a rule, the quantity of fluorescent microtubules cannot be counted up in the internal cytoplasm, but it is possible to estimate microtubules quantity using measuring of their optical density. In living 3T3 and CHO cells the microtubules optical density decreased according to different mathematical dependences that apparently reflected the differences of their microtubule system organization. To determine appropriateness that circumscribe the reduction of microtubules optical density from the centrosome region to the direction of cell margin, we modeled cell contours with the certain ratio and interposition of centrosome-attached and free microtubules in vector schedules CorelDraw program. The decrease of optical density was analyzed in MetaMorph program as it was described earlier (Smurova et al., 2002). It was shown that fluorescent microtubules optical density decreased exponentially (y = ae(-bx)) if the system joined only microtubules growing from the centrosome up to the cell margin. The curve became smoother in the case of not all radial centrosome-attached microtubules reached the margin, and adding of free microtubules into the system led to the sharp fall in optical density in the centrosome area and to its gradual decrease at the cell periphery. The increase in free microtubules quantity changed the character of the curve describing the reduction of optical density microtubule system which included free and centrosome-attached microtubules in proportions of 5 : 1 was described by the equation of linear regression (f= k . x + b). Thus, the mathematical dependence describing the microtubules distribution from the centrosome to the cell periphery, depends on the ratio of microtubules and their relative positioning in the cell volume. The data obtained using model systems have coincided with the results of experiments. The graphs which described the increase in microtubules optical density during microtubule repolymerization after nocodazole treatment, corresponded to the graphs for model cells. Thus, the method we used allows to analyze the microtubule system in the cases when the direct observation of individual microtubules is difficult. 相似文献
20.
Olga N. Zhapparova Artem I. Fokin Nadezhda E. Vorobyeva Sofia A. Bryantseva Elena S. Nadezhdina 《Molecular biology of the cell》2013,24(20):3205-3214
The microtubule- and centrosome-associated Ste20-like kinase (SLK; long Ste20-like kinase [LOSK]) regulates cytoskeleton organization and cell polarization and spreading. Its inhibition causes microtubule disorganization and release of centrosomal dynactin. The major function of dynactin is minus end–directed transport along microtubules in a complex with dynein motor. In addition, dynactin is required for maintenance of the microtubule radial array in interphase cells, and depletion of its centrosomal pool entails microtubule disorganization. Here we demonstrate that SLK (LOSK) phosphorylates the p150Glued subunit of dynactin and thus targets it to the centrosome, where it maintains microtubule radial organization. We show that phosphorylation is required only for centrosomal localization of p150Glued and does not affect its microtubule-organizing properties: artificial targeting of nonphosphorylatable p150Glued to the centrosome restores microtubule radial array in cells with inhibited SLK (LOSK). The phosphorylation site is located in a microtubule-binding region that is variable for two isoforms (1A and 1B) of p150Glued expressed in cultured fibroblast-like cells (isoform 1B lacks 20 amino acids in the basic microtubule-binding domain). The fact that SLK (LOSK) phosphorylates only a minor isoform 1A of p150Glued suggests that transport and microtubule-organizing functions of dynactin are distinctly divided between the two isoforms. We also show that dynactin phosphorylation is involved in Golgi reorientation in polarized cells. 相似文献