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1.
Ikeda K Brown JA Yagi T Norrander JM Hirono M Eccleston E Kamiya R Linck RW 《The Journal of biological chemistry》2003,278(9):7725-7734
Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized "ribbon" of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an approximately 96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility. 相似文献
2.
Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly
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B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components. 相似文献
3.
CHLAMYDOMONAS FLAGELLA : II. The Distribution of Tubulins 1 and 2 in the Outer Doublet Microtubules 总被引:14,自引:9,他引:5
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Quantitative ultrastructural analysis and quantitative gel electrophoresis of preparations of selectively solubilized Chlamydomonas outer doublets indicated that tubulins 1 and 2 were present in both the A tubule and the B tubule, and that only tubulin 1 was present in the three protofilaments which form the wall ("partition") between the lumens of the A and B tubules. The data suggested that the remaining protofilaments of the outer doublet were grouped together in pairs containing the same type of tubulin, pairs containing tubulin 1 alternating with pairs containing tubulin 2. These findings were used to construct models for the arrangement of the two tubulins in the outer doublet. Further analysis by isoelectric focusing resolved tubulins 1 and 2 into at least five bands. 相似文献
4.
Bovine brain tubulin, purified by phosphocellulose chromatography (PC), was assembled in the presence of 10% dimethyl sulfoxide (DMSO), and the reaction was monitored turbidimetrically. Samples were fixed in glutaraldehyde-tannic acid after completion of polymerization, as indicated by no further change in absorbance, and then sectioned and studied electron microscopy, with special attention being given to the arrangement of protofilaments in the walls of formed elements. Samples of PC-tubulin were polymerized in buffer having various pH values from 6.0 to 7.7. At the lower pH values, only branched and flattened ribbons of protofilaments are formed. At intermediate values, the ribbons are unbranched, narrower, and more curved in cross section; complete microtubules are also seen. At the higher pH values, the predominate formed elements are complete microtubules. Most of the complete microtubules examined in this study had 14 wall protofilaments. The effect of pH on tubulin assembly was shown not to be an effect of DMSO. The dimers of associated protofilaments in ribbons and microtubules are conceptually viewed as having trapezoidal profiles in cross section, and, as additional dimers are added, the "C"-shaped ribbon closes to form a tube. The tilt angle of the lateral surfaces of the "trapezoidal" dimers will determine the number of wall protofilaments in the microtubules. At low pH, it is theorized that the trapezoidal profile of the dimer is shifted to a more rectangular configuration such that flat ribbons are formed by the lateral association of dimers. Also, variously shaped ribbon structures are formed at intermediate pH values, including "S"- and "W"-shaped structures, and elements shaped like a figure "6," all representing ribbons viewed in cross section. By visualizing the trapezoidal dimer in three-dimensions, and by arbitrarily indexing its six binding surfaces, it is possible to discuss interdimer binding in terms of preferred and possible binding interactions. 相似文献
5.
Stephens RE 《Cell motility and the cytoskeleton》2000,47(2):130-140
Even in the presence of colchicine or Taxol(R), sea urchin embryonic cilia undergo substantial steady-state turnover, with a rate of tubulin incorporation approaching half that seen in full regeneration [Stephens: Mol Biol Cell 8:2187-2198, 1997]. Preliminary experiments suggest that tubulin incorporates differentially into the most stable portion of the outer doublet, the junctional protofilaments [Stephens: Cell Struct Funct 24:413-418, 1999]. To explore this possibility further, embryos of the sea urchin Tripneustes gratilla, a ciliary length inducible system [Stephens: J Exp Zool 269:106-115, 1994a], were pulse labeled with (3)H leucine during steady-state turnover or induced elongation, followed by regeneration in the presence of unlabeled leucine. Cilia were isolated by hypertonic shock and fractionated into detergent-soluble membrane plus matrix, thermally-solubilized microtubule walls, and insoluble 9-fold symmetric remnants of A-B junctional protofilaments plus associated architectural elements. The fractions were resolved by SDS-PAGE and the specific activity of alpha-tubulin was determined. In cilia undergoing turnover or elongation during an isotope pulse, the specific activity of tubulin in the junctional region approximated that of precursor membrane plus matrix tubulin but surpassed that of the tubule wall by a factor of approximately 1.5. In cilia regenerated during an isotope chase, the specific activity of junctional tubulin exceeded that of both the membrane plus matrix and the tubule wall by a similar factor. These data indicate that tubulin is preferentially incorporated into junctional protofilaments during steady-state turnover, induced elongation and regeneration. A model for directional incorporation based on surface lattice discontinuities in the outer doublet is proposed. 相似文献
6.
Arrangement of tubulin subunits and microtubule-associated proteins in the central-pair microtubule apparatus of squid (Loligo pealei) sperm flagella 总被引:6,自引:4,他引:2
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《The Journal of cell biology》1981,89(2):309-322
This study provides a comprehensive, high-resolution structural analysis of the central-pair microtubule apparatus of sperm flagella. It describes the arrangement of several microtubule-associated "sheath" components and suggests, contrary to previous thinking, that microtubules are structurally asymmetric. The two microtubules of the central pair are different in several respects: the C2 tubule bears a single row of 18-nm-long sheath projections with an axial periodicity of 16 nm, whereas the C1 tubule possesses rows of 9-nm globular sheath components with an axial repeat of 32 nm. The lumen of the C2 tubule always appears completely filled with electron-dense material; that of the C1 tubule is frequently hollow. The C2 tubule also possesses a series of beaded chains arranged around the microtubule; the beaded chains are composed of globular subunits 7.5-10 nm in diameter and appear to function in the pairing of the C1 and C2 tubules. These findings indicate: that the beaded chains are not helical, but assume the form of lock washers arranged with a 16-nm axial periodicity on the microtubule; and that the lattice of tubulin dimers in the C2 tubule is not helically symmetric, but that there are seams between certain pairs of protofilaments. Proposed lattice models predict that, because of these seams, central pair and perhaps all singlet microtubules may contain a ribbon of 2-5 protofilaments that are resistant to solubilization; these models are supported by the results of the accompanying paper (R. W. Linck, and G. L. Langevin. 1981. J. Cell Biol. 89: 323-337. 相似文献
7.
8.
GTP-dependent in vitro polymerization of rat brain microtubular protein is inhibited to 50% by substoichiometric concentrations of the antimitotic drugs colchicine (0.12 mol/mol of tubulin) and podophyllotoxin (0.14 mol/mol of tubulin). Substitution of pp(CH2)pG2 for GTP, however, results in an extensive microtubular protein polymerization at such concentrations. In the presence of pp(CH2)pG, suprastoichiometric concentrations of podophyllotoxin (19 mol/mol of tubulin) are required to inhibit the polymerization process by 50%. Colchicine is very ineffective since 3 × 105 moles/mole of tubulin are required to give a 50% inhibition. Electron microscopical analysis shows that the polymers formed by microtubular protein in the presence of suprastoichiometric concentrations of drugs are not the normal short microtubules typical of pp(CH2)pG-driven polymerization, but are ribbons with three or four protofilaments. The colchicine content of the harvested ribbons has been measured directly and found to be approximately 0.8 moles colchicine/mole of tubulin. Treatment of microtubular protein with substoichiometric concentrations of drugs results in an increase in the number of protofilaments forming the ribbons. Many of the ribbons can close into morphologically normal microtubules when microtubular protein is treated with only 0.05 moles of either colchicine or podophyllotoxin per mole of tubulin. 相似文献
9.
Richard Linck Xiaofeng Fu Jianfeng Lin Christna Ouch Alexandra Schefter Walter Steffen Peter Warren Daniela Nicastro 《The Journal of biological chemistry》2014,289(25):17427-17444
Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems. 相似文献
10.
Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility. 相似文献
11.
In the presence of glycerol, microtubule proteins reassemble into both microtubules and protofilament ribbons with C- and S-shaped cross-section profiles. By means of electron micrographs of cross-sectioned assemblies, we have demonstrated that, during the steady state, the percentage of ribbons, especially of C-shaped ones, decreases in favour of the formation of microtubules. The following conversion modes are discussed: A, closure of the protofilament wall by increasing its curvature; B, lateral association of C-ribbons; C, completion of C-ribbons to microtubules by lateral association of tubulin; D, disassembly of ribbons and elongation of microtubules. We conclude that ribbon disassembly proceeding in an end-wise fashion and microtubule elongation is the favoured mode of conversion. Microtubule-associated proteins were found to be required for the steady-state conversions of ribbons into microtubules. In the absence of microtubule-associated proteins, C-ribbons associate laterally, forming S-ribbons. It is shown that the protofilaments of the counter-curved parts of S-ribbons have the same polarity. 相似文献
12.
FtsZ, the primary cytoskeletal element of the Z ring, which constricts to divide bacteria, assembles into short, one-stranded filaments in vitro. These must be further assembled to make the Z ring in bacteria. Conventional electron microscopy (EM) has failed to image the Z ring or resolve its substructure. Here we describe a procedure that enabled us to image reconstructed, inside-out FtsZ rings by negative-stain EM, revealing the arrangement of filaments. We took advantage of a unique lipid that spontaneously forms 500 nm diameter tubules in solution. We optimized conditions for Z-ring assembly with fluorescence light microscopy and then prepared specimens for negative-stain EM. Reconstituted FtsZ rings, encircling the tubules, were clearly resolved. The rings appeared as ribbons of filaments packed side by side with virtually no space between neighboring filaments. The rings were separated by variable expanses of empty tubule as seen by light microscopy or EM. The width varied considerably from one ring to another, but each ring maintained a constant width around its circumference. The inside-out FtsZ rings moved back and forth along the tubules and exchanged subunits with solution, similarly to Z rings reconstituted outside or inside tubular liposomes. FtsZ from Escherichia coli and Mycobacterium tuberculosis assembled rings of similar structure, suggesting a universal structure across bacterial species. Previous models for the Z ring in bacteria have favored a structure of widely scattered filaments that are not in contact. The ribbon structure that we discovered here for reconstituted inside-out FtsZ rings provides what to our knowledge is new evidence that the Z ring in bacteria may involve lateral association of protofilaments. 相似文献
13.
CHLAMYDOMONAS FLAGELLA : I. Isolation and Electrophoretic Analysis of Microtubules, Matrix, Membranes, and Mastigonemes 总被引:84,自引:62,他引:22
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G. B. Witman K. Carlson J. Berliner Joel L. Rosenbaum 《The Journal of cell biology》1972,54(3):507-539
Methods were developed for the isolation of Chlamydomonas flagella and for their fractionation into membrane, mastigoneme, "matrix," and axoneme components. Each component was studied by electron microscopy and acrylamide gel electrophoresis. Purified membranes retained their tripartite ultrastructure and were shown to contain one high molecular weight protein band on electrophoresis in sodium dodecyl sulfate (SDS)-urea gels. Isolated mastigonemes (hairlike structures which extend laterally from the flagellar membrane in situ) were of uniform size and were constructed of ellipsoidal subunits joined end to end. Electrophoretic analysis of mastigonemes indicated that they contained a single glycoprotein of ~ 170,000 daltons The matrix fraction contained a number of proteins (particularly those of the amorphous material surrounding the microtubules), which became solubilized during membrane removal. Isolated axonemes retained the intact "9 + 2" microtubular structure and could be subfractionated by treatment with heat or detergent. Increasing concentrations of detergent solubilized axonemal microtubules in the following order: one of the two central tubules; the remaining central tubule and the outer wall of the B tubule; the remaining portions of the B tubule; the outer wall of the A tubule; the remainder of the A tubule with the exception of a ribbon of three protofilaments. These three protofilaments appeared to be the "partition" between the lumen of the A and B tubule. Electrophoretic analysis of isolated outer doublets of 9 + 2 flagella of wild-type cells and of "9 + 0" flagella of paralyzed mutants indicated that the outer doublets and central tubules were composed of two microtubule proteins (tubulins 1 and 2) Tubulins 1 and 2 were shown to have apparent molecular weights of 56,000 and 53,000 respectively 相似文献
14.
《The Journal of cell biology》1978,79(2):500-515
Flagellar outer doublet microtubules were solubilized by use of sonication, and the tubulin was reassembled in vitro into single microtubules containing 14 and 15 protofilaments. The tubulin assembly was dependent on both the KCl and tubulin concentrations, exhibiting a critical concentration of 0.72 mg/ml at optimum solvent conditions. Flagellar tubulin was purified by cycles of temperature-dependent assembly-disassembly and molecular sieve chromatography, and characterized by two-dimensional gel electrophoresis. Although doublet microtubules were not formed in vitro, outer doublet tubulin assembled onto intact A- and B-subfibers of outer doublet microtubules and basal bodies of Chlamydomonas; the rate of assembly from the distal ends of these structures was greater than that from the proximal ends. Microtubule-associated proteins (MAPs) from mammalian brain stimulated outer doublet tubulin assembly, decorating the microtubules with fine filamentous projections. 相似文献
15.
Degradation of HMG-CoA reductase-induced membranes in the fission yeast, Schizosaccharomyces pombe 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1995,131(1):81-94
Although the overall structures of flagellar and cytoplasmic microtubules are understood, many details have remained a matter of debate. In particular, studies of the arrangement of tubulin subunits have been hampered by the low contrast of the tubulin subunits. This problem can now be addressed by the kinesin decoration technique. We have shown previously that the recombinant kinesin head domain binds to beta-tubulin, thus enhancing the contrast between alpha- and beta- tubulin in the electron microscope; this allows one to study the arrangement of tubulin dimers. Here we describe the lattices of the four different types of microtubules in eukaryotic flagellar axonemes (outer doublet A and B, central pair C1 and C2). They could all be labeled with kinesin head with an 8-nm axial periodicity (the tubulin dimer repeat), and all of them showed the B-surface lattice. This lattice is characterized by a 0.92-nm stagger between adjacent protofilaments. The B-lattice was observed on the axonemal microtubules as well as on extensions made by polymerizing porcine brain tubulin onto axonemal microtubules in the proximal and distal directions. This emphasizes that axonemal microtubules serve as high fidelity templates for seeding microtubules. The presence of a B-lattice implies that there must be a helical discontinuity ("seam") in the wall. This discontinuity is now placed near protofilaments A1 and A2 of the A- tubule, close to the inner junction between A- and B-microtubules. The two junctions differ in structure: the protofilaments of the inner junction (A1-B10) are staggered roughly by half a dimer, those of the outer junction (A10-B1) are roughly in register. Of the two junctions the inner one appears to have the stronger bonds, whereas the outer one is more labile and opens up easily, generating "composite sheets" with chevron patterns from which the polarity can be deduced (arrow in the plus direction). Decorated microtubules have a clear polarity. We find that all flagellar microtubules have the same polarities. The orientation of the dimers is such that the plus end terminates with a crown of alpha subunits, the minus end terminates with beta subunits which thus could be in contact with gamma-tubulin at the nucleation centers. 相似文献
16.
《The Journal of cell biology》1980,87(2):521-526
The structure of 14-protofilament microtubules reassembled from dogfish shark brain tubulin was analyzed by high resolution electron microscopy and optical diffraction. The simultaneous imaging of the protofilaments from near and far sides of these tubules produces a moire pattern with a period of approximately 96 nm. Optical diffraction patterns show that the 5-nm spots that arise from the protofilaments for the two sides of the tubule are not coincident but lie off the equator by a distance of 1/192 nm-1. These data provide evidence that in reassembled microtubules containing 14 protofilaments, the protofilaments are tilted 1.5 degrees with respect to the long axis of the tubule, giving a left-handed superhelix with a pitch of 2.7 micron. The hypothesis is that the tilt of the protofilaments occurs to accommodate the 14th protofilament. It is determined that when the 14th protofilament is incorporated, the 3-start helix is maintained, but the pitch angle changes from 10.5 degrees to 11.2 degrees, the angle between protofilaments measured from the center of the microtubule changes by 2 degrees, and the dimer lattice is discontinuous. These observations show that the tubulin molecule is sufficiently flexible to accomodate slight distortions at the lateral bonding sites and that the lateral bonding regions of the alpha and beta monomers are sufficiently similar to allow either alpha-alpha and beta-beta subunit pairing or alpha-beta subunit pairing. 相似文献
17.
Amos LA 《Trends in cell biology》1995,5(2):48-51
In 1974, optical diffraction and image analysis indicated that tubulin dimers in the cylindrically complete A-tubule of flagellar doublet microtubules are arranged with helical symmetry, while those in the incomplete B-tubule associate differently. Recently, electron micrographs of reassembled brain microtubules decorated with kinesin heads have shown that the tubulin dimers there are arranged as in the B-tubule. The lack of symmetry of microtubules assembled in vitro prompts Linda Amos to speculate here that the assembly process in vitro may differ from that occurring in the cell. 相似文献
18.
Basal bodies and centrioles play central roles in microtubule (MT)‐organizing centres within many eukaryotes. They share a barrel‐shaped cylindrical structure composed of nine MT triplet blades. Here, we report the structure of the basal body triplet at 33 Å resolution obtained by electron cryo‐tomography and 3D subtomogram averaging. By fitting the atomic structure of tubulin into the EM density, we built a pseudo‐atomic model of the tubulin protofilaments at the core of the triplet. The 3D density map reveals additional densities that represent non‐tubulin proteins attached to the triplet, including a large inner circular structure in the basal body lumen, which functions as a scaffold to stabilize the entire basal body barrel. We found clear longitudinal structural variations along the basal body, suggesting a sequential and coordinated assembly mechanism. We propose a model in which δ‐tubulin and other components participate in the assembly of the basal body. 相似文献
19.
Tendon elongation influences the amplitude of interpolated doublets in the assessment of activation in elderly men. 总被引:1,自引:0,他引:1
Christopher I Morse Jeanette M Thom Karen M Birch Marco V Narici 《Journal of applied physiology》2005,98(1):221-226
This study investigated the influence of tendon elongation (TE) on postcontraction doublet (PCD) torque in the assessment of activation in the plantar flexors of nine elderly men (EM, age 73.7 +/- 3.6 yr) and nine young men (YM, age 24.7 +/- 4.7 yr). Plantar flexion maximal voluntary contractions (MVC) and activation were assessed at ankle joint angles of -20 degrees (dorsiflexion), 0 degrees , and 20 degrees (plantar flexion). Across the ankle joint angles tested, compared with YM, the EM had a 36-49% lower plantar flexion MVC (P < 0.01), TE was greater by 25-31% (P < 0.01), and electromechanical delay was 65-108% greater (P < 0.01). Activation (PCD torque to interpolated doublet torque) was 15% lower in EM compared with YM at -20 degrees (P < 0.05), but no different at 0 and 20 degrees . In the EM, PCD torque relative to MVC torque was significantly lower at 20 degrees compared with 0 degrees (P < 0.05). Electromechanical delay was positively correlated with TE (R(2) = 0.489, P < 0.01). In conclusion, this investigation demonstrates that, although a negative association exists between TE and PCD torque, the consequence of a greater TE on the estimation of activation in EM is negligible. This is due to a greater influence of ankle joint angle on the occlusion of a superimposed doublet, which counteracts the lesser influence of joint angle on TE and PCD torque. However, a greater TE in EM was found to significantly increase electromechanical delay, which is expected to influence the time needed for postural readjustments. 相似文献
20.
Stephens RE 《Cell structure and function》1999,24(5):413-418
Previous pulse-chase labeling studies have shown that structural proteins incorporate into fully assembled sea urchin embryonic cilia at rates approaching those of full regeneration. When all background ciliogenesis was suppressed by taxol, the turnover of most proteins, including tubulin, continued. The present study utilized chemical dissection to explore the route of tubulin incorporation in the presence of taxol and also in steady-state cilia from prism stage embryos. Surprisingly, in cilia from untreated embryos, the most heavily labeled tubulin was found in the most stable portion of the doublet microtubles, the junctional protofilaments. With taxol, this preferential incorporation was suppressed, although control-level turnover still took place in the remainder of the doublet. This paradoxical result was confirmed by pulse-chase labeling and immediately isolating steady-state cilia, then isolating two additional crops of cilia regenerated, respectively, from pools of high and then decreased label. In each case, the level of label occurring in the tubulin from the junctional protofilaments, compared with that from the remainder of the doublet, correlated with the level of pool label from which it must exchange or assemble. These data indicate that ciliary outer doublet microtubules are dynamic structures and that the junctional region is not inert. Plausible mechanisms of incorporation and turnover of tubulin in fully-assembled, fully-motile cilia can now be assessed with regared to recent discoveries, particularly intraflagellar transport, distal tip incorporation, and treadmilling. 相似文献