The lamina propria of the large intestine is rich in macrophages, and they might be one of the first lines of the host defense in enterohemorrhagic Escherichia coli (EHEC) O157 : H7 infection. Although macrophages were infected with them, they can survive the EHEC O157 infection. We examined the structural rearrangements of the actin cytoskeleton during the microbial infection process. Macrophage actin filaments were rearranged in the following sequence ; 1) disappearance of the actin filament bundles in the cytoplasm, 2) accumulation of actin filaments under the cell surface, and 3) construction of actin networks underlying the endosome membrane. Before infection, actin filaments were distributed under the cell surface and in bundles located in the macrophage cytoplasm. Within 2 min, infection caused a rapid and marked loss of the actin filament bundles that had run parallel to the long axis of the cell. Concomitant with the loss, actin filaments became more markedly distributed under the cell surface. In the formation of the endosome, new networks of actin filaments were constructed below the phagosome membrane. The networks contained a large amount of actin as well as α fodrin-like immunoreactivity. The thickness of the networks reached about 400 nm under the phagosome membrane. The actin networks disappeared again after the bacterial digestion. The results of this study showed that actin filaments undergo three major rearrangements of the actin filaments during the infection in macrophages, and suggested that the third rearrangement is mediated by actin-binding proteins, such as α fodrin-like molecules. These morphological changes in macrophages were not clear after infection with other strains of Escherichia coli.

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This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterhemorrhagic Escherichia coli (EHEC) O157 : H7. Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro. The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II(SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor α (TNFα) and interleukin-1(IL-1), in the culture medium. Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes. During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II. The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained. Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium. Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157. The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages.

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Hepatic stellate cells (HSCs) were changed in their morphology, proliferative activity, and functions by culturing on type I collagen gel, as compared to the culture on polystyrene surface. HSCs have been found to produce extracellular matrix components and matrix metalloproteinases (MMPs). In this study, we have assessed the effects of several types of substrata on the expression of MMPs in HSC culture. MMP-1 expression was detectable in HSC culture on polystyrene surface and on type I collagen gel by immunofluorescence staining and reverse transcriptase-polymerase chain reaction (RT-PCR). The results from in situ zymography revealed the presence of interstitial collagenase activity around HSCs and along their cellular processes. Although proMMP-2 and proMMR-9 were detectable by gelatin zymography in the conditioned medium from both cultures using type I collagen gel and Matrigel as substratum, an active form of MMP-2 but not of MMP-9 was detected only in the culture using type I collagen as a substratum. Tissue inhibitor of metalloproteinase-2 expression was observed by RT-PCR in HSCs cultured on or in type I collagen gel, suggesting the suppression of MMP-2 activity detected in HSC culture using type I collagen. These results indicate a differential expression of MMP activity, hence the remodeling of extracellular matrix components is dependent on the substratum used for HSC culture. The HSC culture using several types of substrata appears to be a useful in vitro model to study the mechanism of extracellular matrix remodeling.

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The structure of tubulin, recently solved by electron crystallography, has given a first look at the molecular basis for some of the properties of tubulin and microtubules that have been observed over the last decades. We discuss how the structure relates to some of these properties, and how inferences about drug binding sites can explain some of the effects of the drugs on tubulin. Microbulules can form a highly dynamic system that requires careful tuning of the stability and properties of tubulin and its interactions with its many ligands. Understanding these interactions can provide fundamental information on the regulation of the microtubule system.

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We have studied the structure of microtubules decorated with kinesin motor domains in different nucleotide states by 3D electron microscopy. Having docked the atomic coordinates of both dimeric ADP. kinesin and tubulin heterodimer into a map of kinesin dimers bound to microtubules in the presence of ADP, we try to predict which regions of the proteins interact in the weakly binding state. When either the presence of 5’-adenylyimidodiphosphate (AMP-PNP) or an absence of nucleotides puts motor domains into a strongly-bound state, the 3D maps show changes in the motor domains which modify their interaction with beta-tubulin. The maps also show differences in beta-tubulin conformation compared with undecorated microtubules or those decorated with weakly-bound motors. Strongly-bound ncd appears to produce an identical change.

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Bacterial cell division protein FtsZ assembles into protofilaments, which can adopt a straight or curved conformation, similar to its eukaryotic homolog, tubulin. The straight protofilaments can assemble into sheets with a lattice similar to the microtubule wall. The curved protofilaments can form rings when adsorbed to a lipid monolayer, but in solution they form helices. 4 helices assemble together to make a tube, the characteristic polymer of the curved protofilament. GTP favors the straight conformation, while GDP favors the curved. We show here that addition of EDTA and GTP to tubes causes a rapid transformation to straight protofilament sheets. Apparently when the magnesium is chelated the GDP in the curved protofilaments dissociates rapidly and is replaced with GTP, and this GTP induces the transition to straight protofilaments.

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Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells. Using a computer-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosomes were stained with a DNA-specific fluorescent dye, Hoechst33342, or with rhodamine-conjugated histone. In fission yeast cells, microtubules were stained with α-tubulin fused with the jellyfish green fluorescent protein (GFP), and chromosomes were stained with Hoechst33342.

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The molecular mechanisms that allow elongation of the unstable microtubule lattice remain unclear. It is usually thought that the GDP-liganded tubulin lattice is capped by a small layer of GTP- or GDP-P_i-liganded molecules, the so called “GTP-cap”. Here, we point-out that the elastic properties of the microtubule lattice cause a difference in stability between the elongating tubulin sheet and the completed microtubule wall. The implications of our observations for microtubule structure and dynamics are discussed.

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This minireview summarizes the syntheses of various purinenucleotide analogues and their effects on microtubule (Mt) assembly. 27 analogues were so far synthesized and, together with 3 analogues commercially available (ITP, XTP and dGTP), their effects on Microtubule assembly were investigated. The positions C2, C6, C8, and ribose moiety of purine nucleotides were modified or substituted. It was found that the microenvironments of the purine base and ribose moiety are important for the nucleotides to support Mt assembly. Introduction of amino group into position C2 of ATP, formation of 2-amino ATP, caused Mt assembly substantially. 2-Amino deoxy ATP and deoxy GTP are more potent than GTP in supporting assembly. The introduction of reactive thiol group into C6 (6-SH-GTP) largely reduces the activity of the analogue to support assembly. However, sequestering reactivity of the thiol group by association with methyl group largely recovers the ability of the analogue to promote assembly. Free rotation of the glycosidic linkage was found to be also innevitable in promoting assembly, as the introduction of sulfur atom between C8 of the purine base and C2’ of the ribose moiety (formation of 8, 2’-S-cyclo purine nucleotides) caused total inhibition. Purinenucleoside triphosphate supports assembly better than GTP but the deoxy-type analogues are totally inhibitory. 2-Amino-8-hydroxy ATP and other analogues support assembly much better than does GTP. However, their diphosphate analogues are totally incapable of supporting assembly. Introduction of a bulky fluorescent probes into C3’ can be made to visualize the fluorescent signal in assembled Mts. Together with the suggestions proposed from electron chrystallography of zinc-induced tubulin sheets, interactions of the purine base and ribose moieties with surrounding amino acid residues are discussed.

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E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters, of ensconsin distribution and dynamics ; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.

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In the previous paper {Ookata et al., (1997) Biochemistry, 36: 249-259}, we identified two mitotic cdc2 kinase phosphorylation sites (Ser696 and Ser787) in the proline-rich region of human MAP4. One (Ser696) of them was also phosphorylated during interphase. A protein kinase responsible for interphase phosphorylation of Ser696 could necessarily be distinct from cdc2/cyclin B kinase. To get insights into a physiological role for Ser696 phosphorylation, we searched for a Ser696 kinase and for cellular conditions under which Ser696 is dephosphorylated. Because Ser696 conforms to the MAP kinase phosphorylation consensus motif (PXSP), MAP kinase was tested as a possible kinase phosphorylating Ser696. MAP kinase, in fact, did phosphorylate Ser696 in MTB3, the carboxy-terminal half of human MAP4 in vitro. Phosphorylation of Ser696 in HeLa cell extract was suppressed by a MAP kinase inhibitor, DBTM-0004. Also consistent with the notion that Ser696 is a MAP kinase site were the fact that serum-starvation induced dephosphorylation of Ser696 in HeLa cells, TIG-3 and MRC-5-30 human fibroblasts, while readdition of serum recovered Ser696 phosphorylation, albeit after a surprisingly long interval. Thus, phosphorylation of Ser696 of MAP4, most likely carried out by MAP kinase, may play a role in modulation of MAP4 activity in proliferating versus quiescent cells.

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Microtubules are intrinsically dynamic polymers. Two kinds of dynamic behaviors, dynamic instability and treadmilling, are important for microtubule function in cells. Both dynamic behaviors appear to be tightly regulated, but the cellular molecules and the mechanisms responsible for the regulation remain largely unexplored. While microtubule dynamics can be modulated transiently by the interaction of regulatory molecules with soluble tubulin, the microtubule itself is likely to be the primary target of cellular molecules that regulate microtubule dynamics. The antimitotic drugs that modulate microtubule dynamics serve as excellent models for such cellular molecules. Our laboratory has been investigating the interactions of small drug molecules and stabilizing microtubule-associated proteins (MAPs) with microtubule surfaces and ends. We find that drugs such as colchicine, vinblastine, and taxol, and stabilizing MAPs such as tau, strongly modulate microtubule dynamics at extremely low concentrations under conditions in which the microtubule polymer mass is minimally affected. The powerful modulation of the dynamics is brought about by the binding of only a few drug or MAP molecules to distinct binding sites at the microtubule surface or end. Based upon our understanding of the well-studied drugs and stabilizing MAPs, it is clear that molecules that regulate dynamics such as Kin 1 and stathmin could bind to a large number of distinct tubulin sites on microtubules and employ an array of mechanisms to selectively and powerfully regulate microtubule dynamics and dynamics-dependent cellular functions.

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Bovine microtubule-associated protein 4(MAP4) consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain of MAP4 is further divided into three subdomains : a region rich in proline and basic residues (Pro-rich region), a region containing four repeats of an assembly-promoting (AP) sequence, which consists of 22 amino acid residues (AP sequence region), and a hydrophobic tail region (Tail region). The subdomain structure of MAP4 microtubule binding domain is similar to those of other MAPs (MAP2 and τ). In order to study the function of each subdomain per se of bovine MAP4 microtubule-binding domain, we purified a series of truncated fragments of MAP4, expressed in Escherichia coli. Binding affinity of the PA₄T fragment (containing the Pro-rich region, the AP sequence region and the Tail region) is only four times higher than that of the A₄T fragment (containing the AP sequence region and the Tail region), while the microtubule nucleating activity of the PA₄T fragment is far greater. We propose that the Pro-rich region promotes the nucleation of microtubule assembly. The A₄ fragment (corresponding to the AP sequence region) stimulated the assembly of tubulin into cold-stable amorphous aggregates. The AP sequence region of MAP4 failed to promote microtubule assembly. On the other hand, the fragment has an activity to stimulate microtubule elongation. The function of the MAP4 Tail region is not clear at present. The A₄T fragment (containing the AP sequence region and the Tail region) promote both microtubule nucleation and elongation step, but the A₄ fragment only promotes microtubule elongation, suggesting that the Tail region is indispensable for the nucleation step. However, the fragment containing only the Tail region could not bind to microtubule. Although MAP4 was considered to be long, thin and flexible molecule, never the Tail region may contribute to be the proper folding of MAP4, and/or may interact with other molecules. We concluded that both the Pro-rich region and the AP sequence region take part in the promotion of tubulin polymerization, and that the former is important for the lateral protofilament-protofilament interaction, and the latter is important for the longitudinal affinity between each tubulin dimer in a protofilament.

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Stathmin, also referred to as Op18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in the control of cell proliferation, differentiation and activities. It interacts with several putative downstream target and / or partner proteins. One major action of stathmin is to interfere with microtubule dynamics, by inhibiting the formation of microtubules and / or favoring their depolymerization. Stathmin (S) interacts directly with soluble tubulin (T), which results in the formation of a T2S complex which sequesters free tubulin and therefore impedes microtubule formation. However, it has been also proposed that stathmin's action on microtubules might result from the direct promotion of catastrophes, which is still controversial. Phosphorylation of stathmin regulates its biological action : it reduces its affinity for tubulin and hence its action on microtubule dynamics, which allows for example progression of cells through mitosis. Stathmin is also the generic element of a protein family including the neural proteins SCG10, SCLIP and RB3/RB3’/RB3". Interestingly, the stathmin-like domains of these proteins also possess a tubulin binding activity in vitro. In vivo, the transient expression of neural phosphoproteins of the stathmin family leads to their localization at Golgi membranes and, as previously described for stathmin and SCG10, to the depolymerization of interphasic microtubules. Altogether, the same mechanism for microtubule destabilization, that implies tubulin sequestration, is a common feature likely involved in the specific biological roles of each member of the stathmin family.

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HPC-1/syntaxin 1A (HPC-1), which has been identified as a presynaptic membrane protein, is believed to regulate the synaptic exocytosis as a component of t-SNARE. The distribution of the protein, however, is not restricted to the synaptic terminal, but it has been found to locate on the axonal membrane. When the expression of HPC-1 was suppressed, neurite sprouting was enhanced in cultured neurons. These findings suggest that HPC-1 possesses other functions than the regulation of the membrane fusion in neuro-transmitter release. Rather it may also participate in the morphogenesis of neurons through membrane fusion, and possibly through cytoskeleton. HPC-1 has a sequence resemble to the assembly promoting sequence of heat stable MAPs in residues 89-106, suggesting that it can bind tubulin and be involved in microtubule system. Thus, both the tubulin binding property and the effect on microtubule assembly of HPC-1 were examined in vitro using a mutated HPC-1 lacking the C-terminal transmembrane region (HPC-ΔTM), which was overexpressed in E. coli. Affinity column chromatography showed that tubulin was found to bind HPC-1 directly. Synthetic peptide which corresponds to the residues 89-106 competitively inhibited the tubulin-HPC-1 binding, indicating that the sequence is responsible for the tubulin binding. In addition, chemical cross-linking with EDC revealed that one HPC-1 molecule can bind per one monomeric tubulin molecule. Light scattering measurement of microtubule polymerization showed that HPC-1 decreased the rate of the pure tubulin polymerization. Direct observation of single microtubules under dark-field microscopy showed that the growth rate of microtubule decreased by HPC-1. After shortening stopped, microtubules often spent attenuate phases, in which neither growing nor shortening was detected. When another mutant HPC-1 which is composed of residues 1-97 and lacks tubulin binding activity was used, however, the suppression of microtubule polymerization was not observed. These results suggest that HPC-1 is a potent regulator of microtubule polymerization, which directly bind tubulin subunit and decrease the polymerization activity.

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The existence of γ-tubulin was first reported approximately ten years ago, and it is appropriate to review the progress that has been made in γ-tubulin research and to discuss some of the unanswered questions about γ-tubulin function. γ-Tubulin is ubiquitous in eukaryotes and is generally quite conserved. Two highly divergent γ-tubulins have been discovered, however, one in Saccharomyces cerevisiae and one in Caenorhabditis elegans. Several organisms have two γ-tubulin genes. In Drosophila melanogaster, the two γ-tubulins differ significantly in sequence and expression pattern. In other organisms the two γ-tubulins are almost identical and expression patterns have not been determined. γ-Tubulin is located at microtubule organizing centers in many organisms, and it is also frequently associated with the mitotic spindle. γ-Tubulin is essential for the formation of functional mitotic spindles in all organisms that have been examined to date. In animal cells, complexes containing γ-tubulin are located at microtubule organizing centers where they nucleate the assembly of microtubles. In spite of the considerable progress that has been made in γ-tubulin research important questions remain to be answered. The exact mechanisms of microtubule nucleation by γ-tubulin complexes remain to be resolved as do the mechanisms by which microtubule nucleation from γ-tubulin complexes is regulated. Finally, there is evidence that γ-tubulin has important functions in addition to microtubule nucleation, and these functions are just beginning to be investigated.

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We characterized and compared the diffusion of beads coated with proteins such as cytoplasmic dynein, α-casein, and some immunoglobulins on microtubules. Such weak binding interactions could be common and convenient for concentrating proteins at the surface of cytoplasmic structures such as microtubules. In studying the motile behavior of anionic latex beads coated with limiting dilutions of cytoplasmic dynein, we observed that in addition to active movement, 20-50% of the beads moved back and forth in a random manner. The random movement was inhibited by depletion of ATP or addition of ADP or AMP-PNP. Mean-square-displacement analysis showed that the movement is a one-dimensional diffusion along the microtubule axis with a diffusion coefficient of 2.16×10^-10 cm²/sec. Histogram analysis of off-axis movements suggested that ∼60% of the diffusing beads followed the path of a single microtubule protofilament. Beads coated with proteins such as α-casein or a monoclonal immunoglobulin were also observed to diffuse on microtubules with a similar diffusion coefficient to cytoplasmic dynein. However, α-casein or immunoglobulin-bead diffusion was not ATP dependent and did not follow the paths of single protofilaments. Thus, although the environment of the microtubule surface can trap a variety of different protein-coated beads, cytoplasmic dynein's interaction is unusual in its ATP dependence and tracking on a single protofilament, which is consistent with its specific interaction with microtubules. Diffusive interactions could concentrate associating proteins and still allow for freedom of movement.

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Microtubule (MT) dynamics and organization change markedly during interphase-M phase transition of the cell cycle. This mini review focuses first on p220, a ubiquitous MT-associated protein of Xenopus. p220 is phosphorylated by p34^cdc2 kinase and MAP kinase in M phase, and concomitantly loses its MT-binding and MT-stabilizing activities. A cDNA encoding p220 was cloned, which identified p220 as a Xenopus homolog of MAP4, and p220 was therefore termed XMAP4. To examine the physiological relevance of XMAP4 phosphorylation during mitosis, Xenopus A6 cells were transfected with cDNA encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein (GFP). Mutations of serine and threonine within potential phosphorylation sites for p34^cdc2 kinase to nonphosphorylatable alanine interfered with mitosis-associated reduction in MT-affinity of XMAP4 and their overexpression affected chromosome movement during anaphase A. These results indicated that phosphorylation of XMAP4 by p34^cdc2 kinase is responsible for the decrease in its MT-binding and MT-stabilizing activities during mitosis which are important for chromosome movement during anaphase A. The second focus is on a novel monoclonal antibody W8C3, which recognizes α-tubulin. W8C3 stained spindle MTs but not interphase MTs of Xenopus A6 cells, although tubulin dimers in M phase and interphase were equally recognized by this antibody. The difference in MT staining pattern may be because the W8C3-recognition site on α-tubulin is sterically hidden in interphase MTs but not in spindle MTs.

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Microtubules assembled from pure tubulin in vitro are labile, rapidly depolymerized upon exposure to the cold. In contrast, in a number of cell types, cytoplasmic microtubules are stable, resistant to prolonged cold exposure. During the past years, the molecular basis of this microtubule stabilization in cells has been elucidated. Cold stability is due to polymer association with different variants of a calmodulin-regulated protein, STOP protein. The dynamic and hence the physiological consequences of STOP association with microtubules vary in different tissues. In neurons, STOP seems almost permanently associated with microtubules. STOP is apparently a major determinant of microtubule turnover in such cells and is required for normal neuronal differentiation. In cycling cells, only minor amounts of STOP are associated with interphase microtubules and STOP does not measurably affects microtubule dynamics. However, STOP is associated with mitotic microtubules in the spindle. Recent results indicate that such an association could be vital for meiosis and for the long-term fidelity of the mitotic process.

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The ciliated protozoa Tetrahymena contains two nuclei, a micronucleus and a macronucleus. In the vegetatively growing cell, the macronucleus divides amitotic while the micronucleus divides by mitosis. It has been indicated that microtubules are involved in macronuclear division and microtubules are observed to exist in the dividing macronucleus. To clarify the localization and the organization of microtubules in the amitotic dividing macronuclei, we used immunofluorescent staining technique. The microtubules were observed in the cytoplasm and macronucleus. The microtubules were organized and dynamically changed their distribution throughout the macronuclear division. We suggest a possibility that these microtubules are involved in ‹amitotic’ distribution of chromatin throughout the macronuclear division.

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We tested the stability of microtubules in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under the microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be microtubules by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be microtubules by immunostaining with ant-tubulin antibody. Significant number of microtubules in the established neurites remained longer than 1 hour after membrane removal. To investigate their stabilization mechanism, we transected the exposed microtubules by laser microbeam irradiation and observed their length changes with video-enhanced microscopy. Microtubule fragments started to shorten on both sides of the transection site, more rapidly from the newly generated plus ends than from the minus ends. The maximal rate as well as the pattern of shortening correlated with the time of transection ; microtubules transected later than 30 min after membrane removal shortened at rates less than 20μm-min and typically with intermittent pauses, while the more labile microtubules included in the earlier transections shortened continuously at higher rates. Microtubules in neurites were thus stabilized by (1) stopping disassembly at local sites including the plus ends, and (2) slowing disassembly along the length. Observations of the course of disassembly also suggested the presence of specialized points along microtubules which is involved in anchoring microtubules to the substratum or transiently stopping disassembly.

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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 (23). 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 tububin in fully-assembled, fully-motile cilia can now be assessed with regared to recent discoveries, particularly intraflagellar transport, distal tip incorporation, and treadmilling.

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Effects of kinase inhibitors on the preprophase band of microtubules in onion (Allium cepa L.) root tip cells were examined. Bundled microtubules in preprophase bands were dispersed on the cell cortex when onion seedlings were incubated with 2.5-5.0 mM 6-dimethylaminopurine. Fifteen min was enough for the bundled microtubules to disappear. Although many preprophase bands remained when the seedlings were incubated with 60μM staurosporin, these preprophase band microtubules were loosened and the width of the band became broad. These results sugget that some kinases are involved in the microtubule bundling in the preprophase band development.

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