摘要:

AbstractUsing several in vitro motility assays, we found that motility driven by the microtubule (MT) motors, kinesin and cytoplasmic dynein, could be inhibited by MAP2 but not by tau protein or the MT‐binding proteolytic fragment of MAP2.In MT gliding assays, even the presence of one MAP2 molecule per sixty‐nine tubulin dimers caused an inhibition of about 75% of MT motility at low concentrations of both motors. The percent inhibition of motility decreased with increasing concentration of either motor, suggesting that the inhibition was the result of competition for access to the MT surface. The decrease in the number of moving MTs with MAP2 was correlated with an increase in the frequency of release of moving MTs from the motor‐coated glass. In assays of in vitro vesicular organelle motility and formation of ER networks, the presence of MAP2 inhibited small vesicle movements and to a lesser extent ER network formation.To determine if competition for specific sites on the MT or coating of the MT surface inhibited motility, we used tau protein and the chymotryptic MT‐binding fragments of MAP2 to coat MTs. No inhibition was observed and there was even an increase in the number of attached and moving MTs in the gliding assay with tau‐coated MTs. Because MAP2, tau and the chymotryptic MT‐binding fragments of MAP2 bind to the same domain on tubulin, masking of the MT surface sites does not appear responsible for the inhibition of motility by MAP2. Rather, we suggest that the sidearm of MAP2 interfered with the interaction of motors with MTs and caused a dramatic increase in the rate of MT release. In vivo, MAP2 could play a major role in the generation of cellular polarity even at substoichiometric levels by inhibiting transport on microtubules in specific domains of the cytoplasm. © 1993 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970240102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThree forms of dynein (22S, 19S, and 12S) were purified fromParameciumcilia. Two classes of monoclonal antibodies against purified 22S dynein were generated. One class reacted on immunoblots with the heavy chains of 22S, 19S, and 12S dyneins; the second class reacted with an 88 kD intermediate chain of 22S dynein. Polyclonal antiserum to the heavy chains of 22S dynein reacted with the γ‐heavy chain of 22S and 19S dyneins. A previously described antiserum raised against 22S dynein [Travis et al.: Biochim. Biophys. Acta 966:73–83, 1988] recognized the γ‐heavy chain of 22S dynein which was also present in 19S and 12S dyneins, along with the 88 and 76 kD intermediate chains of 22S dynein. This antiserum was also able to immunoprecipitate dynein from crude extracts of cilia.Electron microscopy revealed that the 22S dynein consisted mainly of two‐headed particles with some three‐headed particles present. The 12S dynein was mainly one‐headed particles. The 19S dynein was a mixture of three‐, two‐, and one‐headed particles. The immunological and electron microscopic studies showed that 19S dynein arises from 22S dynein, and that 12S dynein is heterogeneous, composed of the γ‐heavy chain of 22S dynein and a unique dynein ATPase.The polyclonal antibodies were also used to detect cross‐reactive proteins in other organisms. Both the anti‐heavy chain and the anti‐22S dynein sera reacted strongly with 22S outer arm dynein ofTetrahymena, but not with the 14S dynein of this orga

ISSN:0886-1544    

DOI:10.1002/cm.970240103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe ciliateParamecium tetraureliapresents a powerful system to define the structural basis for dynein functional diversity within a single cell. This analysis will depend on the biochemical resolution of the dynein proteins. As an important first step, the three heavy chains of the ciliary outer arm dynein of paramecium were characterized. Sucrose density gradient centrifugation in a high salt buffer separated the dynein into a 22S species, which contained the α and β heavy chains, and a 12S species, which contained the α chain as well as the inner arm dynein heavy chains. Both the 22S and 12S species retained enzymatic latency as indicated by stimulation of MgATPase activity by 0.1% Triton X‐100. An unusual ATP‐independent V1‐like photolysis of only the β chain provided the basis for estimating that the β chain contributes almost half of the 22S MgATPase activity that is susceptible to V1 photolysis. The combination of the density gradient separation of the partially dissociated dynein and the ATP‐independent V1‐like photolysis of only the β chain led to the unambiguous assignment of the V1 photolytic products to the appropriate parent heavy chains. An estimate of the molecular sizes of the three heavy chains was obtained. The photolytic peptide maps, which define the ATP‐binding domains, were determined for the three heavy chains. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970240104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTolytoxin, a cytostatic, antifungal macrolide produced by blue‐green algae of the genusScytonema, is a potent, reversible inhibitor of cytokinesis in cultured mammalian cells. Treatment of KB cells with 2–16 nM tolytoxin results in profound morphological changes, beginning with the formation of zeiotic processes and culminating in nuclear protrusion. In L1210 cells, cytokinesis is inhibited by as little as 2 nM tolytoxin, while karyokinesis proceeds normally, resulting in polynucleation. Tolytoxin specifically disrupts microfilament organization in A10 cells, while having no apparent effect on microtubules or intermediate filaments. Tolytoxin inhibited actin polymerization in vitro and also caused the depolymerization or fragmentation of F‐actin in vitro. Tolytoxin exhibits effects that closely resemble those of cytochalasin B but is effective at concentrations 1/50−1/1,000 that of cytochalasin B. © 1993 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970240105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractInsect spermatozoa are characterized by having a set of accessory tubules that surrounds the microtubular doublets of the axoneme and that are formed from the B‐subtubules of the doublets. In trichopteran species, the accessory tubules have an unusually large diameter. Those of one species,Odontocerum albicorne, were seen to have a number of protofilaments that is 19 in the main part of the axoneme, but gradually decreasing to 18, 17, and 16 near the distal tip. The accessory tubule of the trichopteran axoneme has an asymmetrical shape and a skewed orientation, which makes it easy to distinguish a tubule that is viewed from its plus‐end from one viewed from the minus‐end. The shape of a cross‐sectioned protofilament in the trichopteran accessory tubules differs from that of microtubules in general, including accessory tubules of other insects, by being polygonal with the most acute angle pointing centripetally. © 1993 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970240106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractEmbryonic quail neural crest cells migrate towards the negative pole of an imposed dc electric field as small as 7 mV/mm (0.4 mV per average cell length). The involvement of protein kinases in the mechanism utilized by these cells to detect and respond to such imposed fields was tested through the use of several kinase inhibitors. Evidence for the involvement of protein kinase C (PKC) included: (1) inhibition of the directed motility by 1 μM sphingosine that was reversed by the addition of the phorbol ester, PMA; (2) stimulation of a faster response to the imposed field by PMA; and (3) inhibition of the directed translocation by 5 μM H‐7. However, another PKC inhibitor, staurosporin, did not inhibit the directed translocation (1 nM‐1 μM). We also found evidence for the involvement of either cAMP‐ or cGMP‐dependent protein kinase. The galvanotactic response was partially inhibited by the addition of 10 μM H‐9 and the response was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. However, the adenylate cyclase stimulant, forskolin, had no significant influence on the directed motility, although it reduced the average cell velocity. While these experiments suggest that cAMP‐ or cGMP‐dependent protein kinase or PKC may be involved in the galvanotaxis response, two other protein kinases appeared not to be required. The myosin light chain kinase inhibitor, ML‐7, had no effect on the directed motility in an imposed field, so myosin light chain kinase may not be required for galvanotaxis. Similarly, 5 μM W‐7 had no significant effect on the directed translocation, suggesting that calmodulin‐dependent protein kinase is not involved.Interestingly, the continuous activity of a protein kinase is apparently not required for the directed translocation response. The addition of the PKC and cAMP‐dependent protein kinase inhibitor, H‐7, after the cells had been exposed to the field for 1 hour, had no effect on the subsequent directed translocation. Thus, for these inhibitors to block the directed translocation, they must be present at the same time as the initial field application. This implies that an integral step in the cellular response mechanism for galvanotaxis involves the stimulation of a protein kinase whose effect is long las

ISSN:0886-1544    

DOI:10.1002/cm.970240107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have investigated the expression of chicken smooth muscle γ‐actin mRNA by isolation and characterization of cDNAs representing this actin isoform and utilizing the cDNA to probe RNA from adult and developing cells. Nucleotide sequence elucidated from an apparent full length smooth muscle γ‐actin cDNA revealed that it contained 94 bp of 5′ non‐translated sequence, an open reading frame of 1131 bp, and 97 bp of 3′ non‐translated sequence. Within the 376 amino acid sequence deduced from the chicken cDNA were diagnostic amino acids at the NH2‐ and COOH‐terminal regions which provided unequivocal identification of the γ‐enteric smooth muscle actin isoform. In addition, the chicken γ‐enteric actin deduced from our cDNA clones was found to differ from the sequence reported in earlier protein studies [J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979] by containing a proline rather than a glutamine at position 359 of the protein, indicating that the avian γ‐enteric actin isoform is identical to its mammalian counterpart. Comparison of the 5′ and 3′ non‐translated sequence determined from the chicken cDNA to that elucidated for rat, mouse, and human showed that there is not a high degree of cross‐species sequence conservation outside of the coding regions among these mRNAs. Northern hybridization analyses demonstrated that the γ‐enteric actin mRNA is expressed in adult aorta and oviduct tissues but not in adult skeletal muscle, cardiac muscle, liver, brain, and spleen tissues. The γ‐enteric actin mRNA was first observed in measurable quantities in gizzard tissue from 4–5 day embryos and increased in content in developing smooth muscle cells through 16–17 embryonic days. Following this initial increase during embryonic development, the γ‐enteric actin mRNA exhibits a decline in content until ∼7 days posthatching, after which there is an increase in content to maximal levels found in adult gizzard tissue. In general, the developmental appearance of the γ‐enteric mRNA parallels that observed for this protein in previous studies indicating that the developmental expression of smooth muscle γ‐actin is regulated, in part, by an increased content of mRNA in chicken visceral smooth

ISSN:0886-1544    

DOI:10.1002/cm.970240108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970240101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY