摘要:

AbstractThis report describes the ontogenesis of cochlear stereocilia using scanning electron microscopy for analysis of cilia appearance, and fluorescence microscopy of phalloidin, a label for F‐actin, to determine the maturation of the cilia framework. Surface and frozen‐sectioned preparations of the otic capsule were obtained from several stages of rat pup development beginning at the 16th gestational day and at various stages until adulthood. In the earliest stage investigated, strong fluorescence labeling was visible on the apical part of Kölliker's organ, revealing a reticular outline of cell junctions. Hair cells started to differentiate at the 18th day of gestation from cells within the primordial receptor area. Phalloidin labeling revealed a sequential appearance of F‐actin as the hair cells differentiated from the cells within the Kölliker's organ. The differentiation of receptor cells occurred first with the appearance of a junctional complex between the hair cell and the surrounding cells. Then a cuticular plate appeared followed by the progressive emergence of stereocilia.The F‐actin labeling also revealed a progressive differentiation of receptors cells from the cochlear base to its apex. There was also an inner to outer hair cell developmental gradient of label. Inner hair cells developed stereocilia before outer hair cells. The third row of outer hair cells was the last to acquire stereocilia. The adult patern of stereocilia was reached around the 6th postnatal day.We conclude that the appearance of actin filaments in developing receptor cells and the emergence of stereocilia can be regarded as markers for correlating function and other structural differentiation. © 1993 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970250302

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe actin‐bundling protein fimbrin is homologous to l‐plastin, a 65kD phosphoprotein expressed in leukocytes and transformed cells [de Arruda et al., J. Cell Biol. 111, 1069–1080]. Because fimbrin is present in cell adhesion sites, we studied the phosphorylation state of fimbrin and its distribution in macrophages sequentially extracted with Triton‐X‐100 (soluble fraction), Tween 40‐deoxy‐cholate (cytoskeletal fraction), and SDS (insoluble cytoskeletal fraction). The approximate distribution of fimbrin and actin among these fractions was found to be: 65% fimbrin/55% actin in the soluble fraction, 30% fimbrin/20% actin in the cytoskeletal fraction, and 5% fimbrin/25% actin in the insoluble cytoskeletal fraction. PMA did not alter this distribution. Fluorescence microscopy of acetone‐extracted macrophages showed that actin is concentrated in podosomes at the substratum interface and is diffusely distributed throughout the remainder of the cell. Fimbrin colocalizes with actin in podosomes and also exhibits a punctate distribution in the cytoplasm that overlaps with actin. In Tween 40/DOC‐extracted cells, podosomes remain, and fimbrin also exhibits a punctate distribution along actin filaments. Metabolic32PO4labeling revealed that fimbrin is constitutively phosphorylated and that phosphorylated fimbrin is concentrated in the insoluble cytoskeletal fraction. PMA increased the relative levels of fimbrin phosphorylation twofold but did not alter the pattern of fimbrin fluorescence or the distribution of phosphorylated fimbrin. Limited trypsin digestion and phosphoamino acid analysis demonstrated that phosphorylation occurs specifically on serine residues within the 10kD headpiece domain of fimbrin. Phosphorylation of the headpiece domain could regulate the actin binding and bundling properties of fimbrin, or it could regulate the interaction of fimbrin with other proteins. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970250303

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe influence of two commonly used sulphonate buffers, PIPES and MES, on the in vitro assembly of bovine brain microtubule protein was examined. Microtubule assembly was monitored by turbimetry and, after centrifugation, the polymerised protein was analysed by SDS‐PAGE and western blotting. Assembly in MES when compared with PIPES resulted in a higher recovery of microtubule proteins at both pH 6.4 and pH 6.9 and in an altered protein composition. The buffer pH affected the total amount of protein polymerized but did not significantly affect the protein composition. At both pH conditions the recovery of HMW‐MAPs was markedly increased in MES buffer and this increase was mostly due to an increase in the amount of MAP1. © 1993 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970250304

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe have cloned and sequenced the two β‐tubulin genes of the ciliated protozoanTetrahymena thermophila.The two genes encode identical 443 amino acid peptides which are 99.7% identical to the β‐tubulin proteins ofT. pyriformisand 95% identical to human β1 tubulin.T. thermophilacontains only one β‐tubulin gene (Callahan et al., 1984:Cell36:441–445). Thus, all of the extremely diverse microtubule structures in this unicellular organism can be formed from a single α‐ and a single β‐tubulin peptide. We have also carried out a phylogenetic analysis of 84 complete β‐tubulin peptide sequences. This analysis supports two hypotheses regarding β‐tubulin evolution and function: (1) Multifunctional β‐tubulins are under greater evolutionary constraint than β‐tubulins present in specialized cells or in cells with very few microtubule related functions, which can evolve rapidly; and (2) Cells which form axonemes maintain a homogeneous population of tub

ISSN:0886-1544    

DOI:10.1002/cm.970250305

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

年代:1993

数据来源: WILEY

摘要:

AbstractParticle motility in cultured rat fibroblasts was studied using video‐enhanced differential interference contrast microscopy. The average velocity of large bright particles (apparent diameter about 0.5–0.7 μm) was measured in control cells and in cells treated with agents which affected targets related to signal transduction pathways. A Rat‐2‐derived fibroblast line transfected with a construct containing multiple copies of the N‐rasproto‐oncogene under the control of dexamethasonesensitive promoter was used as a main experimental model. Dexamethasone treatment was shown to induce high levels of N‐rasexpression in these cells. This treatment greatly increased the average particle velocity. At the same time dexamethasone did not influence the particle motility in the non‐transfected parent cells and in the cells transfected with a construct which did not contain N‐ras.Phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), also induced an approximate eightfold increase in the particle rate after several hours of incubation, while sphingosine, an inhibitor of PKC, prevented this activation. Sphingosine alone reduced the particle motility after a 20 min incubation. The particle movements were inhibited also by colcemid. These data show that the activation of N‐rasand PKC produced dramatic activation of microtubule‐dependent particle motility. A possible role of this activation in signal‐induced alterations of cell morphology is discu

ISSN:0886-1544    

DOI:10.1002/cm.970250306

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

年代:1993

数据来源: WILEY

摘要:

AbstractA tropomyosin‐specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen γgt10 and γgt11 cDNA libraries, which were constructed from poly(A)+RNAs of human fibroblast cell lines HuT‐14 and WI‐38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low Mrisoforms (hTM5a and hTM5b), and a high Mrisoform (hTMsmα). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsmα represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue‐specific expression suggests that different isoforms may perform distinct functions in different tissues. © 1993 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970250307

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

年代:1993

数据来源: WILEY

摘要:

AbstractLimited digestion of pig brain GDP‐tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS‐PAGE, revealed characteristic divalent cation‐dependent changes in the α‐ and β‐tubulin cleavage patterns. Previous studies revealed that the β‐cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992:Arch. Biochem. Biophys.296:152–160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP‐tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+forms sheets of rings. Mn2+induces ring formation in uncleaved GDP‐tubulin. Isotype‐depleted tubulin was generated by the removal of class III β‐tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III β‐tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin‐digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin‐digested tubulin for studying microtubule‐associated prote

ISSN:0886-1544    

DOI:10.1002/cm.970250308

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

年代:1993

数据来源: WILEY

摘要:

AbstractStarving amoebae of the cellular slime moldDictyostelium discoideumreact chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine‐5′‐0‐(2‐thiotriphosphate) (GTPβS) or guanosine‐5′‐0‐(2‐thiodiphosphate) (GDPβS) was scrape‐loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP‐filled glass capillary revealed a reduced capacity of loaded cells to migrate to wards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose‐dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired.In cells loaded with the analogs together with the calcium indicator fura‐2, stimulation with 10 μM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]iat low levels with 1,2‐bis(2‐aminophenoxy) ethane N,N,N′,N′‐tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed.The data suggest that chemotactic migratory activity involves GTP‐dependent processes that participate in the regulation of the Ca2+homeostasis of the cell and in the regulation of membrane traffic that contributes to the d

ISSN:0886-1544    

DOI:10.1002/cm.970250309

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.970250301

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

年代:1993

数据来源: WILEY