ISSN:0886-1544    

DOI:10.1002/cm.970140202

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

年代:1989

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970140203

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

年代:1989

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970140204

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe have previously shown that BHK syncytia have the ability to locomote provided the centrospheres are clustered and located adjacent to the cluster of nuclei. This article reports that experimental reorganizations of the centrospheres or the nuclei change the motile behavior of BHK syncytia in a way that is consistent with our previous observations: When fusion of the multiple nuclei occurred in stationary syncytia whose multiple nuclei encircled the centrosphere cluster, the centrospheres were expelled from the ring of nuclei. Consequently, locomotion was initiated in these syncytia even if they had been previously stationary for up to 5 days. Conversely, when a 2‐hour incubation in 5 μg/ml cytocholasin B caused the cluster of nuclei to surround the centrosphere cluster, the locomotion of the syncytia was inhibited. Similarly, the dispersal of the centrosphere cluster induced by a 4‐hour incubation in 1 μg/ml of colcemid resulted in the long‐term cessation of locomotion in motile s

ISSN:0886-1544    

DOI:10.1002/cm.970140205

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

年代:1989

数据来源: WILEY

摘要:

AbstractSpermatozoa of the rainbow trout,Salmo gairdneri, were demembranated with Triton X‐100. The demembranated spermatozoa showed vigorous motility in the reactivation solution containing Ca2+at the concentrations below 10−8.5M in the presence of cAMP. The motility was lost at 10−8M Ca2+or more. The shape of the immotile flagella in the presence of high concentration of Ca2+was not uniform: Some showed the cane shape and some were almost straight. The change in Ca2+concentration of the extraction solution did not alter the motility of the reactivated spermatozoa. These results were different from those obtained from the sea urchin spermatozoa. When the concentration of cAMP was changed from 0.5 to 100 μM, the concentration of Ca2+for converting the motile to immotile state was not altered. Thus, it is likely that the Ca2+‐dependent regulatory system of flagellar movement is independent of the cAMP‐induced initiation mechanism, which is assumed to require the transient influx of Ca2+in rainbow trout s

ISSN:0886-1544    

DOI:10.1002/cm.970140206

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

年代:1989

数据来源: WILEY

摘要:

AbstractActin and the light chains of myosin were labeled with fluorescent dyes and injected into interphase PtK2 cells in order to study the changes in distribution of actin and myosin that occurred when the injected cells subsequently entered mitosis and divided. The first changes occurred when stress fibers in prophase cells began to disassemble. During this process, which began in the center of the cell, individual fibers shortened, and in a few fibers, adjacent bands of fluorescent myosin could be seen to move closer together. In most cells, stress fiber disassembly was complete by metaphase, resulting in a diffuse distribution of the fluorescent proteins throughout the cytoplasm with the greatest concentration present in the mitotic spindle. The first evidence of actin and myosin concentration in a cleavage ring occurred at late anaphase, just before furrowing could be detected. Initially, the intensity of fluorescence and the width of the fluorescent ring increased as the ring constricted. In cells with asymmetrically positioned mitotic spindles, both protein concentration and furrowing were first evident in the cortical regions closest to the equator of the mitotic spindle. As cytokinesis progressed in such asymmetrically dividing cells, fluorescent actin and myosin appeared at the opposite side of the cell just before furrowing activity could be seen there. At the end of cytokinesis, myosin and actin were concentrated beneath the membrane of the midbody and subsequently became organized in two rings at either end of the midbody.

ISSN:0886-1544    

DOI:10.1002/cm.970140207

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

年代:1989

数据来源: WILEY

摘要:

AbstractQuinacrine, an acridine derivative which competitively binds to ATP binding sites, has previously been shown to cause the reorganization of metaphase spindle microtubules (MTs) due to changes in interactions of non‐kinetochore microtubules (nkMTs) of opposite polarity (Armstrong and Snyder:Cell Motil. Cytoskeleton7:10–19, 1987). In the study presented here, mitotic PtK1cells were treated in early anaphase with concentrations of quinacrine ranging from 2 to 12 μM to determine energy requirements for chromosome motion. The rate and extent of chromosome‐to‐pole movements (anaphase A) were not affected by these quinacrine treatments. The extent of anaphase B (kinetochore‐kinetochore separation) was reduced with increasing concentrations of quinacrine. Five micromolar quinacrine reduced the extent of kinetochore‐kinetochore separation by 20%, and addition of 12 μM quinacrine reduced the kinetochore‐kinetochore separation by 40%. To determine the role of nkMTs in anaphase spindle elongationquinacrine‐treated metaphase cells were treated with hyperosmotic sucrose concentrations, and spindle elongation was measured (Snyder et al.:Eur J. Cell Biol.39:373–379, 1985). Metaphase cells treated with 2–10 μM concentrations of quinacrine for 2–5 min reduced spindle lengths by 10–50% prior to 0.5 M sucrose treatment for 5 min. This treatment showed a significant reduction in the ability of sucrose to induce spindle elongation in cells pretreated with quinacrine. As spindle length and birefringence was reduced by quinacrine treatment, sucrose‐induced elongation was concomitantly diminished. These data suggest that quinacrine‐sensitive linkages are necessary for anaphase B motions. Reduction in these linkages and/or MT length in the nkMT continuum may reduce the ability of the nkMTs to hold compression at metaphase. This form of energy is thought to drive a significant proportion of normal anaphase B in PtK1cells and sucrose‐indu

ISSN:0886-1544    

DOI:10.1002/cm.970140208

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

年代:1989

数据来源: WILEY

摘要:

AbstractProfilin is a G‐actin binding protein that may have a role in controlling the ratio of G/F actin within the cells To devise a way for obtaining large amounts of mammalian profilin in an active state, we transfectedEscherichia coliwith a plasmid containing a full‐length rat spleen profilin cDNA adjacent to a promoter inducible by isopropyl thiogalactoside (IPTG). Upon induction, they synthesized a new protein of 15,000 MW constituting approximately 5% of the total cell protein. This protein bound to poly‐L‐proline Sepharose and could be eluted with 7 M urea, behavior similar to that exhibited by authentic profilin. The protein could be released from the bacteria in soluble form following sonication, and the profilin could then be purified to homogeneity following chromatography on Sephadex G‐75 and DEAE A‐50 Sephadex. The protein began with an unblocked Ala, indicating that the initiating formyl and methionine residues had been removed. The dissociation of the recombinant profilin from chicken skeletal muscle actin was characterized by a Kdof approximately 2 μM based on gel filtration analysis and actin polymerization assays. These results show that purified active mammalian profilin can be made conveniently in large quantities. This study also demonstrates the feasibility of using bacterially synthesized profilin in structure‐function studies involving mutant profilins altered by site‐dire

ISSN:0886-1544    

DOI:10.1002/cm.970140209

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe studied the distribution of acetylated α‐tubulin in the microtubules of spermatogenic cells from the crane flyNephrotoma suturalis(Loew) using a mono‐clonal antibody specific for acetylated α‐tubulin (6‐11B‐1). We found that cells in all stages of spermatogenesis contained acetylated microtubules including primary spermatocytes, meiotic cells, spermatids, and sperm. A subset of the acety‐lated microtubules (those in midbodies and flagella) were resistant to cold depolymerization. Newly polymerized microtubules in nondividing cells were not acetylated for up to 15 min. indicating that acetylation lagged behind polymerization. In spindles, newly polymerized microtubules were acetylated after 5 min. Antibodies to acetylated α‐tubulin selectively stained chromosome‐to‐pole fibers in dividing cells, but the staining appeared to decrease and taper of at the kinetochores. This observation supports the hypothesis that tubulin subunits add at the kinetochore in metaphase and that acetylation occurs subsequent to addition. Further, this taper may be useful as a marker in anaphase, to distinguish between different hypotheses

ISSN:0886-1544    

DOI:10.1002/cm.970140210

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

年代:1989

数据来源: WILEY

摘要:

AbstractWe present evidence that native profilin can be purified from cellular extracts ofAcanthamoeba, Dictyostelium, and human platelets by affinity chromatography on poly‐L‐proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6–8 M.Acanthamoebaprofilin‐I and profilin‐II can subsequently be separated by cation exchange chromatography. The yield ofAcanthamoebaprofilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: (1) dialyzedAcanthamoebaand human profilins rebind quantitatively to poly‐L‐proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; (2) dialyzed profilins form 3‐D crystals under the same conditions as native profilins; (3) dialyzedAcanthamoebaprofilin‐I has an NMR spectrum identical with that of native profilin‐I; and (4) dialyzed human andAcanthamoebaprofilins inhibit actin polymerization. We report the discovery of profilin inDictyosteliumcell extracts using the same method. Based on these observations we conclude that urea elution from poly‐L‐proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin‐II or alpha‐actinin in crude cell extracts, can be bound selectively to the poly‐L‐proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin‐II from bothAcanthamoebaandDictyosteliumcell extracts and alpha‐actinin fromAcanthamoebacell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly‐L‐proline. They can be eluted by dissociating the complexes with ATP and separated fr

ISSN:0886-1544    

DOI:10.1002/cm.970140211

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

年代:1989

数据来源: WILEY