摘要:

AbstractCalcium and calmodulin are believed in play a significant role in the regulation of mitosis, because they are both localized in the mitotic spindle and because they can potentiate microtubule depolymerization in the test tube and in the living cell. It has been hypothesized, specifically, that calcium‐saturated calmodulin drives the shortening of the kinetochore microtubules that must occur during prometaphase, when the chromosomes congress to the metaphase plate, and during anaphase A, when the half‐spindles shorten. We have examined the role of calmodulin in mitosis by observing the consequences of calmodulin microinjection on the progress of mitosis and morphology of the mitotic spindle in PtK2 cells. We have found that the injection of excess calcium‐saturated calmodulin during early prometaphase significantly prolongs the time required for the cell to go into anaphase, and that neither calcium‐depleted calmodulin nor buffer alone produce a similar perturbation. Calcium ion alone produces a similar but much smaller retardation of mitosis. Immunofluorescence and fluorescent analogue cytochemical studies of spindle morphology reveal that the immediate (<5‐min) effect of calcium‐saturated calmodulin on prometaphase spindles is a significant shortening of the kinetochore fibers and “interpolar” microtubules but not the astral microtubules. After this perturbation, however, the spindle quickly recovers its normal form. An equivalent transient shortening of the spindle fibers is seen following the injection of calcium chloride solutions but not after the injection of calciumdepleted calmodulin or buffer alone. Taken together, these observations suggest that calcium‐saturated calmodulin plays a significant role in the regulation of mitosis, and that this regulatory pathway involves more than spindle

ISSN:0886-1544    

DOI:10.1002/cm.970070102

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

年代:1987

数据来源: WILEY

摘要:

AbstractQuinacrine, an acridine derivative which competitively binds to ATP binding sites, has been used to study the role of ATP requiring molecules in microtubule organization in mitotic PtK1cells. Brief treatments of metaphase cells with concentrations of quinacrine ranging from 2 to 10 μM decreased spindle length and birefringence in a concentration‐dependent manner. With either increasing quinacrine concentrations or duration of treatment, metaphase cells demonstrated a specific reorganization of spindle microtubules. Both polarization and electron microscopy showed a substantial loss of non‐kinetochore spindle microtubules with an increase in astral microtubules: this was particularly evident in the region adjacent to the spindle domain. Addition of millimolar concentrations of dinitrophenol to quinacrine‐containing medium did not potentiate the response of metaphase cells to quinacrine treatment. Time‐lapse video analysis demonstrated that the astral microtubules are the result of reorganization of spindle microtubules. These data suggest that functional ATP binding sites are required to maintain stable interactions between microtubules and that these interactions are responsible for maintaining the bowed configuration of non‐kinetochore spindle microtubules which are under compression at

ISSN:0886-1544    

DOI:10.1002/cm.970070103

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

年代:1987

数据来源: WILEY

摘要:

AbstractWe present a high‐resolution electron microscopic study of the sidearms on microtubules and vesicles that are suggested to form the crossbridges which produce the microtubule‐based vesicle transport in squid axoplasm. The sidearms were found attached to the surfaces of the anterogradely transported vesicles in the presence of ATP. These sidearms were made of one to three filaments of uniform diameter. Each filament measured 5–6 nm in width and 30–35 nm in length. The filaments in some of the sidearms had splayed apart by pivoting at their base, thereby assuming a “V” shape. The spread configuration illustrated the independence of the individual filaments. The filaments in other sidearms were closely spaced and oriented parallel to each other, a pattern called the compact configuration. In axoplasmic buffer containing AMP‐PNP, structures indistinguishable from the filaments of the sidearms on the vesicles were observed attached to microtubules. Pairs of filaments, thought to represent the basic functional unit, were observed attached to adjacent protofilaments of the microtubules by their distal tips. These data support a model of vesicle movement in which a pair of filaments within a sidearm forms two crossbridges and moves a vesicle by “walking” along the protofilaments o

ISSN:0886-1544    

DOI:10.1002/cm.970070104

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

年代:1987

数据来源: WILEY

摘要:

AbstractLimited proteolysis of tubulin with subtilisin results in cleavage of both the α and β subunits, releasing small peptides from the C‐terminal ends. At 37°C the digested tubulin assembles into polymorphic structures: microtubules with attached ribbons in the presence of GTP, rings in the presence of GDP, and protofilament spirals in the presence of vinblastine. Undigested tubulin does not assemble under these conditions. Rings and Vinca‐induced spiral structures are assembled from undigested tubulin only when microtubule‐associated proteins, high Mg2+concentrations, or polycations are present. Thus, cleavage with subtilisin affects assembly in a manner similar to the addition of these agents. It appears that binding of positively charged substances may act by neutralizing the charge on the highly acidic C‐terminal regions of the α‐ and β‐subunits, while cleavage with subtilisin produces the same effect by removing these peptides. Undigested and subtilisin‐digested tubulin form sheets of protofilaments in the presence of Zn2+, which indicates that the binding sites for the 2–3 Zn2+ions necessary to induce sheet formation do not reside in the C‐terminal r

ISSN:0886-1544    

DOI:10.1002/cm.970070105

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

年代:1987

数据来源: WILEY

摘要:

AbstractThe glycerinated stalks of the peritrich ciliate,Vorticella, can contract helically and reversibly on the addition of not only Ca2+but also other divalent or trivalent cations having ionic radii not far from 1 Å. In order to investigate the stalk contraction quantitatively in the absence of Ca2+‐chelators, we developed a method to eliminate contaminating Ca2+and other metal ions in KC1 and pHbuffer solutions by using a Ca2+‐and heavy metal ion‐specific ion exchange resin (Eporas MX‐2) Thus, it was possible to measure the relationship between the fractional stalk length ofVorticellaand the free concentration of alkaline earth metal, transition metal, and lanthanide ions in the 0.1 M KC1 and buffer (pH 6.8) solutions. Among these ions, Ca2+, Nd3+, and Eu3+(having ionic radii of about 1 Å) had the highest affinity for the contractile element in the spasmoneme. As the concentration of lanthanide ions (except Nd3+and Eu3+) is increased, theVorticellastalk contracts abruptly at a threshold level; this means that the Hill's parameter is very high, probably more than 6. The results of these experiments and of the co‐mixtures of Ca2+and Tb3+suggest that a contractile element in the spasmoneme contains both contractile Ca2+‐binding and regulatory Ca2+

ISSN:0886-1544    

DOI:10.1002/cm.970070106

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

年代:1987

数据来源: WILEY

摘要:

AbstractThe distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm‐attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discusse

ISSN:0886-1544    

DOI:10.1002/cm.970070107

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

年代:1987

数据来源: WILEY

摘要:

AbstractIn order to analyze the cellular mechanism of shape formation, the shape of individual 3T3 cells was perturbed by micromanipulation resulting in the detachment and relaxation of a cellular extension and the bending of the extension to form an “elbow” at a variable angle β. Finally, the tip of the extension was allowed to reattach to the substrate away from the cell.The cells reacted by drawing the extension tight. If β<90°, the elbow moved laterally for 8–15 min until the extension projected orthogonally at the cell surface. If β ≥90°, the extension remained stationary, Finally, in all cases webs formed between attachment points in the perturbed area. If the tip of the extension was allowed to touch its own cell body, thus forming a loop, the cells invariably closed the loop.The paper interprets the cellular reaction as the result of cortical tension and suggests that it is a major factor in the formation of fibroblast shape and the expressions of fibrobl

ISSN:0886-1544    

DOI:10.1002/cm.970070108

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

年代:1987

数据来源: WILEY

摘要:

AbstractIn the filamentous green algaMougeotia, each daughter nucleus formed by mitosis is then rapidly moved along the recently divided daughter cell to the central cleavage developing in the chloroplast. This movement is brought about by a cone‐shaped array of microtubules (MTs) that ensheath the daughter nucleus and are focused upon a small region, presumably a microtubule‐organising center (MTOC). Movement is completed when the MTOC locates and then resides in the chloroplast cleavage, drawing the nucleus into this position.The mitotic spindle is open with initially broad, ill‐defined poles. Anaphase A contributes minimally, if at all, to chromosome separation since the half spindles remain about the same length during anaphase and telophase. Thus, anaphase is accommodated and probably achieved by spindle elongation; the interzonal MTs also generate a rudimentary phragmoplast at the ingrowing cleavage furrow. The persistent polar MTs become directly transformed into the cone‐shaped array and initiate nuclear movement during early telophase. Various closely or distantly related green algae show this trait of persistent polar MTs. We conclude that this trait has allowed some species to evolve a motility system based directly on the capabilities of astral MTs, for generating the postmitotic nuclear movement essential for the restoration of the interphase cell organ

ISSN:0886-1544    

DOI:10.1002/cm.970070109

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

年代:1987

数据来源: WILEY

摘要:

AbstractThe vegetative giant nucleus of the unicellular and uninucleate marine green algaAcetabulariais a depot of conservative epitopes, homologous to antigenic determinants of vertebrate actin, myosin, actinin, and vinculin. The epitopes appear at the nucleolar surface (actin, actinin, vinculin determinants), in the caryoplasm (actin, myosin determinants), along “caryoplasmic” filaments (actin determinants), and in “nuclear envelope plus perinuclear bodies” (actin, myosin, actinin, vinculin determinants). The structure homologies of the nuclear antigenic determinants to those of the vertebrate muscular and/or microfilamental proteins were deduced from in situ cross‐reaction of anti‐chicken actin (cross‐reaction also with rabbit actin), anti‐chicken alpha actinin, anti‐chicken vinculin, and anti‐bovine myosin (cross‐reaction also with chicken myosin), respectively, by indirect immunofluorescence microscopy. Artifacts which arise from the binding of contaminating unspecific markers or from unspecific adherence of specific ones to the algal nucleus have been overcome by the use of both polyclonal and/or monoclonal immunoglobulins as primary markers and different types of second markers each conjugated with fluorescein isothiocyanate (FITC). Fluorescein staining of primary markers was performed either with fluorescent anti‐immunoglobulin antibodies in a one‐step (AIA‐FITC) or highly sensitive two‐step procedure (AIA/AIA‐FITC), covalently labeled F(ab′)2specific for either Fc or F(ab′)2(the latter anti‐fragment antibody excluded both possible interactions between nuclear “lectins” and glycosidic residues of Fc and staining of glycosidic nuclear antigens by AIAs or anti‐Fc specific for the glycosidic part of the immunoglobulin antigen) or fluorescent complex “protein A‐biotin‐avidin” (PABA‐FITC, a highly sensitive nonimmunoglobulin second reagent). Three of four different AIA‐FITC preparations tested alone and also “F(ab′)2anti‐Fc” showed reactivities with the nucleoli and the nuclear envelope. This indicates the presence of glycosidic determinants at the sites of reaction. Each of the other fluorescent markers, including AIA/AIA‐FITC, reacted with the primary marker only, although they were different in sensitivity.The staining patterns of nuclear actin epitopes differed in certain details if primary marker (monoclonal against polyclonal), second marker (AIA‐FITC against PABA‐FITC), or nuclear preparation (degree of nuclear flattening by the cover slide and salt condition) were changed. It suggests that type and number of actin epitopes, valence, affinity, and number of anti‐actin clones, but also subclass or class specificity of the second marker and accessibility of the nuclear actin determinant(s), were involved. The nuclear actin and myosin epitopes stained most intensively in a “high salt” environment (100 mM PBS, 50 mM/1 KC1; pH 7.2) if compared to “low salt staining.” This indicates local concentrations and/or accessibility of antigenic determinants which were hidden in “low salt” (1.5 mN/1

ISSN:0886-1544    

DOI:10.1002/cm.970070110

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

年代:1987

数据来源: WILEY

摘要:

AbstractChlamydomonascells sucked onto micropipettes were filmed at 500 frames/sec and analyzed as to their forward beating mode. A comparison with freely swimming cells revealed that the flagella of the sucked cells beat in a normal threedimensional manner, with beat frequencies that correspond to those of freely swimming cells. Most beats were synchronous. but not symmetrical; cis‐ and trans‐flagellum appear to beat in a slightly different manner. Some cells beat synchronously throughout, but mostly synchrony was interrupted by a single asynchrony or up to incessant asynchronies, caused by transient accelerations of the trans‐ (fo‐) flagellum. Only rarely did cis‐ and trans‐flagella have different but constant beat frequencies. Helical swimming ofChlamydomonasmore likely is due to the beat asymmetries of the two flagella than to differences of beat frequencies. In our records, the stigma is on the inside of the helical sw

ISSN:0886-1544    

DOI:10.1002/cm.970070111

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

年代:1987

数据来源: WILEY