ISSN:0886-1544    

DOI:10.1002/cm.970270202

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

年代:1994

数据来源: WILEY

摘要:

AbstractUsing a synthetic peptide mimicking the NH2‐terminus of β‐actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the β isoform, by its exclusive recognition of the synthetic β‐actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the β‐actin spot in two‐dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function. © 1994 W

ISSN:0886-1544    

DOI:10.1002/cm.970270203

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe terminal phase of cell division involves tight constriction of the cleavage furrow contractile ring, stabilization/elongation of the intercellular bridge, and final separation of the daughter cells. At first cleavage, the fertilized eggs of the mollusk,Ilyanassa obsoleta, form two contractile rings at right angles to each other in the same cytoplasm that constrict to tight necks and partition the egg into a trefoil shape. The cleavage furrow contractile ring (CF) normally constricts around many midbody microtubules (MTs) and results in cleavage; the polar lobe constriction contractile ring (PLC) normally constricts around very few MTs and subsequently relaxes without cleavage. In the presence of Ag+ions, the PLC 1) begins MT‐dependent rapid constriction sooner than controls, 2) encircles more MTs than control egg PLCs, 3) elongates much more than control PLCs, and 4) remains tightly constricted and effectively cleaves the polar lobe from the egg. If Ag+‐incubated eggs are returned to normal seawater at trefoil, tubulin fluorescence disappears from the PLC neck and the neck relaxes. If nocodazole, a drug that depolymerizes MTs, is added to Ag+‐incubated eggs during early PLC constriction, the PLC is not stabilized and eventually relaxes. However, if nocodazole is added to Ag+‐incubated eggs at trefoil, tubulin fluorescence disappears from the PLC neck but the neck remains constricted. These results suggest that Ag+accelerates and gradually stabilizes the PLC constriction by a mechanism that is initially MT‐dependent, but that progressively becomes MT‐independent. © 1994 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970270204

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

年代:1994

数据来源: WILEY

摘要:

AbstractPrevious immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule‐associated protein of Mr∼210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802–808; DeBrabander et al. (1981) J. Cell Biol. 91:438–455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double‐label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277–288]to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC‐7), and those made by human neuroblastoma cells (IMR‐32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC‐7 cells, we also observed MAP4‐depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity. ©

ISSN:0886-1544    

DOI:10.1002/cm.970270205

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe distinct damped, or attenuated, bending pattern observed when demembranated sperm flagella of the tunicate,Ciona, are reactivated in the presence of 2 mM Li+has been analysed in detail. In these patterns, bends are initiated at the base of the flagellum, but die out after they start to propagate along the flagellum, so that little or no bending is seen in the distal half of the flagellum. A quantitative descriptive analysis shows that the distinctive feature of this attenuation of bending wave amplitude is an asymmetric interbend decay, or slippage, occuring, on average, only at the transitions between a reverse bend and the preceding principal bend. This attenuation is combined with a significant amount of synchronous sliding in the distal half of the flagellum and a decrease in propagation velocity of transitions between bends in the mid‐region of the flagellum.Computer simulations demonstrate that the synchronous sliding in the distal half of these flagella can be an entirely passive consequence of the mechanical interaction between active sliding and bending in the basal third of the flagellum and viscous resistances to movement of the distal region of the flagellum through the fluid environment. The current computer models do not contain a mechanism for asymmetric interbend decay that can reproduce these attenuated bending patterns. © 1994 Wiley‐Liss,

ISSN:0886-1544    

DOI:10.1002/cm.970270206

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

年代:1994

数据来源: WILEY

摘要:

AbstractSubstrate analogs are useful for studying the structures of active sites and for distinguishing between similar enzyme activities. Fluorescent ribose‐modified ATP analogs were used to investigate the functional differences between dynein ATPases. These analogs reactivate (support the movement of) sea urchin sperm axonemes, yet they do not reactivatewild‐type Chalmydomonasaxonemes. Surprisingly, the analogs reactivate the axonemes of mutants completely missing the outer arm dyneins. Competition experiments using ATP and these analogs provide strong evidence that the analogs bind to all dynein active sites but fail to release a subset of dyneins from rigor. We suggest that this subset ofChlamydomonasouter arm dyneins unable to use the analogs remains in rigor in the presence of the analogs and paralyzes the axoneme. © 1994 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970270207

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

年代:1994

数据来源: WILEY

摘要:

AbstractMyonemes are bundles of thin filaments (3‐6 nm in diameter) which mediate calcium‐induced contraction of the whole or only parts of the cell body in a number of protists. InEudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calciumbinding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium‐modulated contractile protein centrin. Western‐blot analysis indicates that the 23 kDa protein crossreacts antigenically with anti‐centrin antibodies. In addition, the 23 kDa protein displays calcium‐induced changes in both electrophoretic and chromatographic behaviour, and contains calcium‐binding domains that conform to the EF‐hand structure, as known for centrin. Based on these observations, we conclude that a calcium‐binding protein with major similarities to centrin occurs in the myonemes ofE. maggii.We postulate that this protein plays an essential role in myonememediated retraction of the ciliature. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970270208

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

年代:1994

数据来源: WILEY

摘要:

AbstractChlamydomonasandTetrahymenaaxonemal dyneins have previously been found to bind to porcine brain microtubules to produce a microtubule‐dynein complex. At appropriate microtubule:dynein concentration, microtubules in the complex became covered to saturation by dynein arms of the same polarity and at a spacing of 24 nm [Haimo et al., 1979; Haimo and Fenton, 1988; Haimo, 1989; Porter and Johnson, 1983a].In the present study, two different types of microtubule‐dynein complexes (α‐and β‐complexes) were prepared fromTetrahymenaciliary 22S dynein and porcine brain tubulin. The characteristics of the adenosine triphosphate (ATP)‐induced extrusion of microtubules from these complexes were analyzed, as a simple and direct in vitro assay for the ATP‐induced extrusion of single microtubules. The α‐complex prepared by adding dynein to microtubules showed an interrupted sliding movement, which would stop and start several times following the addition of ATP. In the β‐complex, prepared by adding dynein bound to DEAE‐tubulin to pre‐assembled microtubules, microtubules became covered with dynein molecules whose orientation and binding were uniform with respect to microtubule polarity. The microtubules in the β‐complex extruded at 12 μm/second following the addition of ATP. Dark‐field and electron microscopy indicated that the extruded microtubules had undergone sliding on a dynein‐track that had become detached from the complexes and had been absorbed onto the surface of the glass slide. At higher light intensity under a dark‐field microscope, the dynein‐track was seen to be composed of rows of dynein molecules arranged densely. The orientation of dynein molecules in rows appeared to be uniform considering the images of bound dynein in the β‐complex under electron microscope. The higher sliding velocity of the microtubules on these dynein‐tracks compared to that seen on slides coated at random with dynein [Vale and Toyoshima, 1988, 1989], may be due to more efficient force generation by this dense arrangement of dynein molecules with the same polari

ISSN:0886-1544    

DOI:10.1002/cm.970270209

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

年代:1994

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970270201

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

年代:1994

数据来源: WILEY