摘要:

AbstractCofilin is a small actin‐binding protein which reguiates actin polymerization in a pH‐dependent manner. Immunofluorescence microscopy with a monoclonal antibody for cofilin revealed that this protein is temporarily concentrated at the contractile ring during cytokinesis. Cofilin appeared to accumulate rapidly at the contractile ring during late stages of furrowing, and was finally enriched at the midbody. The concentration of cofilin at the contractile ring was observed in several kinds of cultured cells. Furthermore, cofilin introduced into living cells by a microinjection method was also concentrated at the contractile ring. These results suggest that cofilin is involved in actin reorganization during cytokinesis. © 1995 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970300102

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

年代:1995

数据来源: WILEY

摘要:

AbstractBull sperm extracted with 0.1% Triton X‐100 can be reactivated to full motility with 0.33 mM Mg‐ATP (sperm models). When motile sperm models are treated with 0.66 mM NiSO4, spontaneous motility is lost. During the transition to motility arrest, the beat becomes progressively more asymmetric, finally arresting at one extreme of the beat cycle. After spontaneous motility has been lost, the flagellum retains the ability to respond to mechanical stimulation. If a microprobe is used to bend the flagellum in the direction opposite to its own prevailing curvature and released, the recoil is rapid and overshoots the equilibrium position. When the same flagellum is manipulated in the opposite direction (into a tighter bend of the existing curve), the recoil is slower and does not exceed the initial bend. If a microprobe is used to carefully bend the whole flagellum into a curve, the flagellum will resume continuous beating, but only if the imposed bend is in the direction opposite the natural curvature. The reinstated beating activity (mechanical reactivation) is sustained as long as the flagellum is held by the microprobe. The rate of change of the shear angle in these mechanically reactivated, Ni2+‐inhibited sperm suggests an impaired rate of sliding on one side of the axoneme compared to similarly restrained control sperm. It appears that Ni2+has a selective inhibitory effect on the dynein arms that bend the flagellum in one direction. Furthermore, the remaining functional arms activate only when the flagellum is bent in the direction opposing their own action. © 1995 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970300103

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

年代:1995

数据来源: WILEY

摘要:

AbstractApplying a new in vitro motility assay system for microtubules and 22S dynein, we recently reported on an ATP‐induced extrusion of microtubules from microtubule‐dynein α‐ and β‐complexes [Mimori and Miki‐Noumura, 1994:Cell Motil. Cytoskeleton27:180–191]. In the present study, we prepared a γ‐complex by copolymerizing porcine brain tubulin andTetrahymenaciliary 22S dynein, and examined the ATP‐induced microtubule movement from the γ‐complex. The extrusion process appeared quite similar to that of the β‐complex. The sliding velocity was 18.39 ± 2.20 m̈m/sec, which was a value comparable to that of trypsin‐digested flagellar axonemes [Yano and Miki‐Noumura, 1980:J. Cell Sci.44:169–186]. Higher velocity may be due to a densely arranged dynein‐track with the same polarity, which was detached from the γ‐complex and absorbed in rows on a glass surface of the slide. Sometimes a free‐floating microtubule in the perfusion chamber was observed riding and sliding on the dynein‐track remaining on the slide after extrusion.Unexpectedly, we found that when the front part of the microtubule was fixed to a glass surface, a continuous sliding microtubule at the rear part on the dyneintrack often transformed into a left‐handed helix, and subsequently a twisted helix with several turns. The helix formation may be due to some rigidity in the microtubule and a right‐handed torque component in the sliding force of 22S dynein. The addition of ATP may release some distortion accumulated in the complex structure during copolymerization of tubulin and 22S dynein, inducing reverse rotati

ISSN:0886-1544    

DOI:10.1002/cm.970300104

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

年代:1995

数据来源: WILEY

摘要:

AbstractBrush border myosin‐I (BBMI) is associated with the membrane of intestinal epithelial cells where it probably plays a structural role. BBMI also has been identified on Golgi‐derived vesicles in intestinal epithelial cells where it may translocate vesicles into the brush border. However, the mechanochemical activity of BBMI bound to a phospholipid membrane has not been described. This study reports that phospholipid membrane‐associated BBMI displays ATPase activity when bound to phospholipids, but does not move actin filaments when associated with a phospholipid bilayer. BBMI does not bind significantly to brush border membrane lipids, which contain about 16% phosphatidylserine (PS), in either a pelleting or planar membrane assay. Similarly, planar membranes containing 20% PS do not bind a significant amount of BBMI. Increasing the concentration of PS to 40% does result in the binding of BBMI to both vesicles and planar membranes. This binding is enhanced with increased Ca2+concentrations. BBMI retains its ATPase activity when bound to phospholipid vesicles containing 40% PS. However, BBMI attached to a phospholipid bilayer surface does not move actin filaments, even though the amount of BBMI bound to the lipid surface, as reflected by the number of actin filaments associated with bilayer‐bound BBMI, is sufficient to observe motility in control experiments. When membrane fluidity is reduced by adding cholesterol to the membrane lipids containing 40% PS, BBMI still binds to the membrane, but again no actin filament motility is observed. The lack of binding by BBMI to brush border membrane lipids and the absence of membrane‐associated BBMI mechanical activity suggest that factors in addition to membrane lipids are necessary for membrane‐associated myosin‐I motility. © 1995 W

ISSN:0886-1544    

DOI:10.1002/cm.970300105

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

年代:1995

数据来源: WILEY

摘要:

AbstractInfection of host cells byListeria monocytogenesresults in the recruitment of cytoplasmic actin into a tail‐like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthenes, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results inListeriacell‐to‐cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial‐actin tail interface. We have studied the consequences of microinjecting three different actin monomer‐binding proteins on the actin tail assembly andListeriaintracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11–22 m̈M) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer‐sequenstering proteins, Vitamin D‐binding protein (DBP; 1–2 m̈M) and DNase I (6–7 m̈M) completely block bacterial‐induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10–20 min) indicating that the mechanisms by which these proteins interfere withListeria‐induced actin assembly are likely to be complex. To exclude the possibility thatListeriarecruits preformed actin filaments to generate the tails and that these monomer‐binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 m̈M). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate thatListeria‐induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and thatListeria'sability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer‐bindi

ISSN:0886-1544    

DOI:10.1002/cm.970300106

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

年代:1995

数据来源: WILEY

摘要:

AbstractPlatelets circulate in the blood as discoid cells which, when activated, change shape by polymerizing actin into various structures, such as filopodia and stress fibers. In order to understand this process, it is necessary to determine how many other proteins are involved. As a first step in defining the full complement of actin‐binding proteins in platelets, filamentous (F)‐actin affinity chromatography was used. This approach identified>30 different proteins from ADP‐activated human blood platelets which represented 4% of soluble protein. Although a number of these proteins are previously identified platelet actin‐binding proteins, many others appeared to be novel. Fourteen different polyclonal antibodies were raised against these apparently novel proteins and used to sort them into nine categories based on their molecular weights and on their location in the sarcomere of striated muscle, in fibroblasts and in spreading platelets. Ninety‐three percent of these proteins (13 of 14 proteins tested) were found to be associated with actin‐rich structures in vivo.Four distinct actin filament structures were found to form during the initial 15 min of activation on glass: filopodia, lamellipodia, a contractile ring encircling degranulating granules, and thick bundles of filaments resembling stress fibers. Actin‐binding proteins not localized in the discoid cell became highly concentrated in one or another of these actin‐based structures during spreading, such that each structure contains a different complement of proteins. These results present crucial information about the complexity of the platelet cytoskeleton, demonstrating that four different actin‐based structures form during the first 15 min of surface activation, and that there remain many as yet uncharacterized proteins awaiting further investigation that are differentially involved in this process. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970300107

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

年代:1995

数据来源: WILEY

摘要:

AbstractChanges in the tubulin‐protein and ‐poly(A)+RNA contents were monitored by means of Western and Northern blot analyses, respectively, during growth and maturation of leaves of a dicotyledonous (tobacco) and monocotyledonous (barley) plant. It was recently argued from immunofluorescence and preliminary biochemical data that the density of microtubular networks and concomitantly the tubulin content are distinctly reduced after cessation of cell growth in leaves [Jung et al., 1993]. The results presented now confirm and extend this view. There appeared to be clear differences between the monocot and the dicot: (1) the loss of tubulin during leaf development was much slower in the dicot than in the monocot leaves (within months instead of days); (2) the degree of loss was more dramatic in the monocot leaf and only very low threshold levels of tubulin were retained in fully differentiated tissues; and (3) the loss of tubulin in the monocot leaf tissue appeared to be correlated with the decrease in the mRNA content, whereas the high level of tubulin‐RNA in fully differentiated or even almost senescent dicot leaves indicated a gene expression control at the posttranscriptional level.The comparatively rapid and very distinct tubulin‐protein and ‐RNA disappearance during development of the monocot leaf tissues confirm at the molecular level that differentiation proceeds much faster and is much more determinative in these leaves, as was postulated from histological and physiological data. The differences in the behaviour of the microtubular cytoskeleton perhaps even reflect the differences in the ability of the differentiated leaf cells to dedifferentiate, i.e., to establish new sets of microtubules and to reenter the mitotic cell cycle, e.g., during would response, tumour induction or in vitro culture. © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970300108

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe family of myosin motors is comprised of numerous classes distributed among a diverse set of organisms and cell types. We have identified an unconventional myosin gene (MYO3) in the yeastSaccharomyces cerevisiaeand show that it is member of a subclass of unconventional myosin proteins originally found only in the amoeboid organismsDictyosteliumandAcanthamoeba.Identification of this protein in these genetically and morphologically divergent organisms suggests that it will be ubiquitous in eukaryotes and that it has a role in the basic functions of the eukaryotic cell. We have constructed a strain of yeast missing 99% of the MYO3 coding sequence. This mutation has no observable phenotypic effect, placing MYO3 into a growing class of yeast genes which are dispensable under laboratory conditions, perhaps due to genetic redundancy. Alignment of MYO3 with other unconventional myosins shows that it shares with a subset of them a previously unrecognized region of homology in the tail; this region falls within a domain identified as important for mediating nonspecific electrostatic interactions with membranes. The existence of this region suggests that it may be involved in mediating specific protein‐protein interactions, possibly helping to localize this myosin to specific membranes or membrane regions. In addition, we show that “classic” myosin I proteins share a region of hyper‐proline‐richness 10 amino acids before the SH3 domain. Proline‐rich regions have recently been implicated as SH3 binding sites, which suggests that this region might be involved with regulating or in other ways interacting with SH3 domains. © 1995 Wil

ISSN:0886-1544    

DOI:10.1002/cm.970300109

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

年代:1995

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970300101

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

年代:1995

数据来源: WILEY