摘要:

AbstractWe have shown, by indirect immunofluorescence microscopy using an antiserum against the mouse keratin subunit K2 and by electron microscopy, that transformed (PAM) and primary (PME) mouse epidermal cells possess extensive net works of IF bundles. Following trypsinization and replating of PAM cells, IF bundles are seen to move as a continuous net work from a perinuclear zone into the peripheral cytoplasmic regions. In PAM cells lysed in high‐ionic‐strength solutions containing Triton ×‐100 and DNAase‐1, IF bundles appear to be closely associated with nuclear envelope remnants and, in some cases, appear to be attached to nuclear pore complexes. PME cells cultivated in low Ca2+‐containing medium possess perinuclear birefringent arrays of IF bundles. Within 2 hours of switching the cells to normal Ca2+levels, the PME IF bundle network moves towards and establishes contact with the cell surface as desmosomes form. Live cells observed by phase contrast and fixed cells observed by immunofluorescence microscopy demonstrate that desmosomes can be distinguished as dark bands separating neighboring cells. There is little difference between the major proteins seen in SDS‐polyacrylamide gel profiles of isolated IF bundle net works from PME cells before and after the Ca2+switch. Therefore, a reorganization of relatively insoluble membrane‐associated protein following the Ca2+switch may be involved in desmosome formation. The isolated IF networks from PAM cells differ in protein composition compared to the PME IF networks. This may be related to the greatly reduced number of desmosomes in PAM cells. The IF bundle system in epidermal cells appears to be involved in shape formation, shape maintenance, the establishment of desmosomes, nuclear centration, and cel

ISSN:0271-6585    

DOI:10.1002/cm.970020302

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe lateral (L) cilia on an isolated filament from the gill of Mytilus edulis remain arrested at the end of their recovery stroke (hands up) when perfused with artificial sea water (ASW). The laterofrontal (LF) cilia continue to be active. The addition of 10% ethanol (ETOH) to the ASW perfusate arrests the LF cilia in a hands‐up posture; the L cilia remain undisturbed. By contrast, 10−6M 5‐hydroxytryptamine (5HT) in ASW activates the L cilia and arrests the LF cilia at the end of their effective stroke (hands down). Continued perfusion with 10% ETOH (v/v) in ASW/5HT restores activity to the LF cilia but arrests the L cilia (hands up). These effects are reversible and independent of external Ca2+.Following the detergent extraction of the filament, all gill cilia are inactive. The addition of 0.2 mM ATP in the presence of low Ca2+(10−3M) induces hands‐up arrest of the LF cilia, confirming that the Ca2+threshold of the two ciliary types is different by several orders of magnitude.The addition of 10% ETOH in low Ca2+to demembranated reactivated cilia arrests the L cilia hands up while the LF cilia continue to beat. Ten percent ETOH appears to interact with the axoneme, mimicking the effect of high Ca2+and with the membrane to increase Ca2+permeability and possibly to inactivate 5HT receptors. These results are discussed in terms of axonemal switching mechanisms and the physiological control of filter feeding in the lamel

ISSN:0271-6585    

DOI:10.1002/cm.970020303

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

年代:1982

数据来源: WILEY

摘要:

AbstractBehavioral responses of mature spermatozoa treated with neurotropic factors suggest that calcium entry and intracellular transport may be regulated by a cholinergic mediated program. To test the validity of this proposed mechanism, the effect of several agents on Ca distribution in the sperm cell was examined cytochemically.Sites of Ca accumulation were visualized in thin sections of bull spermatozoa by the application of a modification of Gomori's histochemical procedure for phosphatases. Intact bull sperm cells incubated at room temperature in a buffered balanced salt solution containing 5 mM/liter of CaCl2showed small, randomly scattered deposits of the reaction product. Similarly treated sperm cells, plasmolyzed in hypoosmotic KCl, revealed a greatly increased amount of deposit associated with the cell membranes (mitochondrial surfaces and plasmalemma), the axonemal complex components, and satellite fibers adjacent to the outer dense fibers. Preincubation of intact cells in nicotine or eserine considerably enhanced the entry of calcium into the cell and its association with the membranes and other intracellular organelles. Decamethonium, an irreversible depolarizer and blocker of cholinergic receptors, interfered with the uptake and intracellular distribution of the calcium. Ouabain, the digitalis glycoside that decreases progressive motility of bull sperm and inhibits Na‐, K‐ATPase, appears to block Ca efflux, causing an intense accumulation of electron‐opaque particles in the plasma membrane while smaller numbers of particles are distributed sparsely throughout the cell interior.The cytochemical results showing enhanced calcium entry in the presence of cholinergic agents, depressed intracellular calcium in cells treated with cholinergic receptor blocker, and intense accumulation of calcium within the cell membrane in the presence of ouabain are consistent with spermatozoan behavioral responses to these agents. These observations support the concept that neurotropic factors may be involved in regulating transmembrane and intracellular transport of ions in control of sperm cell fun

ISSN:0271-6585    

DOI:10.1002/cm.970020304

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

年代:1982

数据来源: WILEY

摘要:

AbstractExtracellularly applied electric fields (<12 V/cm) strongly influence murine resident peritoneal macrophages (Mø) to undergo directional protrusive pseudopodial activity to wards the positive pole of the electric fields in the absence of exogenously applied chemotactic ligands. Internal and external morphological features were not grossly disrupted by the fields. Directional motility induced by the electric fields was inhibited in the presence of 1.0 mM La3+or 2.5 mM Mg2+and 5.0 mM EGTA. Effects of the fields were latent in the inhibited cells and directional motility was expressed after termination of the field and removal of the inhibitors. Receptors for the lectins concanavalin A (Con A) and phytohemagglutinin (PHA‐L) were uniformly distributed on the surfaces of Mø with no exposure to electric fields. After exposure to the fields, Con A receptors were preferentially distributed on regions of the Mø surface facing the negative pole and PHA‐L receptors were preferentially distributed on those regions facing the positive pole. The possibility that directional Mø motility is regulated by the molecular topography of the cell surface is di

ISSN:0271-6585    

DOI:10.1002/cm.970020305

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

年代:1982

数据来源: WILEY

摘要:

AbstractNucleus associated bodies (NABs) were isolated from Dictyostelium discoideum or Dictyostelium mucoroides and their ability to nucleate microtubules in vitro was examined.NABs were localized at the tapered ends of the nuclei and released from lysed cells in complex with the nuclei. Microtubules radiating from the NAB could also be isolated with the complex under microtubule stabilizing conditions. The ultrastructure of the isolated NAB showed it to be composed of a core structure surrounded by an amorphous matrix.The ability of isolated NABs to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. Microtubule assembly was easily visualized by dark‐field or immunofluorescence microscopy. Polymerization of microtubules seemed to be initiated not from the core structure but from the surrounding matrix.The number of microtubules polymerized from the NAB was directly counted in whole‐mount preparations by electron microscopy, which provided a quantitative assay for the NAB activity. The nucleating activity of NAB was quite unstable and its half‐life was calculated as about 5 hours. The activity was sensitive to protease digestion and was also temperature sensitive but could be stabilized by addition of glycerol or storage at – 80°C or in liquid nitrogen. These characteristics are analogous to those of the centrosomes in cultured mammalian cells and a possible explanation of their similarity is d

ISSN:0271-6585    

DOI:10.1002/cm.970020306

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

年代:1982

数据来源: WILEY

摘要:

AbstractA new actin binding protein has been purified to homogeneity from amoebae of Dictyostelium discoideum. This protein is a single polypeptide with a molecular weight of 120,000 upon sodium dodecyl sulfate gel electrophoresis. It is soluble and trypsin‐sensitive, contains no carbohydrate, increases the viscosity and sedimentation rate of F actin, and inhibits the actin‐stimulated Mg ATPase of rabbit muscle heavy meromyosin. The interaction of 120,000‐dalton protein with F actin is not inhibited by millimolar ATP, pyrophosphate, or micromolar calcium. The 120,000‐dalton actin binding protein increases the initial rate of actin polymerization and decreases the critical concentration of actin at steady state.These properties demonstrate that 120,000‐dalton protein from Dictyostelium discoidum is not a myosinlike protein. Rather, this protein is probably involved in regulating the assembly of the actin cyto

ISSN:0271-6585    

DOI:10.1002/cm.970020307

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

年代:1982

数据来源: WILEY

摘要:

AbstractA protein from Dictyostelium discoideum with an apparent subunit molecular weight of 95,000 daltons (95K protein) was previously identified as an actin‐binding protein ‘Hellewell and Taylor, 1979’. In this paper, we present a method for purifying the protein, and characterize some important aspects of its structure and function. Purification of the 95K protein is achieved by fractionation with ammonium sulfate followed by chromatography on DEAE‐cellulose, gel filtration on 6% agarose, and final purification on hydroxyapatite. The 95K protein is a dimer, composed of apparently identical subunits. It is a rod‐shaped molecule, 38 nm in length, with a Stokes radius of 74 Å. In these structural properties, the 95K protein is similar to muscle and nonmuscle α‐actinins. The 95K protein and filamin are equally competent, when compared on a weight basis, to enhance the apparent viscosity of actin as determined by falling ball viscometry. The apparent viscosity of mixtures of the 95K protein and actin is dramatically reduced at pH greater than 7.0 or free ‘Ca2+’ greater than 10−7M. We also examine the mechanism by which calcium regulates the interaction of the 95K protein and actin. A change in free ‘Ca2+’ induces no detectable change in the quaternary structure of the 95K protein. Our experiments indicate that the 95K protein does not dramatically alter the length distribution of actin filaments in the presence of micromolar free ‘Ca2+’. A large fraction of the 95K protein cosediments with actin in the presence of low free ‘Ca2+’ (ca. 3 × 10−8M), but not in the presence of high free ‘Ca2+’ (ca. 4 × 10−6M). We conclude that increased free ‘Ca2+’ inhibits gelation of actin by the 95K protein by reducing the affinity of the 95K protein for actin. We propose that 95K protein is an important component of the cytoskeleta

ISSN:0271-6585    

DOI:10.1002/cm.970020308

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe isotype pattern of tropomyosin was investigated in normal and dystrophic avian pectroal muscle using two‐dimensional gel electrophoresis. Previous reports have shown that adult pectoral muscle of chickens contains only the α‐subunit of tropomyosin and a breast‐type troponin‐T (TN‐T), whereas pectoral fetal muscle contains both α‐ and β‐tropomyosin and leg‐type TN‐T. The change from the fetal to the adult forms begins shortly after hatching. It has been previously reported that avian dystrophic pectoral muscle contains both the leg‐ and breast‐type TN‐T; we show that in avian dystrophic muscle there is also persistent expression of th

ISSN:0271-6585    

DOI:10.1002/cm.970020309

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

年代:1982

数据来源: WILEY

ISSN:0271-6585    

DOI:10.1002/cm.970020301

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

年代:1982

数据来源: WILEY