摘要:

AbstractBased on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle‐specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0–24 h postmitosis) by video time‐lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x‐h‐old to define their age relative to their mitotic “birthdate.” All 6 h myoblasts that emerged in a mitogen‐rich medium were desmin+but only 50% were positive for a α‐actin, troponin‐I, α‐actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up‐regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were α‐actinin+or zeugmatin+but MyHC−or titin−whereas others were troponin‐I+or MyHC+but α‐actinin−or α‐actin−. In 15‐h‐old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle‐shaped cells; and (2) the sarcolemma became fusion‐competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP‐C (C protein). The emergence of one, or a string of ∼ 2 μ long sarcomeres, was invariably characterized by the localization of myomesin and MyBP‐C to their mature positions in the developing A‐bands. The latter group of A‐band proteins may be rate‐limiting in the assembly program. The great majority of rnyoblasts stained positively for desmin and rnyofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle‐specific proteins in vitro fully recapitulates myofibrillar assembly steps in rnyoblasts of the myotome and limb bud in vivo, as well as in nonrnuscle cells converted to myoblasts by MyoD. We suggest that this cell‐autonomous myoblast differentiation program may be blocked at different contro

ISSN:0886-1544    

DOI:10.1002/cm.970290102

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

年代:1994

数据来源: WILEY

摘要:

AbstractSchwann cells (SC), the myelinating cells of the peripheral nervous system, show a remarkable capacity to switch from a differentiated state to a proliferative state both during development and peripheral nerve regeneration. In order to better understand the regulatory mechanisms involved with this change we are studying a Schwann cell line transfected with the SV‐40 large T gene (TSC). Serum‐free medium combined with elevating intra‐cellular cAMP levels produced a slower proliferating TSC whose morphology changed from pleiomorphic to process bearing, reminiscent of primary SC in culture. This change was abrogated by colcemid but was unaltered by cytochalasin D, indicating a major role for microtubules. Ultrastructural studies demonstrated numerous microtubules in the cellular extensions which correlated with strong immunocytochemical staining for tubulin in the processes. Analysis of cytoskeletal fractions from the treated cells revealed a greater proportion of tubulin in the polymerized state compared with untreated cells which closely resembled the distribution in primary SC. The cytoskeletal changes observed in the TSC as a result of elevating the intra‐cellular cAMP levels may reflect the earliest cellular changes in the induction of myelination. © 1994 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970290103

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

年代:1994

数据来源: WILEY

摘要:

AbstractIn this report, we have compared the physical properties and actin‐binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin‐II and brush border (BB) myosin‐I. All of the bacterially produced nonmuscle tropomyosins bind to F‐actin with the expected stoichiometry and with affinities comparable to that of a tissue produced α‐tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity of F‐actin than a tissue‐purified striated muscle α tropomyosin. The bacterially produced isoforms also protect F‐actin from severing by villin as effectively as tissue‐purified striated muscle α‐tropomyosin. The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin‐activated MgATPase activity of muscle myosin S1 by ∼ 70–85%, comparable to the inhibition seen with tissue‐purified striated muscle α tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin‐activated MgATPase activity of muscle myosin S1 approximately two‐ to threefold. The in vitro sliding of actin filaments translocated by muscle myosin‐II (2.4 μm/sec at 19°C, 5.0 μm/s at 24°C) increased 25–65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin‐II motility. The actin‐activated MgATPase activity of BB myosin‐I was inhibited 75–90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin‐I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulati

ISSN:0886-1544    

DOI:10.1002/cm.970290104

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

年代:1994

数据来源: WILEY

摘要:

AbstractDictyostelium discoideumamoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, α‐actinin and the 30 kD actin‐bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for α‐actinin is not apparent in this region. By contrast, α‐actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of α‐actinin with the contractile vacuole was confirmed by colocalization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and α‐actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but disassociates from phagosomes soon after internalization [Furukawa et al., 1992:Protoplasma169: 18–27]. α‐actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has disassociated. These results support the hypothesis that α‐actinin and the 30 kD protein play distinct roles in cell structure and movement inDictyostelium

ISSN:0886-1544    

DOI:10.1002/cm.970290105

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

年代:1994

数据来源: WILEY

摘要:

AbstractMicrotubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22°C. The bundles, which were readily visible by video‐enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6–11 μm in length and 2–5 MTs in thickness. No inter‐MT cross‐bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS‐PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP‐2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt‐extractable and heat‐stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were “unbundled” by subtilisin or by high salt (0.5–1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt‐induced unbundling was reversible with desalting. However, reversibility was observed only after salt‐induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly f

ISSN:0886-1544    

DOI:10.1002/cm.970290106

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

年代:1994

数据来源: WILEY

摘要:

AbstractTo better understand the solution structure of spectrin, the environments of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its α and β subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the β subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated β chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2‐p‐toluidinyl napthalene‐6‐sulfonic acid (TNS). It therefore appears that low ionic strength, α‐β association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solutions does not involve exposure of significant numbers of hydrophobic sites. © 1994

ISSN:0886-1544    

DOI:10.1002/cm.970290107

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe production of monoclonal antibodies and the use of biochemical techniques revealed that B‐type costa proteins in trichomonads are composed of several major polypeptides with molecular weight detected between 100 and 135 kDa similar to those found in the A‐type costae. Although differences were observed between the two types in their fine structure, we tested whether proteins composing the two costa types belong to the same protein family. A polyclonal antibody produced against the 118 kDa costa protein ofTrichomonas vaginalisalso recognized a 118 kDa costa protein in all other trichomonad genera studied so far whether they have A‐ or B‐type costae. Moreover biochemical characteristics of costa proteins indicated that these proteins might represent a novel class of striated root‐forming proteins in addition to centrin, giardin, and assemblin. © 1994 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970290108

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.970290101

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

年代:1994

数据来源: WILEY