摘要:

AbstractA series of deletions was made from the C‐terminal end of actin by inserting termination codons into a full length cDNA of human α‐skeletal muscle actin. These included deletions of 2, 3, 10, 20, 30, and 40 amino acids. The cDNA clones were transcribed and the resulting mRNA were translated in vitro using35S‐labeled methionine. The35S‐labeled actin and actin mutants were then tested for the ability to coassemble with carrier actin, bind DNAse I, bind myosin S‐1, bind a 27 kDa proteolytic fragment of α‐actinin, and incorporate into myofibrils in vitro. Removal of the C‐terminal two or three amino acids did not grossly alter the properties of actin tested. Deletion of an additional 7 amino acids (10 amino acids total) significantly decreased coassembly, binding to DNAse I, and incorporation into myofibrils, but did not dramatically reduce binding to myosin S‐1 or the 27 kDa fragment of α‐actinin. Deletion of 20 or more amino acids virtually abolished all normal actin function tested. By examining the structure of actin, we propose that the effect of removing residues 356–365 is due to the important role Trp356plays in maintaining hydrophobic bonds between three non‐contiguous segments of actin. We also suggest that removal of residues 366–372 adversely affected the structure or orientation of the DNAse I binding loop and that this change can account for defects in actin binding to DNAse I, coassembly with wild type actin, and incorporation into myofibri

ISSN:0886-1544    

DOI:10.1002/cm.970320302

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe Study of microtubule (MT) dynamics in cells has largely been restricted to events occurring over relatively short periods in nonmotile or stationary cell in culture. By using the antioxidant, Oxyrase, we have reduced the sensitivity of fluorescent MTs to photodamage and this has allowed us to image fluorescent MTs with good temporal resolution over much longer periods of time. We have used our enhanced imaging capabilities to examine MT dynamics in fibroblasts moving directionally into a wound. We found that MTs in these cells exhibited dynamic instability similar to that reported for other cells. More interestingly, we found a novel dynamic behavior of the MTs in wihch entire MTs were moved inward from the leading edge toward the cell nucleus. This centripetal transport (CT) of MTs only occurred to those MTs that were oriented with their long axis parallel to the leading edge; radially oriented MTs were not transported centripetally. Both small bundles of MTs and individual MTs were observed to undergo CT at a rate of 0.63 × 0.37 μm/min. This rate was similar to the rate of CT of latex beads applied to the cell surface and of endogenous pinocytotic vesicles in the cytoplasm. When we imaged both MTs and pinocytotic vesicles, we found that the pinocytotic vesicles were ensheathed by a small group of parallel MTs that moved centripetally in concert with the vesicles. Conversely, we found many instances of MTs moving centripetally without associated vesicles. When cells were treated with nocodazole to depolymerize MTs rapidly, the rate of pinocytotic vesicle CT was inhibited by 75%. This suggests that centripetal transport of MTs may be involved in the movement of pinocytotic vesicles in cells. In conclusion, our results show that MTs in motile cells are redistributed by a novel mechanism, CT, that does not require changes in polymer length. The centripetally transported MTs may play a role in transporting pinocytotic vesicles in the cell. © 1995 Wiley‐Liss,

ISSN:0886-1544    

DOI:10.1002/cm.970320303

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe neuronal growth cone plays a fundamental role in nerve development and regeneration. A sensory‐motile structure, it determines the path of axonal extension through its interactions with the extracellular environment, ultimately directing the formation of functional connections in the nervous system. Though several mechanisms of interaction have been proposed, these have been difficult to describe quantitatively due to the complexity of growth cone behavior, as manifested in the randomly and rapidly changing shape of the growth cone. The application of mathematical techniques to model growth cone shape and motility in terms of underlying processes represents a promising approach with untapped potential for helping to unravel this complexity while revealing new insights into axonal pathfinding events. This paper presents a simulation model for filopodial dynamics, a primary feature of the motile growth cone. The model produces realizations of dynamic filopodial structure on representative growth cones for a given set of model parameters, which include the rates of filopodial initiation, extension, and retraction, filopodial length at maximum extension, and angular orientation. These parameters are based on recent experimental characterization of filopodial dynamics [Buettner et al., 1994: Dev. Biol. 163:407–422]. The mathematical relationship between the model parameters and average filopodial number and length per growth cone is described, and the contribution of individual parameters to overall filopodial morphology is illustrated both visually and numerically. In addition, the model is used to simulate filopodial encounter with a target for various conditions of filopodial dynamics. The result is characterized in terms of a mean encounter time for a population of growth cones and provides an indication of the effect of individual parameters of filopodial dynamics on the encounter process. Future experimental testing will be required to develop the model further. However, in its current form, the model enables a first approximation analysis of many hypotheses of growth cone migration and pathfinding and offers insight into the the underlying mechanismes of nerve growth and regeneration. © 1995 Wiley‐Lis

ISSN:0886-1544    

DOI:10.1002/cm.970320304

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

年代:1995

数据来源: WILEY

摘要:

AbstractA 107‐kD protein has been identified in primary cultures of chicken embryonic cardiomyocytes by immunoprecipitations with certain anti‐nebulin monoclonal antibodies (mAbs). These mAbs, prepared against a fragment of human skeletal muscle nebulin located near the carboxyl terminus, detect a 107‐kD protein in extracts of adult chicken heart, adult mouse heart, and adult rabbit heart by immunoblot analysis. A partial cDNA corresponding to this protein has been isolated by immunological screening of a chicken heart cDNA expression vector library. The partial cDNA encodes a 380‐amino acid open reading frame composed entirely of nebulin‐like 35‐residue modules marked by the highly conserved sequence motifs: SXXXYK and TPD. The open reading frame exhibits 60–85% homology with skeletal muscle nebulins from a variety of species. This cDNA recognizes an ˜8‐kb transcript in cardiac RNA and does not hybridize to skeletal muscle RNAs by northern analysis. Immunofluorescence localization of this nebulin‐like protein in primary cultures of chicken cardiomyocytes and embryonic chicken cardiac myofibrils indicates that the protein is localized to the I‐Z‐I complex of the myofibrils, extending approximately 25% of the thin filament length. Comparisons of the distribution of this protein relative to actin, myosin, and titin in spreading cardiomyocytes suggest that the cardiac nebulin‐like protein becomes aligned with the nascent myofibrils early during myofibrillogenesis. To distinguish this petite nebulin‐like protein from the 600–900 kD skeletal muscle nebulin, we have named it nebul

ISSN:0886-1544    

DOI:10.1002/cm.970320305

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

年代:1995

数据来源: WILEY

摘要:

AbstractLocated at the level of the Z‐line, the transverse cytoskeletal network of insectflight muscle interconnects adjacent myofibrils with one another, and interconnects peripheral myofibrils with the cell membrane. This network has been presumed to keep myofibrils in register, or to distribute tension laterally among myofibrils. In this study, we used scanning‐electron microscopy to reveal details of the three‐dimensional arrangement of this network. The network is seen to interconnect longitudinal elements of the cytoskeletal network which surround each myofibril. The arrangement is not unlike that seen in vertebrate skeletal muscle. Interestingly, the transverse network makes contact with cell components such as dense bodies and mitochondria. Such contacts imply potential roles over and above those noted above. The network may be involved not only in mechanical function, but possibly also in intracellular communication. © 1995 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970320306

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

年代:1995

数据来源: WILEY

摘要:

AbstractCaldesmon is an actin, calmodulin, tropomyosin, and myosin binding protein implicated in the regulation of actomyosin interactions. We have invesigated the effect of overexpression of the higher molecular weight smooth muscle isoform of caldesmon on mouse L cell physiology. Mouse L(TK−) cell were transfected stably with plasmids carrying the TK+gene and a full length human smooth muscle caldesmon cDNA under control of the adenovirus major late promoter. Two clones displaying four and eight times the level of the endogenous mouse high molecular weight caldesmon were isolated. These cells acquire a distinct phenotype characterized by an altered morphology, including an increased number of processes and larger area due to enhanced cell spreading, and a significantly slower growth rate than that of untransfected control cells, or cells transfected with the TK+gene alone. The majority of the overexpressed caldesmon appears to be active and localized on cytoskeleton structures as determined by detergent lysis. Immuno‐fluorescence analysis of the clones revealed that the caldesmon is localized as punctate staining on stress‐fibers and in membrane ruffles. The immunofluores‐cence images suggest that caldesmon overexpressing cells have more total filaments than control cells. The effects of excess caldesmon on cell mobility are ambiguous: one clone displayed increased motility compared to the control, while the motility of the second clone was decreased relative to the control. © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970320307

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.970320301

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

年代:1995

数据来源: WILEY