ISSN:0886-1544    

DOI:10.1002/cm.970260402

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

年代:1993

数据来源: WILEY

摘要:

AbstractTo understand the mechanism of dynamic instability of microtubule growth and shortening, one needs a means of reliably determining the polarity of the microtubules under investigation. Sea urchin sperm‐tail axonemal fragments nucleate the growth of both plus‐ended and minus‐ended microtubules, but their polarity is not apparent by video‐enhanced DIC microscopy. The polarity of a microtubule is usually assessed by observing differences between the rates and lengths of growth and shortening excursions of the two ends. In practice, though, a significant fraction of the population of microtubules displays characteristics intermediate between the average characteristics of either end, thereby escaping classification. Excluding these “intermediate” microtubules from the measured populations introduces bias into the understanding of microtubule dynamic instability. We circumvent this problem by making use of the plus‐end directed movement of the microtubule‐dependent molecular motor kinesin to determine the polarity of any given microtubule unambiguously. Carboxylated‐microspheres coated with kinesin, which are clearly visible by DIC microscopy, were used to determine the polarity of a microtubule. The dynamics were then observed. Kinesin was found to have no marked effect on dynamic instability. By this technique, we show that the distributions of properties that describe microtubule dynamic instability (rates and lengths of growth and shortening as well as frequencies of interconversion between these phases) of plus‐ends overlap to a significant extent with those of minus‐ends. It is this overlap that obscures the usual classification of the ends. Therefore, models describing microtubule dynamic instability need to incorporate the broad and overlapping range of properties of the two ends.

ISSN:0886-1544    

DOI:10.1002/cm.970260403

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe 10S→6S (Flexed→Extended) transition in smooth muscle myosin is related to increased ATPase activity, but there is controversy over whether the analogous 9S→7S transition in HMM is also associated with ATPase activity. We therefore studied the association of ionic strength, phosphorylation, and ATPase activity for HMM as compared to S1 which has no apparent flexed conformation. In addition, we performed both steady state and single turnover analyses, to control for artifacts due to multiple subfragment populations that might skew steady state results.At low ionic strength where myosin and HMM are in the flexed conformation, HMM had a near zero ATPase activity while S‐1 had a high ATPase rate (0.07 s−1). At 400 mM ionic strength, where both myosin and HMM are in the extended conformation, S1 and HMM had the same ATPase rate (0.04 s−1). Phosphorylation did not affect S1 significantly, but shifted the HMM curve to higher rates at lower ionic strengths. Both steady state and single turnover experiments gave the same results, indicating that steady state results were not skewed by multiple subfragment populations. These data indicate that HMM has a conformation‐ATPase relation similar to that observed with myosin. Furthermore, these findings suggest that the S1 ATPase rate corresponds to that of HMM in the extended conformation. © 1993 W

ISSN:0886-1544    

DOI:10.1002/cm.970260404

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe purpose of this study was to determine how quickly contractile proteins are incorporated into the myofibrils of freshly isolated cardiomyocytes and to determine whether there are regions of the cells that are more dynamic than others in their ability to incorporate the proteins. Paired cardiomyocytes joined at intercalated discs and single cells were isolated from adult rats, and microinjected 3 hours later with fluorescently labeied actin, alpha‐actinin, myosin light chains, and vinculin. The cells were fixed and permeabilized at various period, 5 seconds and longer, after microinjection. Actin became incorporated throughout the I‐Bands in as short a time as 5 seconds. The free edges of the cells, which were formerly intercalated discs, exhibited concentrations of actin greater than that incorporated in the I‐Bands. This extra concentration of actin was not detected, however, at intact intercalated discs connecting paired cells. Alpha‐actinin was incorporated immediately into Z‐Bands and intercalated discs. Vinculin, also, was localized at the Z‐Bands and at intercalated discs, but in contrast to alpha‐actinin, there was a higher concentration of vinculin in the region of the intact intercalated discs. Both alpha‐actinin and vinculin were concentrated at the free ends of the cells that were formerly parts of intercalated discs. Myosin light chains were observed to incorporate into the A‐Bands in periods as short as 5 seconds. These results suggest that the myofibrils of adult cardiomyocytes may be capable of rapid isoform transitions along the length of the myofibrils. The rapid accumulation of fluorescent actin, alpha‐actinin, and vinculin in membrane sites that were previously parts of intercalated discs, may reflect the response to locomotory activity that is initiated in these areas as cells spread in culture. A similar response after an injury in the intact heart could allow repair to occur. ©

ISSN:0886-1544    

DOI:10.1002/cm.970260405

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

年代:1993

数据来源: WILEY

摘要:

AbstractSquid giant axons were used to obtain axonal cytoskeletons that had been separated from the confines of their plasma membranes. To remove the plasma membrane, axoplasm was extruded from the giant axon directly into an artificial axoplasm solution (AAS). This procedure produces a smooth axoplasmic cylinder in which neurofilaments (NFs) are the most prevalent cytological elements. The NFs scatter light strongly and thus dark‐field light microscopy can be used to quantify the volume occupied by these polymers. Measurements of the widths of the dark‐field images of the axoplasmic cylinders showed that the cross‐sectional area of the NF population increased by 60–110% (n = 8) between 1–100 min after plasma membrane removal, and then continued to increase more slowly for many hours. After 1,000 min, the cross‐sectional area was 75–160% (n = 8) larger than at 1 min. These light microscopic measurements of axoplasm suggest that the NF population disperses to occupy a continuously increasing volume after removal of the plasma membrane and immersion in AAS. This inference was confirmed by quantitative ultrastructural studies of NFs in axoplasmic cross‐sections, which demonstrated that the spacing between the NFs increased between 1–1,000 min after plasma membrane removal. Comparison of the NF density distribution after 1,000 min with a theoretical distribution calculated using the Poisson theorem indicated that the NFs dispersed randomly. These studies on NFs in isolated axoplasm suggest that ordinary thermal forces of Brownian motion are sufficient to move axonal NFs apart independently and thereby to disperse them. We propose that, in the intact axon, the dispersive movements of the NFs spread the NF cytoskeleton radially and expansively to fill out the cylindrical space contained by the axonal plasma membrane and its surrounding connective tissue elements. © 19

ISSN:0886-1544    

DOI:10.1002/cm.970260406

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

年代:1993

数据来源: WILEY

摘要:

AbstractCytokeratin and vimentin intermediate filaments (IFs) possess relatively stable polymeric properties which can be affected by phosphorylation. The present study, using cultures of thyroid epithelial cells, shows by indirect immunofluorescence that these cells contain both keratin tonofilament and vimentin IF complexes. Immunoblots of Triton X‐100 insoluble cytoskeletal fractions show vimentin, and ∼52 kDa type II and 40/38 kDa type I keratins. Under “basal” conditions, following prelabeling of cells with [32PO4], vimentin is not significantly phosphorylated, while both type II and I keratins are phosphorylated. Treatment of cells for 20 min with 1 mM dbcAMP or 0.4 μM 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), to stimulate protein kinase A and C, respectively, has no effect on either the phosphorylation state or cytoplasmic filament integrity of vimentin. However, while dbcAMP also does not affect keratin filaments, TPA increases both type II and I phosphorylation ∼3‐fold, and concomitantly disrupts tonofilament complexes associated with the nucleus, cytoplasm, and desmosomes. TPA‐treated cells also show dramatic shape changes and protrusive activity. Tryptic peptide mappings show phosphorylations of at least 6 and ∼2 additional sites for type II and I keratins, respectively, vs. [32P]‐peptides from control cells. Treatment of [32PO4]‐labeled cells with 0.4 μM calyculin A to inhibit types 1 and 2A phosphatase activity causes hyperphosphorylation of both vimentin and keratin, disruption of IF complexes, and actomyosin/cell contraction within 20 min. Quantitatively, ∼50% of the type II/I keratin hyperphosphorylations are at some sites apparently also phosphorylated after TPA treatment. Thus, in these cells, IFs are specifically and differentially affected and regulated by the activity of several

ISSN:0886-1544    

DOI:10.1002/cm.970260407

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe have extended our previous studies of adherent neutrophils and compared actin depolymerization and intracellular calcium changes induced by adherence to laminin and fibronectin. In order to accurately assess cellular actin changes, F‐actin depolymerization in the cell lysates must be inhibited. We found that phalloidin or 3.7% formaldehyde treatment effectively inhibited the depolymerization of F‐actin fragments following cell lysis. Formaldehyde and phalloidin treatment reduced G‐actin levels 75–80% in suspended cells, 35–73% in cells adherent for 1 min, and about 50% for cells adherent for 3 min. When the actin was fixed, there were highly significant differences in G‐actin levels between the suspended and adherent cells as compared with unfixed cells. Adhesion to both laminin and fibronectin initiated a rapid rise in G‐actin with a corresponding decrease in F‐actin. However, the changes were more pronounced in cells adherent to laminin. The peak of depolymerization occurred by 1 min and, thereafter, G‐actin decreased and F‐actin increased reaching a steady state at 5 min. Adhesion to both laminin‐ and fibronectin‐coated surfaces was accompanied by an increase of [Ca2+]i with a peak at 3 min, followed by a decrease from 3–5 min and a steady state attained between 5 and 10 min. The rise of [Ca2+]i in laminin‐adherent cells was about twice that in fibronectin‐adherent cells at 3 min (P<0.02). Pertussis toxin, H‐7, and staurosporin treatments did not alter the dynamic changes of actin in adherent cells, suggesting that these metabolic events are transduced by a G‐protein and Protein Kinase C independent mechanism. The results support the hypothesis that a transient mobilization of F‐actin to a monomeric pool, which then serves as a source for further repolymerization, is induced by adherence of neutrophils to extracellular matr

ISSN:0886-1544    

DOI:10.1002/cm.970260408

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

年代:1993

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970260401

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

年代:1993

数据来源: WILEY