ISSN:0886-1544    

DOI:10.1002/cm.970200402

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe have employed video‐enhanced light microscopy to study alterations of the overall shape of microtubules that are produced by the aldehyde fixation methods commonly employed to study them in vitro. Changes brought about by these methods include deformation and breakage. The severity of the effects depends on the fixative employed and increases with its concentration, and with the time of fixation. The changes are observed under a variety of conditions, such as brief exposure to 3.7% formaldehyde, or somewhat longer exposure to glutaraldehyde at concentrations as low as 0.05%. The observed distortion explains why microtubules usually appear curved or sinuous in electron micrographs while appearing relatively rigid and linear in video‐enhanced light microscopy. The observed breakage implies that caution must be used in inferring length distributions from measurements of aldehyde‐fixed microtu

ISSN:0886-1544    

DOI:10.1002/cm.970200403

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

年代:1991

数据来源: WILEY

摘要:

AbstractABP‐50 is the elongation factor‐1 alpha (EF‐1 alpha) ofDictyostelium discoideum(Yang et al.:Nature347:494–496, 1990). ABP‐50 is also an actin filament binding and bundling protein (Demma et al.:J. Biol. Chem. 265:2286–2291, 1990). In the present study we have investigated the compartmentalization of ABP‐50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F‐actin in surface extensions in unstimulated cells, ABP‐50 exhibits a diffuse distribution throughout the cytosol. Upon addition of cAMP, a chemoattractant, ABP‐50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP‐50 in Triton‐insoluble and‐soluble fractions of resting cells indicates that 10% of the total ABP‐50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABP‐50 into the Triton cytoskeleton. The peak of incorporation of ABP‐50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP‐50 from unstimulated cells using affinity‐purified polyclonal anti ABP‐50 results in the coprecipitation of non‐filamentous actin with ABP‐50. Purified ABP‐50 binds to G‐actin with a Kd of approximately 0.09 μM. The interaction between ABP‐50 and G‐actin is inhibited by GTP but not by GDP, while the bundling of F‐actin by ABP‐50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP‐50 is bound to either G‐ or F‐actin in vivo and that the interaction between ABP‐50 and F‐ac

ISSN:0886-1544    

DOI:10.1002/cm.970200404

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

年代:1991

数据来源: WILEY

摘要:

AbstractStable subsets of microtubules (MTs) are often enriched in detyrosinated α‐tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time‐dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated α‐tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA‐resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA‐sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT‐associated proteins (MAPs) may organize and stabilize the juxtanuclear dety

ISSN:0886-1544    

DOI:10.1002/cm.970200405

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

年代:1991

数据来源: WILEY

摘要:

AbstractCellular and intracellular motility are compared between normalDictyosteliumamoebae and amoebae lacking myosin IB (DMIB−). DMIB−cells generate elongated cell shapes, form particulate‐free pseudopodia filled with F‐actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB−cells also exhibit a normal response to the addition of the chemoattractant cAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F‐actin in pseudopodia, and a concomitant increase in cortical F‐actin. DMIB−cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB−cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB−cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhi

ISSN:0886-1544    

DOI:10.1002/cm.970200406

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

年代:1991

数据来源: WILEY

摘要:

AbstractRat sperm, demembranated with 0.1% Triton X‐100, were used to explore the reversal in flagellar curvature induced by calcium ion. As reported earlier (Lindemann and Goltz, Cell Motil. Cytoskeleton, 10:420–431, 1988), the radius of curvature of the flagellar midpiece of rat sperm is controlled by the free Ca2+concentration. A reversal of the direction of curvature (judged by the asymmetric sperm head) takes place at ≈ 2.5 + 10−6M free Ca2+.In our current study, the time course of the curvature change, after elevating free Ca2+to 3.5 ± 10−4M, was utilized to assess the effects of the cAMP‐kinase A pathway on the calcium response. In addition, calmodulin's involvement in this response was explored using anti‐calmodulin and Cd2+. The activity state of the sperm models (which could be directly influenced through cAMP) was found to control the rate of curvature change in response to increased free Ca2+. In the most extreme case, fully quiescent sperm did not respond to Ca2+at all, and cAMP‐primed sperm models completed the response to Ca2+in two minutes or less.Anti‐calmodulin demonstrated strong inhibitory effects on the curvature reversal. Cadmium ion was also extremely potent at blocking the response to Ca2+, completely eliminating the curvature reversal at 2 × 10−10M free Cd2+.Based on these findings, it appears that the Ca2+‐activated curvature reversal of rat sperm is potentiated by cAMP‐dependent kinase and may be me

ISSN:0886-1544    

DOI:10.1002/cm.970200407

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

年代:1991

数据来源: WILEY

摘要:

AbstractIn this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium‐modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100α, slow‐twitch skeletal muscle fibers contained predominantly S100α, vascular smooth muscle contained both S100α and S100β, and fast‐twitch skeletal muscle fibers contained low but detectable levels of S100α and S100β. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100α staining was associated with muscle cells, while S100β staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100α at postnatal day 1 and that as development proceeded the S100α levels increased. In contrast to adult muscle, S100α expression as confined to fast‐twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100α positive, but no staining periodicity was detectable. At postnatal day 21, S100α exhibited the same subcellular localization as seen in the adult: colocalization with the A‐band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100α‐binding protein profiles in fast‐ and slow‐twitch fibers of various species revealed few, if any, species‐ or fiber type‐specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myo fibrils contained multiple S100α‐hinding proteins. The colocalization of S100α and S100α‐binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100α may regulate excitation

ISSN:0886-1544    

DOI:10.1002/cm.970200408

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

年代:1991

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970200401

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

年代:1991

数据来源: WILEY