摘要:

AbstractCell locomotion depends on polymerization and depolymerization of filamentous actin. Net polymerization at the cell front occurs fast enough to fill the extending lamellipod, and since total F‐actin is essentially constant over time, depolymerization must equal polymerization. Indeed, the fastest moving cell types have the highest rates of depolymerization. Accounting for the high rate of depolymerization raises several problems. One is that net depolymerization requires the concentration of G‐actin to be low (below the critical concentration), but rapid polymerization (occurring<1 μm away) requires the concentration of G‐actin to be high (well above the critical concentration). This may be accomplished by spatial compartmentalization of factors that favor polymerization or depolymerization, and/or by proteins that bind G‐actin and prevent spontaneous polymerization while allowing barbed‐end elongation. A second problem is that depolymerization proceeds faster than would seem possible from studies of F‐actin in vitro (as calculated from number and lenghts of filaments present and in vitro rate constants). Rapid depolymerization may be accomplished by filament cutters or by cytoplasmic components (as yet undiscovered) that increase the rate of depolymerization. © 1993 Wi

ISSN:0886-1544    

DOI:10.1002/cm.970250402

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

年代:1993

数据来源: WILEY

摘要:

AbstractActin filaments undergo dramatic changes in their organization during myofibrilloenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z‐discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to “cap” each actin filament of the sacromere. Double inmmunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, α‐actinin, titin, and myosin during myofibrilloenesis. Of particular interest was the assembly of CapZ at nascent Z‐discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non‐striated actin filaments called stress fiber‐like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z‐discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I‐bands, CapZ was distributed in a periodic pattern characteristic of mature Z‐discs. The finding that CapZ was assembled at nascent Z‐discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I‐band formation in skeletal muscle cells. The assembly of CapZ at nascent Z‐disc structures also was observed relative to the assembly of sarcomeric α‐actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z‐discs. The distribution of CapZ and sarcomeric α‐actinin in young myotubes was not coincident; in older myotubes, both CapZ and α‐actinin were co‐localized at Z‐discs. In cardiac myocytes, CapZ was detected at Z‐discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co‐localized with A‐CAM and vinculin at cell‐cell junctio

ISSN:0886-1544    

DOI:10.1002/cm.970250403

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

年代:1993

数据来源: WILEY

摘要:

AbstractCentrosomes are unique cytoplasmic structures which serve as microtubule organizing centers (MTOC). In most animal cells centrosomes consist of one or more pair of centrioles surrounded by electron dense amorphous pericentriolar material (PCM) responsible for nucleation of microtubules. In the present study we analyzed the pattern of induction and localization of proteins of the PCM at different stages of neuronal development in cell cultures prepared from the embryonic hippocampus. For this purpose we used a human polyclonal antibody that recognizes two proteins of the PCM (100 kd and 60 kd, respectively). The results indicate that in mature neurons, pericentriolar immunoreactive material is preferentially localized in dendritic processes, and that throughout the course of neurite development and differentiation it is systematically excluded from the neuron's axon. Western blot analysis showed that during neuronal development in situ, there is an increase in he immunoreactivity for both proteins recognized by this antibody. In contrast, in hippocampal pyramidal neurons that develop in culture, there is an increase in the 60 kd polypeptide, while the 100 kd one is not detected after 7 days in vitro. © 1993 Wiley‐Liss, I

ISSN:0886-1544    

DOI:10.1002/cm.970250404

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe have examined the effects of various means of photobleaching on the recovery of fluorescene, movement, and morphology of the microtubules in the neurites of rhodamine‐tubulin‐injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different patterns of fluorescence recovery in regenerating PC12 neurites. If bleaching is performed with a relatively low‐power beam for an extended period, fluorescence in polymer recovers very little after 1 hours. Under these conditions, however, tubulin immunostaining is seen extending through the bleach zone, and microtubules are present through the bleached zone in thin section electron micrographs. If bleaching is performed with a high‐power laster, for 0.5–5 seconds, fluorescence recovery also is quite slow, but electron microscopic observations reveal that no microtubules extend through the bleached region of the neurite, and the uranyl acetate‐stained cytoplasm appears more electron lucent than in the unbleached neurite. Finally, if bleaching is performed by very brief exposure to a high‐intensity laser beam, resulting in an incomplete reduction of fluorescence intensity through the bleach zone, fluorescence recovery occurs within 20–30 minutes, and immunostained microtubules appear intact through the bleach zone; electron microscopy confirms that microtubules extended through the bleached zone of such neurites. In all three cases, movement of the bleach zone is observed in approximately half of the experimental neurites. These results indicate that highly variable microtubule behaviors can be obtained with photobleach technology, presumably due to different levels and pathways of photodamage generated by different bleach protocols. Nevertheless, it is clear that both turnover and movement of microtubules occur in FC12 neurites, and both are likely to be involved in neurite maintenance and growth. © 1993

ISSN:0886-1544    

DOI:10.1002/cm.970250405

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

年代:1993

数据来源: WILEY

摘要:

AbstractIn smooth muscle tissue, two or three isoforms of myosin heavy chain (MHC) have been reported (SM1, SM2, and/or NM). In mouse uterus tissue, four bands in the region of the MHC's can be resolved on high resolution SDS polyacrylamide gels. Western blots using smooth muscle (SM) MHC‐specific and nonmuscle (NM) MHC‐specific polyclonal antibodies show the upper two bands in the MHC region are SM isoforms, whereas the lower two bands are NM isoforms. One‐dimensional peptide maps of these four bands show each to have a unique pattern of polypeptide fragments following α‐chymotrypsin digestion. Developmental expression of myosin heavy chains (MHC) in mouse uterus, aorta, bladder, and stomach (6 ages, 10–150 days) was determined using tissue homogenates. In the uterus, both SM MHC's show an increase in relative content with increasing age, whereas the NM MHC's show a decrease. The mouse aorta shows a significant increase in the SM MHC's and a significant decrease in the NM MHC from day 10 to day 30, which is similar to data reported for the rat aorta. Whereas both the bladder and stomach contain relatively small amounts of NM MHC's (∼ 10% or less), these quantities do show decreases with development. The SM1:SM2 ratio for the uterus remains high (3.4 at 150 days) through development; the aorta, bladder, and stomach also start out high, but tend toward 1.0 in the 150‐day animals. The presence of four MHC isoforms in the uterus with unique developmental regulation of expression is consistent with hypotheses of unique functional roles for these isoforms. © 1993

ISSN:0886-1544    

DOI:10.1002/cm.970250406

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

年代:1993

数据来源: WILEY

摘要:

AbstractMyosin heavy chain (MHC) phosphorylation was examined throughout the period of first cleavage in developing sea urchin embryos. MHC was found to be phosphorylated in these cells and, furthermore, the relative state of myosin phosphorylation was found to decrease as cells progressed through cytokinesis. Following the completion of cytokinesis, the relative state of MHC phosphorylation returned to levels observed in precytokinesis cells. The above results were obtained with myosin immunoprecipitated from whole cell lysates. In order to specifically examine the phosphorylation, state of MHC in the cleavage furrow, a protocol was developed for the isolation of intact contractile rings from dividing sea urchin embryos. MHC was immunoprecipitated from isolated contractile rings and the relative phosphorylation state of this MHC was compared with that of MHC isolated from whole cell lysates prepared at the same time. MHC from isolated contractile rings was found to be significantly less phosphorylated than total cellular MHC, suggesting a role for MHC dephosphorylation during cytokinesis. © 1993 Wiley‐Liss, I

ISSN:0886-1544    

DOI:10.1002/cm.970250407

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe have identified a 67 kDa heat‐stable protein among the proteins which bind specifically to brain microtubules immobilized on a chromatographic support. Its relationship to tubulin and to the cytoskeleton using polyclonal antibodies has been studied. This 67 kDa protein is present in cytoskeleton and microtubule preparations from pancreas. This heat‐stable microtubule‐associated protein (MAP) copolymerized with phosphocellulose purified brain tubulin. The 67 kDa polypeptide was immunoreactive to antibodies against the 210 kDa MAP from HeLa cells; it also reacted with antibodies against an oligopeptide whose sequence corresponded to the second repeat of mouse brain tau. © 1993 Wiley‐L

ISSN:0886-1544    

DOI:10.1002/cm.970250408

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.970250401

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

年代:1993

数据来源: WILEY