摘要:

AbstractThe relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole‐mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814–821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5–20 μg/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of3H‐thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of ea

ISSN:0886-1544    

DOI:10.1002/cm.970060402

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

年代:1986

数据来源: WILEY

摘要:

AbstractBundles of microtubules (MTs) are readily visualized in vivo by videomicroscopy in highly flattened reticulopodia of the foraminiferan protozoanAllogromiasp. strain NF. In this report we use videomicroscopy, immunocytochemistry, and high‐voltage electron microscopy to characterize the dynamic changes that occur in this extensive MT cytoskeleton, and in the associated cytoplasmic transport, during induced withdrawal and subsequent reextension of reticulopodia. Within seconds after application of the withdrawal stimulus (seawater substitute made hypertonic with MgCl2) intracellular bidirectional transport along linear MT‐containing fibrils ceases and is replaced by an inward, constant‐velocity flow of cytoplasm along the fibrils. As withdrawal continues, most fibrils become wavy and coalesce to form phase‐dense pools. These wavy fibrils and phase‐dense pools contain a paracrystalline material and few if any MTs. Same‐section correlative immunofluorescence and high‐voltage electron microscopy reveal that the paracrystalline material contains tubulin. During recovery linear fibrils (MTs) rapidly extend from the phase‐dense pools (paracrystals), which concurrently shrink in size, thus reestablishing normal network morphology and motility. We conclude that the MT cytoskeleton inAllogromiareticulopodia is transfonned during withdrawal into a tubulin‐containing paracrystal, which serves as a temporary reservoir of MT protein and an initiation sit

ISSN:0886-1544    

DOI:10.1002/cm.970060403

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

年代:1986

数据来源: WILEY

摘要:

AbstractPlatinum‐carbon (Pt‐C) replicas of freeze‐dried erythrocyte cytoskeletons of the toad,Bufo marinus, were prepared using a modified Balzers 300 system. Examination in stereo of replicas of the microtubule‐containing marginal band revealed filaments projecting from the microtubule walls to form links between adjacent microtubules. These cross‐bridging proteins may bundle the microtubules into the configuration of the marginal band (MB) and may also serve to stabilize the structure. The MB appears to have linkages to components of the surface‐associated cytoskeleton (SAC). The SAC forms a continuous matrix that spreads across the upper and lower surfaces of the cell adjacent to the plasma membrane and extends around the outer perimeter of the MB. Thus, the SAC encapsulates the MB and the central nucleus. After lysis, the elements of the cytoskeleton remain in a configuration similar to that found in the whole cell. Spectrin (fodrin) and actin were identified by immunofluorescence in the region of the SAC. When labeled with antibodies specific for vimentin and synemin, a network of intermediate filaments can be detected in the region between the nucleus and the MB. These vimentin filaments are also enclosed within the SAC and appear in Pt‐C replicas to emerge from the area of the nuclear envelope. As the filaments extend toward the periphery of the cell, they form attachments to the SAC. Attachments of intermediate filaments to both the nucleus and the SAC thus appear to anchor the nucleus in its central position within the

ISSN:0886-1544    

DOI:10.1002/cm.970060404

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

年代:1986

数据来源: WILEY

摘要:

AbstractIntermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK‐21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a “cell membrane complex” defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces.In order to investigate the possibility that components of the membrane complex may co‐isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a ∼220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to

ISSN:0886-1544    

DOI:10.1002/cm.970060405

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

年代:1986

数据来源: WILEY

摘要:

AbstractSpreading and fully spread chick embryo fibroblasts (CEF) were examined by double‐label fluorescence microscopy using the actin‐specific probe rhodamine‐phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase‐dense, phalloidin‐binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin‐containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers.Ultrastructural analysis confirmed the presence of non‐membrane‐bound out‐pocketings along the length of stress fibers from which 10‐nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment‐1. Other IF‐MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine‐treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D‐treated CEF exhibited loose aggregates of actin‐containing material that appeared to be associated with IF.These results suggest the possibility of an interaction between actin‐containing structures and IF, particularly during cell sp

ISSN:0886-1544    

DOI:10.1002/cm.970060406

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

年代:1986

数据来源: WILEY

摘要:

AbstractReversal of meiotic arrest in crane‐fly spermatocytes by U.V. irradiation of Colcemid‐arrested cells or by rinsing Nocodazole‐arrested cells in fresh buffer results in the induction of chromosome malorientation. Malorientations observed among Colcemid‐recovering and Nocodazole‐recovering spermatocytes at frequencies higher than normally observed in untreated cells included associations of sister kinetochores of half‐bivalents with both spindle poles (amphitely), in contrast with associations of sisters with only one pole (syntely) as is usually found during the first meiotic division. In several cases, prior to anaphase onset, maloriented bivalents appeared unusually tilted with respect to the spindle axis, and during anaphase they gave rise to laggard half‐bivalents that did not segregate during anaphase along with half‐bivalents having proper syntelic orientation. The results parallel previous findings obtained during cold recovery, and the properties of the drugs used here suggest that their action on microtubules, although reversible, induces malorientation during recovery from

ISSN:0886-1544    

DOI:10.1002/cm.970060407

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

年代:1986

数据来源: WILEY

摘要:

AbstractWe investigated the involvement of kinetochore microtubules (kMTs) in mediating chromosome‐to‐pole connections in crane‐fly (Nephrotoma suturalisandNephrotoma ferruginea) spermatocytes. Two experimental treatments were used to yield spindles with reduced numbers of nonkinetochore microtubules (nkMTs). Short‐term (10–15 min) exposure of spermatocytes to 2°C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long‐term (24h) exposure to 2°C followed by recovery at 6°C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2°C‐treated spindle but fewer than an untreated spindle, in which the kMT:(kMT + nkMT) ratio was 0.27. The numbers and lengths of kMTs in 6°C‐grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6°C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (>3 μm), and many extended to the polar regions of the spindle. Thus, the crane‐fly spindle appears not to be as atypical as it was pr

ISSN:0886-1544    

DOI:10.1002/cm.970060408

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

年代:1986

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970060401

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

年代:1986

数据来源: WILEY