摘要:

AbstractThe effects of colcemid (0.16–1.0 μM) and taxol (10 μM) on the primary cilia cycle in PtK1cells were studied by antitubulin immunofluorescence microscopy and by high‐voltage electron microscopy of serial 0.25‐μm sections. Although these dings induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubulcs, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5–7 h at 37°C and then form 4N “micronucleated” restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration‐dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16–0.25 μM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (≥ 1.0 μM) concentrations. However, even in the presence of 1.0 μM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell‐cycle–dependent assembly and disassembly of a

ISSN:0886-1544    

DOI:10.1002/cm.970070302

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

年代:1987

数据来源: WILEY

摘要:

AbstractTransient ciliary movement during responses to electric stimulation of embryos of the sea urchin,Hemicentrotus pulcherrimus, was analyzed in terms of angular direction with a time resolution of approximately 2 ms with high‐speed microcinematography. In the primitive response, which can be induced only in the early stages of development of the embryo, bending transients always started with a short pause in the middle of the effective stroke, irrespective of beat position on stimulation. In the reversal response, induced only in the late stages of development, bending transients occurred with a delay as short as some 10 ms from stimulation, and with a transient sharp deviation from the normal beat before the cilium took the position of the beginning of the recovery stroke of the reversed beat. The delay was significantly shorter at the base than at the tip, suggesting that some form of signal travels along the cilium; the speed was ten times higher than that of propagating bends in the normal beat. These facts indicate that the sensitivity to internal changes resulting from stimulation of the axoneme may vary with development, ciliary beat positions, and regions along the ciliu

ISSN:0886-1544    

DOI:10.1002/cm.970070303

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

年代:1987

数据来源: WILEY

摘要:

Abstractα‐Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. α‐Actinin was incorporated into stress fibers within 5 minutes after injection and remained present in the fibers for up to 11 days. The pattern of incorporation was the same regardless of whether the α‐actinin was isolated from muscle or nonmuscle tissues or whether it was labeled with fluorescein, Lucifer Yellow, or rhodamine dyes. In contrast, neither labeled ovalbumin nor bovine serum albumin were incorporated into stress fibers. When the injected cells entered prophase, all stress fibers disassembled, resulting in a distribution of the fluorescent α‐actinin throughout the cytoplasm. During cytokinesis, the fluorescent α‐actinin was concentrated in the broad area between the separated chromosomes and along the edge of the cell in the cleavage area. Within 10 minutes after the completion of cleavage, the first fluorescent stress fibers reformed parallel to the spreading edges of the daughter cells and in close association with the midbody with a concomitant loss of α‐actinin in the former cleavage furrow. Additional fibers formed adjacent to these first stress fibers. In some cases, new stress fibers formed between two existing stress fibers and some stress fibers moved up to 4 μm apart from one another in the course of 2 hours. Thus, fluorescent α‐actinin, injected into living cells, undergoes the same cyclical changes in distribution as endogenous α‐actinin during the cell cycle: from stress fibers to cleavage furrow a

ISSN:0886-1544    

DOI:10.1002/cm.970070304

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

年代:1987

数据来源: WILEY

摘要:

AbstractWe have demonstrated the presence of MAP‐2 in squirrel fish erythrophores using SDS‐PAGE, immunobolt, and immunoprecipitation techniques. The monoclonal antibodies used (AP‐9, ‐13, ‐14) were raised against distinct antigenic sites on Chinese hamster brain MAP‐2. Immunoprecipitation studies demonstrated that all three antibodies bind a 300 K protein found in crude cell extracts and in partially purified MAP fractions isolated from erythrophores of the squirrel fishHolocentrus rufus. Immunofluorescent studies confirmed that the 300 K protein was present in cultured erythrophores. Studies of cells induced to aggregate and disperse their pigment granules revealed that the 300 K protein comigrated with the pigment, suggesting that the 300 K protein may constitute part of the “α‐cytomatrix” involved in pigm

ISSN:0886-1544    

DOI:10.1002/cm.970070305

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

年代:1987

数据来源: WILEY

摘要:

AbstractWe tested diethylstilbestrol (DES) and 17 β‐estradiol as mitotic arrestants to determine their effects on chromosome distribution, spindle microtubules, and the cytoplasmic microtubule complex (CMTC) in the Chinese hamster strain Don. Cytological experiments assessed micronuclei induction, chromosome displacement, and anaphase recovery Indirect immunofluorescence microscopy with antibody to tubulin and electron microscopy were used to illustrate effects on microtubules. Both DES and estradiol were potent inhibitors of mitosis when applied to cells in vitro. Estradiol induced micronuclei at a greater frequency than did DES. Estradiol‐arrested metaphases often contained misaligned chromosomes despite the presence of a bipolar spindle and an equatorial plate. Equatorial plates were not observed in DES‐arrested cells. Cells recovered quickly from estradiol exposure upon removal of the steroid. The frequency of abnormal metaphases and abnormal anaphases declined as the recovery period increased. Microtubule experiments showed that DES inhibited spindle assembly and disassembled the CMTC, whereas estradiol, at similar concentrations, arrested mitosis in a manner that allowed spindle assembly. A definite effect on the CMTC by estradiol could not be determined. However, changes in cell morphology were observed. In the presence of estradiol, centrosomes organized microtubules that joined with kinet‐ochores of chromosomes at the equatorial plate as well as with those of misaligned chromosomes. Misaligned chromosomes appeared predominantly at polar regions of mitotic cells. Following drug removal, the pole‐oriented chromosomes reoriented at the equatorial plate. The unique arresting properties of estradiol may prove useful in studies of chromosome migration and segregation duri

ISSN:0886-1544    

DOI:10.1002/cm.970070306

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

年代:1987

数据来源: WILEY

摘要:

AbstractEvidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP‐dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP‐dependent kinase during mitosis, dividing PtK1cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP‐dependent protein kinase (PKI) and an affinity‐purified polyclonal antiserum (anti‐C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early‐ to mid‐prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti‐C during early‐ to mid‐prophase also caused a significant delay in the completion of mitosis, with many cells becoming “hung up” in prometaphase. Anti‐C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti‐C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP‐dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic sp

ISSN:0886-1544    

DOI:10.1002/cm.970070307

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

年代:1987

数据来源: WILEY

摘要:

AbstractWe recorded live, undifferentiated amebae ofDictynstelium discoideumby video microscopy and analyzed the behavior of cytoplasmic particles and granules. Cytoplasmic streaming and saltatory movements are the two major types of particle movements that occur in interphase amebae. Saltatory movements predominated in an area around the nucleus‐associated body (NAB) and many were radial toward or away from it, the velocity being very similar in both directions. Some saltations were simple forward movements, and others were complex to‐and‐fro movements with as many as seven turnabouts. For a given leg of movement the velocity was not uniform along the path. Small particles (1 μm;X= 1.4 μm/s) particles, but the smallest particles were visible only in the running image and could not be analyzed. Ultrastructurally, saltating particles are digestive vacuoles and vesicles of various sizes, appearances, and contents, which are numerous particularly in the vicinity of the NAB. Several lines of evidence pointed to a role of microtubules (MTs) in saltatory particle movements. Composites of particle tracks corresponded closely to MT arrays visualized by immunofluorescence. No saltations occurred in mitotic amebae that lack cytoplasmic MTs, but the movements resumed toward the end of division, concurreduced with the rebuilding of the complex of cytoplasmic MTs. Nocodazole reduced and eventually slopped saltatory movements over a period of 3 h, when aberrant MT patterns were the rule. Saltations in slime mold amebae may be an eye‐catching feature of intracellular transport functioning in endo‐ and exocytosis in the shuffling of vesicles containing factors involved in ameboid movement, and in the transduction of external signals t

ISSN:0886-1544    

DOI:10.1002/cm.970070308

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

年代:1987

数据来源: WILEY

摘要:

AbstractTubulin synthesis in the naturally synchronous plasmodium ofPhysarum polycephalumis a markedly periodic event restricted to the late G2period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle.We have shown that all four tubulin isotypes identified in the plasmodium (α1, α2, β1 and β2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: β2>α1>α2>β1. We have also shown that tubulins synthesized in the G2period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin‐containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucl

ISSN:0886-1544    

DOI:10.1002/cm.970070309

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

年代:1987

数据来源: WILEY

摘要:

AbstractAfter fusion of BHK cells with polyethylene glycol, the resulting syncitia contained in 77% of the cases multiple microtubule organizing centers (MTOCs), which were aggregated into a common centrosphere. Based on the observation of phagokinetic tracks, we found that the syncitia were able to locomote if (1) the MTOCs aggregated into a common centrosphere cluster, and (2) the clustered centrospheres were excluded from the cluster of nuclei of the syncitium. The results suggest that each individual pair of one nucleus and one centrosphere contributes, in a process of vectorial addition, its individual polarity to the polarity of the syneitium. Thus the widely accepted idea that the centrosphere is involved in the determinatinn of cell polarity can be generalized beyond the case of single cells.

ISSN:0886-1544    

DOI:10.1002/cm.970070310

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

年代:1987

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970070311

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

年代:1987

数据来源: WILEY