The effect of the UV microirradiation of the centrosome on cell behavior. III. The ultrastructure of the centrosome after irradiation]

One of the spindle poles of mitotic PK cells was irradiated with UV microbeam in metaphase or in anaphase. Electron microscopy showed that immediately after irradiation the microtubules around the centrosome were maintained, and that the ultrastructure of both irradiated and nonirradiated poles was similar. After microirradiation of the centrosome in metaphase, the mitotic halo around this centrosome was retained, but in due time the number of microtubules was getting less compared to that around the nonirradiated centrosome. When daughter cells with irradiated centrosomes are passing into the interphase, their centrioles are not separated from each other, no primary cilia are formed, and no replication of centrioles occurs. In the interphase cells with irradiated centrosomes, satellites are formed on the active centriole, but centrosome-attached microtubules are practically absent.     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK1. When the laser power delivered to sample surface was 90-190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360-420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50-70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK₁. When the laser power delivered to sample surface was 90–190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360–420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50–70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.     

Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy

The formation of mitotic centrosomes is a complex process in which a number of cellular proteins translocate to mitotic poles and play a critical role in the organization of the mitotic apparatus. The 238-kDa nuclear mitotic apparatus protein NuMA is one of the important proteins that plays a significant role in this process. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps of phosphorylations. The role of NuMA in the interphase nucleus is not well known but it is clear that NuMA responds to external signals (such as hormones) that induce cell division, or heat shock that induces apoptosis. In order to determine the function of NuMA it is important to study its localization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen responsive LNCaP prostate cancer cells using immunoelectron microscopy, and on correlation to MPM-2 monoclonal phosphoprotein antibody. These results show that NuMA is present in speckled and punctate form associated with distinct material corresponding to a speckled or punctate immunofluorescence appearance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules while MPM-2 is uniformly distributed in the cytoplasm. During metaphase or anaphase anti-NuMA labeling is associated with spindle fibers. During telophase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MCF-7 and LNCaP cells in the log phase of cell culture growth.     

A class of nonribosomal nucleolar components is located in chromosome periphery and in nucleolus-derived foci during anaphase and telophase

The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1–2 μm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp 140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis. Edited by: S. A. Gerbi     

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.     

Structure of the nucleoli of developing microsporangiate strobili and root tips of scotch pine

Summary The characteristics of the nucleoli of the microsporangiate strobili and the root tips of Scotch pine (Pinus sylvestris L.) vary both during the course of the cellular cycle and, with regard to the pattern and stage of organ and tissue differentiation. Nucleologenesis takes place in interphase and the nucleoli last until prophase. Several types of nucleoli occur during the nucleolar cycle, the pattern and age of tissues determining which type or types dominate. In the strobilus primordia collected at the end of July and in August, the mitotic frequency is high. Nucleoli remain small throughout the nucleolar cycle, and at the electron microscopic level, they display intermingled fibrillar and fibrillogranular components. Strobilus primordia collected in September contain larger nucleoli in the sporogenous nuclei than in the nuclei of the tapetum or of the wall cells. Amongst the nucleoli with completely intermingled fibrous and granular material, nucleoli with nucleolonema or with vacuoles occur frequently. Small balls of fibrous material are seen on the nucleolar surface and in the nucleoplasm. In October, the mitotic frequency of strobilal cells is low. Nucleoli with completely intermingled fibrillar and granular components have vanished whereas a new, compact type of nucleolus with a dense fibrillogranular main portion and with nucleolonema, has developed. The nucleoli of the sporogenous cells have enlarged continuously whereas those of the wall cells are small. The nucleoli of the root tip cell resemble, to a certain extent, those of the strobilus primordia collected in September. In squashed preparations, the nucleoli of the strobilal cells bind the common nucleolar stains poorly whereas the nucleoli of the root cells can be stained with all the methods used. In certain cases, DNase treatment improves the stainability of the strobilal nucleoli. AgNO₃-staining is successful after acetic acid: alcohol fixation but not after formalin: hydrochinone fixation.     

The RNA binding activity of a ribosome biogenesis factor,nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.     

The E2-C vihar is required for the correct spatiotemporal proteolysis of cyclin B and itself undergoes cyclical degradation

BACKGROUND: Proteolytic degradation of mitotic regulatory proteins first requires these targets to be ubiquitinated. This is regulated at the level of conjugation of ubiquitin to substrates by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin-protein ligase. Substrate specificity and temporal activity of the APC/C has been thought to lie primarily with its two activators, Cdc20/Fizzy and Cdh1/Fizzy-related. RESULTS: Here, we show that reduction in the E2 ubiquitin-conjugating enzyme (UBC) of the E2-C family that is encoded by the Drosophila gene vihar (vih), by either mutation or RNAi, leads to an accumulation of cells in a metaphase-like state. Cyclin B accumulates to high levels in all mitotic vih cells, particularly at the spindle poles. Vihar E2-C is present in the cytoplasm of mitotic cells but also associates with centrosomes, and its own degradation is initiated at the metaphase-anaphase transition. Expression of destruction D box mutants of vihar in the syncytial embryo results in mitotic arrest at late anaphase. In contrast to hypomorphic mutants, Cyclin B is degraded at the spindle poles and accumulates in the equatorial region of the spindle. CONCLUSIONS: In Drosophila, the Vihar E2 UBC contributes to the spatiotemporal control of Cyclin B degradation that first occurs at the spindle poles. APC/C-mediated proteolysis of Vihar E2-C autoinactivates the APC/C at the centrosome before a second wave of proteolysis to degrade Cyclin B on the rest of the spindle and elsewhere in the cell.     

Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells

The cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen has been evaluated immunocytochemically in L-132 human fetal lung cells. The cells were synchronized and cell cycle phases were determined: G1 = 6.7 h, S = 5.4 h, G2 = 8.5 h and mitosis = 1.3 h. The Ki-67 patterns were strictly correlated with the cell cycle phases. In late G1-phase, Ki-67 antigen was present only in the perinucleolar region. In the S-phase, Ki-67 staining was found homogeneously in the karyoplasm and in the perinucleolar region. G2-phase cells contained a finely granular Ki-67 staining in the karyoplasm with Ki-67-positive specks and perinucleolar staining. In early mitotic cells (pro- and metaphase) an intense perichromosomal Ki-67 staining was observed in addition to a homogeneously stained karyoplasm in prophase, and cytoplasm in metaphase. During ana- and telophase the Ki-67 antigen disappeared rapidly. In resting cells there was no Ki-67 staining.     

KL1 is a novel microtubule severing enzyme that regulates mitotic spindle architecture

Katanin is a microtubule severing enzyme with demonstrated roles in a variety of cellular activities including mitosis. Here we identify the closely related, but relatively uncharacterized human protein, Katanin-like 1 (KL1), as a novel mitotic regulator. Over expression of KL1 in tissue culture cells results in the complete disassembly of cellular microtubules strongly suggesting that it is an active microtubule severing protein. During mitosis, the localization of KL1 is restricted to spindle poles and is notably absent from centrosomes. This is in clear contrast to conventional Katanin whose localization extends from centrosomes onto poles. Consistent with its localization, siRNA depletion of KL1 from U2OS cells results in a specific and significant reduction in the density of microtubules at spindle poles and significantly increases spindle length. Depletion of KL1 also alters the distribution of gamma-tubulin at centrosomes/spindle poles. Despite its impact on spindle morphology, we could find no evidence that KL1 influences anaphase chromosome motility. Based on our findings, we propose that KL1-mediated microtubule severing is utilized to generate microtubule seeds within the poles and that loss of this activity alters the normal balance of motor-generated forces that determine spindle length.     

THE NUCLEOLI IN MITOTIC DIVISIONS OF MAMMALIAN CELLS IN VITRO

In a number of mammalian cell strains nucleoli persisted through mitosis. This phenomenon was especially pronounced in several cell lines derived from Chinese hamster tissues. All the methods employed, including radioautography with tritiated uridine, cytochemical stains (methyl green-pyronin and azure B), fluorescent microscopy (coriphosphine O), ribonuclease digestion, and electron microscopy, demonstrated that the bodies identified as persistent nucleoli in the mitotic stages had the same characteristics as did the nucleoli in the interphase. Persistent nucleoli may attach to the chromosomes or may be free in the cytoplasm. In cells where no persistent nucleoli as such were noted, nucleolar material was observed to attach to the chromosomes in shapeless masses which moved with the chromosomes during anaphase. At least a portion of the nucleolar material was included in the daughter nuclei, presumably for immediate use for protein synthesis after cell division.     

Construction of centrosomes and spindle poles by molecular motor-driven assembly of protein particles

Centrosomes and other microtubule organizing centers are the largest non-membranous organelles in most cells. This morphologically diverse class of organelles shares a common ability to nucleate and organize microtubules in interphase and participates in the formation of mitotic spindles during cell division. This review summarizes recent evidence suggesting that assembly of centrosomes and mitotic spindle poles require transport of large protein particles along microtubules by the molecular motor cytoplasmic dynein.     

Localization of the centrin-related 165,000-Mr protein of PtK2 cells during the cell cycle

In this study, we follow changes in localization of the centrin-related 165,000-Mr protein of PtK2 cells during the cell cycle. This protein is a component of a pericentriolar lattice that consists of pericentriolar satellites, pericentriolar matrix, and basal feet (Baron A.T., and J.L. Salisbury, J. Cell Biol. 107:2669-2678, 1988). By immunofluorescence microscopy, the 165,000-Mr protein is seen as a constellation of pericentrosomal spots. We observe that cells in late G1 and S are characterized by a dense centrosomal focus of spots with additional spots dispersed throughout the cytoplasm. In G2, one bright centrosomal focus of clustered spots is observed. As the cells proceed through prophase this single focus divides, forming two foci that move toward opposite sides of the nucleus. During prometaphase, each polar focus of spots disperses. At metaphase, the spots are distributed throughout each half-cytoplast from the poles to the chromosomes. During anaphase chromosome movement, some spots are seen beside and behind the trailing chromosome arms while others are clustered at the poles. At telophase, pericentrosomal spots radiate from the poles to surround each mass of chromatin. In early G1, pericentrosomal spots surround each newly formed nucleus. We conclude that the 165,000-Mr protein is a dynamic component of both the centrosome (pericentriolar matrix) and the mitotic apparatus (spindle matrix).     

Downregulation of protein 4.1R, a mature centriole protein, disrupts centrosomes, alters cell cycle progression, and perturbs mitotic spindles and anaphase

Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G(1) accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.     

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.