Separase-dependent cleavage of pericentrin B is necessary and sufficient for centriole disengagement during mitosis

Centriole disengagement is considered an essential step for licensing a new round of centriole duplication in the next cell cycle. Separase is critical for centriole disengagement. Here, we showed that pericentrin B (PCNTB) is specifically cleaved by separase at the exit of mitosis. The cleavage-resistant PCNTB mutant blocks the centriole disengagement and duplication. We also observed that an artificial cleavage of PCNTB during M phase induced premature disengagement of centrioles. Based on these results, we concluded that the separase-dependent cleavage of PCNTB is necessary and sufficient for centriole disengagement during mitosis.     

Kinetochores and microtubules in multipolar mitosis and chromosome orientation.

The ultrastructure of the kinetochore and the orientation of kinetochore microtubules were studied in multipolar divisions of cultured rat-kangaroo cells (Pt-K1). The metaphase kinetochore exhibited a lamellar structure and most of the chromosomes expressed a bipolar microtubule orientation. A multipolar kinetochore orientation was observed in some chromosomes. Equal or unequal portions of a chromatid's kinetochore and a corresponding number of kinetochore microtubules may be oriented to two different poles. Curved or bent continuous microtubules were observed in the vicinity of chromosomes showing multipolar orientation. The findings are in accordance with Östergren's theory of ‘auto-orientation’ [8]. It is speculated that orientation of a chromatid kinetochore to more than one pole might be a possible regular event during the process of chromosome orientation prior to full metaphase in bipolar mitosis.     

Mode of centriole duplication and distribution

Centriole stability and distribution during the mammalian cell cycle was studied by microinjecting biotinylated tubulin into early G1 cells and analyzing the pattern of incorporation into centrioles. Cells were extracted and cold treated to depolymerize labile microtubules, allowing the fluorescent microscopic visualization of the stable centrioles. The ability to detect single centrioles was confirmed by use of correlative electron microscopy. Indirect hapten and immunofluorescent labeling of biotinylated and total tubulin permitted us to distinguish newly formed from preexisting centrioles. Daughter centrioles incorporated biotinylated tubulin, and at mitosis each cell received a centrosome containing one new and one old centriole. We conclude that in each cell cycle tubulin incorporation into centrioles is conservative, and centriole distribution is semiconservative.     

Nuclear membrane behavior during mitosis in normal and heteroploid Myxomycetes

Summary Photomicrographic evidence is presented of the difference in behavior of nuclear membranes during mitosis in amoebae, zygotes and plasmodia of Myxomycetes. One of the species was cultured on bacteria and possessed a normal cycle of plasmogamy and karyogamy between the amoebal and plasmodial phases. The second species was axenically grown in liquid media and had become highly heteroploid and lacked the ability to develop into plasmodia, existing only in the amoeboid form. The significance of the amoeboid form of mitosis in the heteroploid axenically cultured strain is discussed in relation to the difference in nuclear membrane behavior and the possible significance of such behavior.     

Nucleus behavior during the closed mitosis of Tritrichomonas foetus

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.     

Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole.     

The fate of chrysotile-induced multipolar mitosis and aneuploid population in cultured lung cancer cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.     

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G₁/S boundary) or the Cdk1 inhibitor, RO3306 (G₂/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.     

Metabolic inhibitors and mitosis: II. Effects of dinitrophenol/deoxyglucose and nocodazole on the microtubule cytoskeleton

Summary To examine the effects exerted on the microtubule (MT) cytoskeleton by dinitrophenol/deoxyglucose (DNP/DOG) and nocodazole, live PtK₁ cells were treated with the drugs and then fixed and examined by immunofluorescence staining and electronmicroscopy. DNP/DOG had little effect on interphase MTs. In mitotic cells, kinetochore and some astral fibers were clearly shortened in metaphase figures by DNP/DOG. Nocodazole rapidly broke down spindle MTs (except those in the midbody), while interphase cells showed considerable variation in the susceptibility of their MTs. Nocodazole had little effect on MTs in energy-depleted (DNP/DOG-treated) cells. When cytoplasmic MTs had all been broken down by prolonged nocodazole treatment and the cells then released from the nocodazole block into DNP/DOG, some MT reassembly occurred in the ATP-depleted state. MTs in permeabilized, extracted cells were also examined with antitubulin staining; the well-preserved interphase and mitotic arrays of MTs showed no susceptibility to nocodazole. In contrast, MTs suffered considerable breakdown by ATP, GTP and ATPS; AMPPNP had little effect. This susceptibility of extracted MT cytoskeleton to nucleotide phosphates was highly variable; some interphase cells lost all MTs, most were severely affected, but some retained extensive MT networks; mitotic spindles were diminished but structurally coherent and more stable than most interphase MT arrays.We suggest that: 1. in the living cell, ATP or nucleotide triphosphates (NTPs) are necessary for normal and nocodazole-induced MT disassembly; 2. the NTP requirement may be for phosphorylation; 3. shortening of kinetochore fibers may be modulated by compression and require ATP; 4. many of these results cannot be accomodated by the dynamic equilibrium theory of MT assembly/disassembly; 5. the use and role of ATP on isolated spindles may have to be reevaluated due to the effects ATP has on the spindle cytoskeleton of permeabilized cells.     

Chromosome malsegregation and embryonic lethality induced by treatment of normally ovulated mouse oocytes with nocodazole

The mouse egg is ovulated with its nucleus arrested at the metaphase-II stage of meiosis. Sperm entry triggers the completion of the second meiotic division. It has been speculated that damage to the meiotic spindle of normally ovulated eggs at around the time of sperm entry could result in chromosome malsegregation and the death of conceptuses with numerical chromosome anomalies. This hypothesis was tested using nocodazole, a microtubule inhibitor. Nocodazole was administered either to maturing preovulatory oocytes or to normally ovulated eggs at one of the following stages: (1) the time of sperm entry, (2) early pronuclear stage, (3) pronuclear DNA synthesis, (4) prior to first cleavage division, (5) early 2-cell stage, or (6) prior to the second cleavage division. Little or no effect was observed for treatment times other than the time of sperm entry, when the egg is being activated to complete the second meiotic division. Remarkably high frequencies of embryonic lethality, expressed at around the time of implantation, were induced at this stage. Cytogenetic analysis of first cleavage metaphases of zygotes treated at the time of sperm entry revealed a high incidence of varied numerical chromosome anomalies, with changes in ploidy being predominant.     

Metabolic inhibitors and mitosis: I. Effects of dinitrophenol/deoxyglucose and nocodazole on the live spindle

Summary Dinitrophenol and deoxyglucose (DNP/DOG) were used to investigate the effects of ATP depletion on mitotic PtK₁ cells. Direct determination of cellular ATP levels showed that the drop of ATP induced by DNP/DOG was rapid; recovery to normal ATP levels was equally rapid once DNP/DOG was removed. On addition of DNP/DOG to live cells, cytoplasmic activity ceased; interphase and prophase cells showed little other response to DNP/DOG. During prometaphase, DNP/DOG induced a pronounced movement of oscillating, monopolar chromosomes towards the spindle poles. As chromosomes became bipolarly attached, DNP/DOG caused the spindle poles themselves to move together. By metaphase, DNP/DOG-treatment led to significant shortening of the spindle which remained intact. DNP/DOG rapidly stopped anaphase chromosome movement and cytokinesis.Nocodazole (NOC) caused the rapid breakdown of the mitotic spindle; prometaphase chromosomes clustered at the poles and in metaphase cells, the poles were drawn towards the chromosomes as the spindle became disorganized. When cells were pretreated with DNP/DOG and then NOC/DNP/DOG, nocodazole did not break down the spindle. When nocodazole was applied first to break down spindle MTs then DNP/DOG was added to the nocodazole, a second contraction was often induced by the DNP/DOG in the absence of spindle microtubules (MTs). Chromosomes expanded appreciably outwards from the poles when the DNP/DOG was removed, even when the cells remained in nocodazole.     

Asymmetric distribution of EGFR receptor during mitosis generates diverse CNS progenitor cells

It has been debated whether asymmetric distribution of cell surface receptors during mitosis could generate asymmetric cell divisions by yielding daughters with different environmental responsiveness and, thus, different fates. We have found that in mouse embryonic forebrain ventricular and subventricular zones, the EGFR can distribute asymmetrically during mitosis in vivo and in vitro. This occurs during divisions yielding two Nestin+ progenitor cells, via an actin-dependent mechanism. The resulting sibling progenitor cells respond differently to EGFR ligand in terms of migration and proliferation. Moreover, they express different phenotypic markers: the EGFRhigh daughter usually has radial glial/astrocytic markers, while its EGFRlow sister lacks them, indicating fate divergence. Lineage trees of cultured cortical glioblasts reveal repeated EGFR asymmetric distribution, and asymmetric divisions underlie formation of oligodendrocytes and astrocytes in clones. These data suggest that asymmetric EGFR distribution contributes to forebrain development by creating progenitors with different proliferative, migratory, and differentiation responses to ligand.