MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1–1 mutation. Strains containing mob2Δ also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.

The Golgi and Endoplasmic Reticulum Remain Independent during Mitosis in HeLa Cells

Partitioning of the mammalian Golgi apparatus during cell division involves disassembly at M-phase. Despite the importance of the disassembly/reassembly pathway in Golgi biogenesis, it remains unclear whether mitotic Golgi breakdown in vivo proceeds by direct vesiculation or involves fusion with the endoplasmic reticulum (ER). To test whether mitotic Golgi is fused with the ER, we compared the distribution of ER and Golgi proteins in interphase and mitotic HeLa cells by immunofluorescence microscopy, velocity gradient fractionation, and density gradient fractionation. While mitotic ER appeared to be a fine reticulum excluded from the region containing the spindle-pole body, mitotic Golgi appeared to be dispersed small vesicles that penetrated the area containing spindle microtubules. After cell disruption, M-phase Golgi was recovered in two size classes. The major breakdown product, accounting for at least 75% of the Golgi, was a population of 60-nm vesicles that were completely separated from the ER using velocity gradient separation. The minor breakdown product was a larger, more heterogenously sized, membrane population. Double-label fluorescence analysis of these membranes indicated that this portion of mitotic Golgi also lacked detectable ER marker proteins. Therefore we conclude that the ER and Golgi remain distinct at M-phase in HeLa cells. To test whether the 60-nm vesicles might form from the ER at M-phase as the result of a two-step vesiculation pathway involving ER–Golgi fusion followed by Golgi vesicle budding, mitotic cells were generated with fused ER and Golgi by brefeldin A treatment. Upon brefeldin A removal, Golgi vesicles did not emerge from the ER. In contrast, the Golgi readily reformed from similarly treated interphase cells. We conclude that Golgi-derived vesicles remain distinct from the ER in mitotic HeLa cells, and that mitotic cells lack the capacity of interphase cells for Golgi reemergence from the ER. These experiments suggest that mitotic Golgi breakdown proceeds by direct vesiculation independent of the ER.

Swi5 Controls a Novel Wave of Cyclin Synthesis in Late Mitosis

We have shown previously that the Swi5 transcription factor regulates the expression of the SIC1 Cdk inhibitor in late mitosis. This suggests that Swi5 might control other genes with roles in ending mitosis. We identified a gene with a Swi5-binding site in the promoter that encoded a protein with high homology to Pcl2, a cyclin-like protein that associates with the Cdk Pho85. This gene, PCL9, is indeed regulated by Swi5 in late M phase, the only cyclin known to be expressed at this point in the cell cycle. The Pcl9 protein is associated with a Pho85-dependent protein kinase activity, and the protein is unstable with peak levels occurring in late M phase. PCL2 is already known to be expressed in late G1 and we find that, in addition, it is also regulated by Swi5 in telophase. The expression of PCL2 and PCL9 at this stage of the cell cycle implies a role for the Pho85 Cdk at the end of mitosis. Consistent with this a synthetic interaction was observed between pho85Δ and strains deleted for SIC1, SWI5, and SPO12. These and other studies support the notion that the M/G1 switch is a major cell cycle transition.

Yeast Dam1p Is Required to Maintain Spindle Integrity during Mitosis and Interacts with the Mps1p Kinase

We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5′-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5′-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3′-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli.

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.

The Protein Kinase Cdr2, Related to Nim1/Cdr1 Mitotic Inducer, Regulates the Onset of Mitosis in Fission Yeast

Cdc2–Cyclin B, the protein kinase that catalyzes the onset of mitosis, is subject to multiple forms of regulation. In the fission yeast Schizosaccharomyces pombe and most other species, a key mode of Cdc2–Cyclin B regulation is the inhibitory phosphorylation of Cdc2 on tyrosine-15. This phosphorylation is catalyzed by the protein kinases Wee1 and Mik1 and removed by the phosphatase Cdc25. These proteins are also regulated, a notable example being the inhibition of Wee1 by the protein kinase Nim1/Cdr1. The temperature-sensitive mutation cdc25–22 is synthetic lethal with nim1/cdr1 mutations, suggesting that a synthetic lethal genetic screen could be used to identify novel mitotic regulators. Here we describe that such a screen has identified cdr2+, a gene that has an important role in the mitotic control. Cdr2 is a 775 amino acid protein kinase that is closely related to Nim1 and mitotic control proteins in budding yeast. Deletion of cdr2 causes a G2-M delay that is more severe than that caused by nim1/cdr1 mutations. Genetic studies are consistent with a model in which Cdr2 negatively regulates Wee1. This model is supported by experiments showing that Cdr2 associates with the N-terminal regulatory domain of Wee1 in cell lysates and phosphorylates Wee1 in vitro. Thus, Cdr2 is a novel mitotic control protein that appears to regulate Wee1.

Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsBrad3 and uvsDrad26 have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIMEcyclinB, but ectopic expression of active nondegradable NIMEcyclinB does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMXcdc2 and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

Cell cycle-dependent regulation of RNA polymerase I transcription: The nucleolar transcription factor UBF is inactive in mitosis and early G1

Transcription of ribosomal RNA genes by RNA polymerase (pol) I oscillates during the cell cycle, being maximal in S and G2 phase, repressed during mitosis, and gradually recovering during G1 progression. We have shown that transcription initiation factor (TIF)-IB/SL1 is inactivated during mitosis by cdc2/cyclin B-directed phosphorylation of TAFI110. In this study, we have monitored reactivation of transcription after exit from mitosis. We demonstrate that the pol I factor UBF is also inactivated by phosphorylation but recovers with different kinetics than TIF-IB/SL1. Whereas TIF-IB/SL1 activity is rapidly regained on entry into G1, UBF is reactivated later in G1, concomitant with the onset of pol I transcription. Repression of pol I transcription in mitosis and early G1 can be reproduced with either extracts from cells synchronized in M or G1 phase or with purified TIF-IB/SL1 and UBF isolated in the presence of phosphatase inhibitors. The results suggest that two basal transcription factors, e.g., TIF-IB/SL1 and UBF, are inactivated at mitosis and reactivated by dephosphorylation at the exit from mitosis and during G1 progression, respectively.

Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

Characterization and cell cycle regulation of the related human telomeric proteins Pin2 and TRF1 suggest a role in mitosis

Telomeres are essential for preserving chromosome integrity during the cell cycle and have been specifically implicated in mitotic progression, but little is known about the signaling molecule(s) involved. The human telomeric repeat binding factor protein (TRF1) is shown to be important in regulating telomere length. However, nothing is known about its function and regulation during the cell cycle. The sequence of PIN2, one of three human genes (PIN1-3) we previously cloned whose products interact with the Aspergillus NIMA cell cycle regulatory protein kinase, reveals that it encodes a protein that is identical in sequence to TRF1 apart from an internal deletion of 20 amino acids; Pin2 and TRF1 may be derived from the same gene, PIN2/TRF1. However, in the cell Pin2 was found to be the major expressed product and to form homo- and heterodimers with TRF1; both dimers were localized at telomeres. Pin2 directly bound the human telomeric repeat DNA in vitro, and was localized to all telomeres uniformly in telomerase-positive cells. In contrast, in several cell lines that contain barely detectable telomerase activity, Pin2 was highly concentrated at only a few telomeres. Interestingly, the protein level of Pin2 was highly regulated during the cell cycle, being strikingly increased in G2+M and decreased in G1 cells. Moreover, overexpression of Pin2 resulted in an accumulation of HeLa cells in G2+M. These results indicate that Pin2 is the major human telomeric protein and is highly regulated during the cell cycle, with a possible role in mitosis. The results also suggest that Pin2/TRF1 may connect mitotic control to the telomere regulatory machinery whose deregulation has been implicated in cancer and aging.

Serine Phosphorylation of Focal Adhesion Kinase in Interphase and Mitosis: A Possible Role in Modulating Binding to p130Cas

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130Cas.

Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression

Although neurogenesis in the embryo proceeds in a region- or lineage-specific fashion coincident with neuropeptide expression, a regulatory role for G protein-coupled receptors (GPCR) remains undefined. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates sympathetic neuroblast proliferation, whereas the peptide inhibits embryonic cortical precursor mitosis. Here, by using ectopic expression strategies, we show that the opposing mitogenic effects of PACAP are determined by expression of PACAP receptor splice isoforms and differential coupling to the phospholipase C (PLC) pathway, as opposed to differences in cellular context. In embryonic day 14 (E14) cortical precursors transfected with the hop receptor variant, but not cells transfected with the short variant, PACAP activates the PLC pathway, increasing intracellular calcium and eliciting translocation of protein kinase C. Ectopic expression of the hop variant in cortical neuroblasts transforms the antimitotic effect of PACAP into a promitogenic signal. Furthermore, PACAP promitogenic effects required PLC pathway function indicated by antagonist U-73122 studies in hop-transfected cortical cells and native sympathetic neuroblasts. These observations highlight the critical role of lineage-specific expression of GPCR variants in determining mitogenic signaling in neural precursors.

Meristem-Specific Suppression of Mitosis and a Global Switch in Gene Expression in the Root Cap of Pea by Endogenous Signals1

Two functionally distinct sets of meristematic cells exist within root tips of pea (Pisum sativum): the root apical meristem, which gives rise to the body of the root; and the root cap meristem, which gives rise to cells that differentiate progressively through the cap and separate ultimately from its periphery as border cells. When a specific number of border cells has accumulated on the root cap periphery, mitosis within the root cap meristem, but not the apical meristem, is suppressed. When border cells are removed by immersion of the root tip in water, a transient induction of mitosis in the root cap meristem can be detected starting within 5 min. A corresponding switch in gene expression throughout the root cap occurs in parallel with the increase in mitosis, and new border cells begin to separate from the root cap periphery within 1 h. The induction of renewed border cell production is inhibited by incubating root tips in extracellular material released from border cells. The results are consistent with the hypothesis that operation of the root cap meristem and consequent turnover of the root cap is self-regulated by a signal from border cells.