The genetic analysis of mitosis in Aspergillus nidulans

We describe here recent work on the molecular genetics of mitosis in the filamentous fungus Aspergillus nidulans. Aspergillus is one of three simple eukaryotes with powerful genetic systems that have been used to analyze mitosis. The modern molecular biological techniques available with this organism have made it possible to use mutations to identify genes and proteins that play an important role in mitosis. Three Aspergillus genes that affect mitosis are described. One gene, nimA, is specifically expressed late in the cell cycle and codes for a putative protein kinase that induces mitosis, even in cells blocked in S-phase. The second gene, bimG, codes for a putative phosphatase that interacts functionally with the nimA kinase. The third gene, bimE, codes for a protein that suppresses mitosis during interphase, apparently by keeping nimA turned off. None of these genes appear to be similar to any of the genes affecting mitosis that have been characterized in other eukaryotes, but rather appear to be elements of a system that prevents mitosis from occurring during interphase.     

Golgi membranes are absorbed into and reemerge from the ER during mitosis

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.     

Schizosaccharomyces pombe Bir1p, a nuclear protein that localizes to kinetochores and the spindle midzone, is essential for chromosome condensation and spindle elongation during mitosis

The inhibitor of apoptosis (IAP) family of proteins contains a subset of members characterized by the presence of highly conserved baculoviral IAP repeat (BIR) domains. Recent work has shown that some of these BIR-domain proteins play a prominent role in the regulation of cell division, in particular at the stage of chromosome segregation and cytokinesis. We and others have shown that the Schizosaccharomyces pombe BIR-domain protein, Bir1p/Pbh1p/Cut17p, is important for the regulation of mitosis. Here we further characterize S. pombe Bir1p using methods of cell biology and genetics. We show that Bir1p is dispersed throughout the nucleus during the cell cycle. In addition, a significant part of Bir1p is also detected at the kinetochores and the spindle midzone during mitosis and meiosis. Time-lapse microscopy studies suggest that Bir1p relocates from the kinetochores to the spindle at the end of anaphase A. Bir1p colocalizes with the S. pombe Aurora kinase homolog Aim1p, a protein essential for mitosis, at the kinetochores as well as the spindle midzone during mitosis, and functional Bir1p is essential for localization of Aim1p to the kinetochores and the spindle midzone. Analyses of bir1 conditional mutants revealed that Bir1p is essential for chromosome condensation during mitosis. In addition, anaphase cells show the presence of lagging chromosomes and a defect in spindle elongation. We conclude that Bir1p is important for multiple processes that occur during mitosis in S. pombe.     

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.     

Effect of roscovitine,a selective cyclin B-dependent kinase 1 inhibitor,on assembly of the nucleolus in mitosis

It is well known that at the beginning of mitosis the nucleolus disassembles but then reassembles at the end of mitosis. However, the mechanisms of these processes are still unclear. In the present work, we show for the first time that selective inhibition of cyclin B-dependent kinase 1 (CDK1) by roscovitine induces premature assembly of the nucleolus in mammalian cells in metaphase. Treatment of metaphase cells with roscovitine induces formation of structures in their cytoplasm that contain major proteins of the mature nucleolus participating in rRNA processing, such as B23/nucleophosmin, C23/nucleolin, fibrillarin, Nop52, as well as partially processed (immature) 46-45S pre-rRNA. This effect is reproducible in cells of various types; this indicates that general mechanisms regulate early stages of the nucleolus reassembly with CDK1 participation in mammalian cells. Based on our and literature data, we suggest that inactivation of the CDK1-cyclin B complex at the end of mitosis results in dephosphorylation of B23/nucleophosmin and C23/nucleolin; this facilitates their interaction with pre-rRNA and leads to formation of insoluble supramolecular complexes--nucleolus-derived foci.     

A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis

The upstream of N-Ras (Unr) protein is involved in translational regulation of specific genes. For example, the Unr protein contributes to translation mediated by several viral and cellular internal ribosome entry sites (IRESs), including the PITSLRE IRES, which is activated at mitosis. Previously, we have shown that translation of the Unr mRNA itself can be initiated through an IRES. Here, we show that UNR mRNA translation and UNR IRES activity are significantly increased during mitosis. Functional analysis identified hnRNP C1/C2 proteins as UNR IRES stimulatory factors, whereas both polypyrimidine tract-binding protein (PTB) and Unr were found to function as inhibitors of UNR IRES-mediated translation. The increased UNR IRES activity during mitosis results from enhanced binding of the stimulatory hnRNP C1/C2 proteins and concomitant dissociation of PTB and Unr from the UNR IRES RNA. Our data suggest the existence of an IRES-dependent cascade in mitosis comprising hnRNP C1/C2 proteins that stimulate Unr expression, and Unr, in turn, contributes to PITSLRE IRES activity. The observation that RNA interference-mediated knockdown of hnRNP C1/C2 and Unr, respectively, abrogates and retards mitosis points out that regulation of IRES-mediated translation by hnRNP C1/C2 and Unr might be important in mitosis.     

Identification and characterization of a new set of nucleolar ribonucleoproteins which line the chromosomes during mitosis.

We investigated the perichromosomal architecture established during mitosis. Entry into mitosis brings about a dramatic reorganization of both nuclear and cytoplasmic structures in preparation for cell division. While the nuclear envelope breaks down, nuclear proteins are redistributed during chromosome condensation. Some of these proteins are found around the chromosomes, but little is known concerning their nature and function. Ten autoimmune sera were used to study the microenvironment of chromosomes and, in particular, the chromosome periphery. They were selected for their anti-nucleolar specificity and were found to recognize three nucleolar proteins that coat the chromosomes during mitosis. The distribution of these antigens was followed through the cell cycle by confocal laser scanning microscopy. The antigens dispersed very early during prophase and simultaneously with the chromosome condensation suggesting a correlation between these two processes. The antigens have apparent molecular weights of 53, 66, and 103 kDa on SDS-PAGE migration. Elution of the antibodies and immunopurification showed that they are RNA-associated proteins. The coimmunoprecipitating RNA moiety involved in these RNPs appeared to be U3, but the antigens are not related to the fibrillarin family. Therefore, small nucleolar RNPs follow the same distribution during mitosis as that described for small nuclear RNPs. Possible functions for these antigens are discussed.     

Induction of nuclear envelope formation around individual chromosomes under impact of hypotonic shock

In the present work we have studied the distribution of some proteins participating in the nuclear envelope assembly (lamins A/C, B and LAP2 alpha) in mitotic cells and after hypotonic treatment with 15% Hank's solution. In untreated cells, these proteins are localized in the nuclei of interphase cells migrate to the cytoplasm during mitosis. Hypotonic treatment of interphase, prophase and telophase cells does not lead to considerable relocalization of lamins A/C and B. However, unlike normal mitosis, in prometaphase and metaphase cells their chromosomes acquire affinity to lamins and LAP2 alpha. Comparative analysis of lamins and LAP2 alpha distribution have revealed that chromosomes have special sites for binding with different proteins.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Septin1, a new interaction partner for human serine/threonine kinase aurora-B

Several families of kinases work together to ensure the rate and precision of mitosis. Aurora-B is an important serine/threonine kinase required for chromosome segregation and cytokinesis. Identification of Aurora-B substrates will help to enhance our understanding of the molecular mechanism of mitosis. Through a yeast two-hybrid screen, we found a novel partner of Aurora-B, Septin1, belonging to a conserved family of GTPase proteins that localize to the cleavage furrow and are involved in cytokinesis. We confirmed this interaction using Co-immunoprecipitation experiments in mammalian cells and GST-pull-down analysis in vitro. Moreover, Aurora-B can phosphorylate Septin1 in vitro. We identified that Ser248, Ser307, and Ser315 are the main phosphorylation sites in Septin1. These two proteins partially co-localize to the midbody during cytokinesis. So, it is possible that Septin1's role in the regulation of cytokinesis is related to its phosphorylation by Aurora-B. Unlike previous reports that Septins function in cytokinesis and localize to the cleavage furrow, we found that Septin1 localizes to the spindle pole throughout mitosis, indicating that Septin1 may function in chromosome segregation as well.     

Tyrosine phosphorylations specific to mitosis in human and hamster cells

Changes in protein tyrosine phosphorylation are known to be important for regulating cell cycle progression. With the aim of identifying new proteins involved in the regulation of mitosis, we used an antibody against phosphotyrosine to analyze proteins from synchronized human and hamster cells. At least seven proteins were found that displayed mitosis-specific tyrosine phosphorylation in HeLa cells (pp165, 205, 240, 250, 270, 290, and ～ 400) and one such protein in hamster BHK cells (pp155). In synchronized HeLa and BHK cells, all proteins except HeLa pp165, pp205, and pp250 were readily detectable only in mitosis. Tyrosine phosphorylation of pp165, pp205, and pp250 was apparent during arrest in S phase, suggesting that cell cycle perturbations can affect the phosphorylation state of some of these proteins. In a related finding in BHK cells, pp155 underwent tyrosine phosphorylation when cells were forced into premature mitosis by caffeine treatment. Only one protein (pp135 in HeLa cells) was found to be dephosphorylated on tyrosine during mitosis. The above findings may prove helpful for isolating new cell cycle proteins that are important for both the normal regulation of mitosis and the mitotic aberrations associated with cell cycle perturbations and chemical treatments.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

p68 Sam is a substrate of the insulin receptor and associates with the SH2 domains of p85 PI3K.

The 68 kDa Src substrate associated during mitosis is an RNA binding protein with Src homology 2 and 3 domain binding sites. A role for Src associated in mitosis 68 as an adaptor protein in signaling transduction has been proposed in different systems such as T-cell receptors. In the present work, we have sought to assess the possible role of Src associated in mitosis 68 in insulin receptor signaling. We performed in vivo studies in HTC-IR cells and in vitro studies using recombinant Src associated in mitosis 68, purified insulin receptor and fusion proteins containing either the N-terminal or the C-terminal Src homology 2 domain of p85 phosphatidylinositol-3-kinase. We have found that Src associated in mitosis 68 is a substrate of the insulin receptor both in vivo and in vitro. Moreover, tyrosine-phosphorylated Src associated in mitosis 68 was found to associate with p85 phosphatidylinositol-3-kinase in response to insulin, as assessed by co-immunoprecipitation studies. Therefore, Src associated in mitosis 68 may be part of the signaling complexes of insulin receptor along with p85. In vitro studies demonstrate that Src associated in mitosis 68 associates with the Src homology 2 domains of p85 after tyrosine phosphorylation by the activated insulin receptor. Moreover, tyr-phosphorylated Src associated in mitosis 68 binds with a higher affinity to the N-terminal Src homology 2 domain of p85 compared to the C-terminal Src homology 2 domain of p85, suggesting a preferential association of Src associated in mitosis 68 with the N-terminal Src homology 2 domain of p85. This association may be important for the link of the signaling with RNA metabolism.     

Prostate-derived sterile 20-like kinases (PSKs/TAOKs) are activated in mitosis and contribute to mitotic cell rounding and spindle positioning

Prostate-derived sterile 20-like kinases (PSKs) 1-α, 1-β, and 2 are members of the germinal-center kinase-like sterile 20 family of kinases. Previous work has shown that PSK 1-α binds and stabilizes microtubules whereas PSK2 destabilizes microtubules. Here, we have investigated the activation and autophosphorylation of endogenous PSKs and show that their catalytic activity increases as cells accumulate in G(2)/M and declines as cells exit mitosis. PSKs are stimulated in synchronous HeLa cells as they progress through mitosis, and these proteins are activated catalytically during each stage of mitosis. During prophase and metaphase activated PSKs are located in the cytoplasm and at the spindle poles, and during telophase and cytokinesis stimulated PSKs are present in trans-Golgi compartments. In addition, small interfering RNA (siRNA) knockdown of PSK1-α/β or PSK2 expression inhibits mitotic cell rounding as well as spindle positioning and centralization. These results show that PSK catalytic activity increases during mitosis and suggest that these proteins can contribute functionally to mitotic cell rounding and spindle centralization during cell division.     

Fresh WNT into the regulation of mitosis

Canonical Wnt signaling triggering β-catenin-dependent gene expression contributes to cell cycle progression, in particular at the G1/S transition. Recently, however, it became clear that the cell cycle can also feed back on Wnt signaling at the G2/M transition. This is illustrated by the fact that mitosis-specific cyclin-dependent kinases can phosphorylate the Wnt co-receptor LRP6 to prime the pathway for incoming Wnt signals when cells enter mitosis. In addition, there is accumulating evidence that various Wnt pathway components might exert additional, Wnt-independent functions that are important for proper regulation of mitosis. The importance of Wnt pathways during mitosis was most recently enforced by the discovery of Wnt signaling contributing to the stabilization of proteins other than β-catenin, specifically at G2/M and during mitosis. This Wnt-mediated stabilization of proteins, now referred to as Wnt/STOP, might on one hand contribute to maintaining a critical cell size required for cell division and, on the other hand, for the faithful execution of mitosis itself. In fact, most recently we have shown that Wnt/STOP is required for ensuring proper microtubule dynamics within mitotic spindles, which is pivotal for accurate chromosome segregation and for the maintenance of euploidy.     

p34cdc2: the S and M kinase?

In the yeast cell cycle, the induction of two very different processes, DNA synthesis (S-phase) and mitosis (M-phase), requires the same serine/threonine-specific protein kinase p34cdc2, which has been highly conserved through evolution. On the basis of work conducted largely in multicellular eukaryotes, it has recently been suggested that p34cdc2 is able to perform these two mutually exclusive roles by phosphorylating different sets of substrates through a cell cycle-dependent association with other proteins that dictate the substrate specificity of the protein kinase. To recognize its mitotic substrates, p34cdc2 associates with one of the cyclins--a family of proteins of two distinct but related types (A and B) characterized by their periodic destruction at each mitosis. In interphase, the formation of a complex between p34cdc2 and another protein (or proteins) would allow the phosphorylation of a different set of proteins involved in the G1 to S transition. This review focuses on the evidence for this appealing simple model and the nature of the putative substrates proposed.     

Change in phosphorylation of nucleolar proteins of Physarum polycephalum during the cell cycle in vivo and in vitro

We compared the phosphorylation of nucleolar proteins during the cell cycle of Physarum polycephalum labeled by pulse and continuous labeling methods in vivo with that obtained by in vitro labeling of isolated nucleoli. Both the phosphorylating activity of nucleoli and total incorporation of radioactive phosphate into nucleolar proteins increased and reached a maximum about 1.5-2.0 h before mitosis, confirming our previous observation. Analyses of labeled nucleolar proteins by SDS-polyacrylamide gel electrophoresis and by autoradiography indicated that most of the phosphoproteins labeled by in vitro labeling were labeled by in vivo pulse labeling. At least 10 nucleolar proteins underwent phosphorylation, which closely followed the cell cycle-dependent changes of the total phosphate incorporation into the nucleolar proteins. When mitosis was delayed by UV-irradiation, the maximal incorporation of radioactive phosphate into nucleolar proteins in vivo was not observed at the usual time, it shifted to about 2 h before the delayed mitosis, and the same set of nucleolar proteins that were phosphorylated without UV-irradiation were most heavily phosphorylated at this time. These results suggest the possibility that the increased phosphorylation of nucleolar proteins of Physarum just before mitosis is related to the onset of subsequent mitosis.     

Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C

Nuclear events of mitosis are initiated when the protein kinase cyclin-B1-Cdk1 is translocated into the nucleus during prophase. Recent work has unveiled many of the mechanisms that govern the localization of cyclin-B1-Cdk1 and its regulator Cdc25C. Phosphorylation-dependent changes in the rate of nuclear import and export of these proteins help to control the onset of mitosis both in normal cells and in cells delayed before mitosis by DNA damage.     

Mass spectrometric analysis identifies a cortactin-RCC2/TD60 interaction in mitotic cells

Cortactin is an F-actin binding protein that functions as a scaffold to regulate Arp2/3 mediated actin polymerization in lamellipodia and invadopodia formation as well as functioning in cell migration and endocytosis of many different cell types. In light of the fact that regulated actin polymerization is critical for many cellular processes we launched a search for novel cortactin interactions with cellular proteins that might indicate heretofore undescribed biological activities supported by cortactin. Using a modified stable isotope labeling in cell culture (SILAC) approach in HEK293 cells and Flag-tagged cortactin (F-cortactin) as bait, we identified a limited set of cortactin interactions including several proteins which have not previously been identified as cortactin associated proteins. Among these were serine/threonine-protein phosphatase 2A subunit beta (PP2A-beta) and RCC2/TD60, a Rac guanine nucleotide exchange factor (GEF) required for completion of mitosis and cytokinesis. The interaction between cortactin and RCC2/TD60 was verified in cell lysates immunoprecitated with anti-RCC2/TD60 antibody. Furthermore, cortactin was localized by immunofluorescence in the equatorial plane of dividing HeLa cells in the region where RCC2/TD60 has previously been localized thus providing support for a complex containing cortactin and RCC2/TD60 complex that may play a functional role in cells undergoing mitosis.