Mitotic phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial fission

Organelles are inherited to daughter cells beyond dynamic changes of the membrane structure during mitosis. Mitochondria are dynamic entities, frequently dividing and fusing with each other, during which dynamin-related GTPase Drp1 is required for the fission reaction. In this study, we analyzed mitochondrial dynamics in mitotic mammalian cells. Although mitochondria in interphase HeLa cells are long tubular network structures, they are fragmented in early mitotic phase, and the filamentous network structures are subsequently reformed in the daughter cells. In marked contrast, tubular mitochondrial structures are maintained during mitosis in Drp1 knockdown cells, indicating that the mitochondrial fragmentation in mitosis requires mitochondrial fission by Drp1. Drp1 was specifically phosphorylated in mitosis by Cdk1/cyclin B on Ser-585. Exogenous expression of unphosphorylated mutant Drp1S585A led to reduced mitotic mitochondrial fragmentation. These results suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells.     

Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability

Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.     

Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Entry into mitosis depends on the activity of cyclin‐dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55δ subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle—high in interphase and suppressed during mitosis. Depletion of PP2A–B55δ (in interphase) from ‘cycling’ frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A–B55δ was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A–B55δ in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.     

RALA and RALBP1 regulate mitochondrial fission at mitosis

Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission. Equal distribution of mitochondria to daughter cells during mitosis requires fission. Mitotic mitochondrial fission depends on both the relocalization of the large GTPase DRP1 to the outer mitochondrial membrane and phosphorylation of Ser 616 on DRP1 by the mitotic kinase cyclin B-CDK1 (ref. 2). We now report that these processes are mediated by the small Ras-like GTPase RALA and its effector RALBP1 (also known as RLIP76, RLIP1 or RIP1; refs 3, 4). Specifically, the mitotic kinase Aurora A phosphorylates Ser 194 of RALA, relocalizing it to the mitochondria, where it concentrates RALBP1 and DRP1. Furthermore, RALBP1 is associated with cyclin B-CDK1 kinase activity that leads to phosphorylation of DRP1 on Ser 616. Disrupting either RALA or RALBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B-CDK1 converge on RALA and RALBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function.     

CELL CYCLE-SPECIFIC CHANGES IN HISTONE PHOSPHORYLATION ASSOCIATED WITH CELL PROLIFERATION AND CHROMOSOME CONDENSATION

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G₁-arrested and early G₁-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G₂/M boundary and traversing prophase. Since these phosphorylation events do not occur in G₁, S, or G₂ and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled ³²PO₄ from those specific histone fractions during transition of metaphase cells into interphase G₁ cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.     

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation

We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.     

Cell-cycle-dependent cortical localization of pEg3 protein kinase in Xenopus and human cells

BACKGROUND INFORMATION: Protein kinase pEg3 belongs to the evolutionarily conserved KIN1/PAR-1/MARK family, whose members are involved in a variety of functions, including cell polarity, microtubule stability, intracellular signalling and the cell cycle. Activity and phosphorylation of pEg3 are cell-cycle dependent and rise to maximum levels during mitosis. pEg3 was shown to interact with and phosphorylate phosphatase CDC25B, and to potentially control cell-cycle progression. Subcellular localization of pEg3 was investigated in Xenopus and human cultured cells. RESULTS: By expression of GFP (green fluorescent protein)-tagged pEg3 and indirect immunofluorescence with specific antibodies, pEg3 was found to be localized in the cytoplasm and the nucleus in interphase cells. During mitosis pEg3 was also found in the cytoplasm. From anaphase to telophase, a proportion of the protein was detected at the cell cortex. The cortical distribution in mitotic cells was dependent on F-actin, because the actin-depolymerization-inducing drugs cytochalasin D or latrunculin A prevented pEg3 cortical localization. The protein lacking the conserved C-terminal domain was not detected at the cell cortex, whereas the C-terminal domain was targeted to the cell periphery. In contrast with full-length pEg3, the cortical localization of the C-terminal domain and construct lacking the N-terminal domain was cell-cycle independent, and these constructs were found at the cell periphery in interphase cells. CONCLUSIONS: pEg3 is localized at the cell periphery specifically during mitosis. The C-terminal domain is the only pEg3 domain found to be necessary and sufficient for cortical targeting. Cortical distribution of pEg3 also requires the F-actin cytoskeleton. The cell-cycle-independent cortical localization of the pEg3 C-terminal domain and a construct lacking the N-terminal domain indicates that a negative control mechanism involving the pEg3 catalytic N-terminal domain probably acts to prevent pEg3 cortical distribution during interphase. These results suggest that pEg3 might play a role at the cell cortex during mitosis.     

p34cdc2 acts as a lamin kinase in fission yeast

The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.     

Restraint of apoptosis during mitosis through interdomain phosphorylation of caspase‐2

The apoptotic initiator caspase‐2 has been implicated in oocyte death, in DNA damage‐ and heat shock‐induced death, and in mitotic catastrophe. We show here that the mitosis‐promoting kinase, cdk1–cyclin B1, suppresses apoptosis upstream of mitochondrial cytochrome c release by phosphorylating caspase‐2 within an evolutionarily conserved sequence at Ser 340. Phosphorylation of this residue, situated in the caspase‐2 interdomain, prevents caspase‐2 activation. S340 was susceptible to phosphatase 1 dephosphorylation, and an interaction between phosphatase 1 and caspase‐2 detected during interphase was lost in mitosis. Expression of S340A non‐phosphorylatable caspase‐2 abrogated mitotic suppression of caspase‐2 and apoptosis in various settings, including oocytes induced to undergo cdk1‐dependent maturation. Moreover, U2OS cells treated with nocodazole were found to undergo mitotic catastrophe more readily when endogenous caspase‐2 was replaced with the S340A mutant to lift mitotic inhibition. These data demonstrate that for apoptotic stimuli transduced by caspase‐2, cell death is prevented during mitosis through the inhibitory phosphorylation of caspase‐2 and suggest that under conditions of mitotic arrest, cdk1–cyclin B1 activity must be overcome for apoptosis to occur.     

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.     

The mitochondrial fission regulator DRP3B does not regulate cell death in plants

BACKGROUND AND AIMS: Recent reports have described dramatic alterations in mitochondrial morphology during metazoan apoptosis. A dynamin-related protein (DRP) associated with mitochondrial outer membrane fission is known to be involved in the regulation of apoptosis. This study analysed the relationship between mitochondrial fission and regulation of plant cell death. METHODS: Transgenic plants were generated possessing Arabidopsis DRP3B (K56A), the dominant-negative form of Arabidopsis DRP, mitochondrial-targeted green fluorescent protein and mouse Bax. KEY RESULTS: Arabidopsis plants over-expressing DRP3B (K56A) exhibited long tubular mitochondria. In these plants, mitochondria appeared as a string-of-beads during cell death. This indicates that DRP3B (K56A) prevented mitochondrial fission during plant cell death. However, in contrast to results for mammalian cells and yeast, Bax-induced cell death was not inhibited in DRP3B (K56A)-expressing plant cells. Similarly, hydrogen peroxide-, menadione-, darkness- and salicylic acid-induced cell death was not inhibited by DRP3B (K56A) expression. CONCLUSIONS: These results indicate that the systems controlling cell death in animals and plants are not common in terms of mitochondrial fission.     

Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are involved in the control of cell cycle progression. Plant A-type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle. In contrast, B-type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown. Here, we identified two B2-type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1. The CDKB2;1-CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2. We then expressed green fluorescent protein (GFP)-fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis. Surprisingly, the fluorescence signal of CDKB2;1-GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase. During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast. On the other hand, the CycB2;2-GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase. Co-localization of CDKB2;1-GFP and CycB2;2-GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2-CycB2 complex may function in retaining chromosomes at the metaphase plate. Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.     

Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase

DNA damage by double-strand breaks induces arrest during interphase in mammalian cells. It is not clear whether DNA damage can arrest cells in mitosis. We show here that three human cell lines, HeLa, U2OS, and HCT116, do not delay in mitosis in response to double-strand breaks induced during mitosis by gamma irradiation or by adriamycin. Durable arrest at metaphase occurs, however, with ICRF-193, a topoisomerase II inhibitor that does not damage DNA. Arrest with ICRF-193 is not accompanied by recruitment of Mad2 or Bub1 to kinetochores, nor by phosphorylation of the histone H2AX, indicating arrest by ICRF-193 is not due to activation of the spindle assembly checkpoint, nor is it a response to DNA damage. VP-16, another decatenation inhibitor, induces metaphase arrest only at concentrations well above those that induce DNA damage. We conclude that decatenation failure, but not DNA damage, creates metaphase arrest in mammalian cells.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo

Fertilization of clam oocytes initiates a series of cell divisions, of which the first three--meiosis I, meiosis II, and the first mitotic division--are highly synchronous. After fertilization, protein synthesis is required for the successful completion of every division except meiosis I. When protein synthesis is inhibited, entry into meiosis I and the maintenance of M phase for the normal duration of meiosis occur normally, but the chromosomes fail to interact correctly with the spindle in meiosis II metaphase. By contrast, inhibition of protein synthesis immediately after completion of meiosis or mitosis stops cells entering the next mitosis. We describe the behavior of cyclins A and B in relation to these "points of no return." The cyclins are synthesized continuously and are rapidly destroyed shortly before the metaphase-anaphase transition of the mitotic cell cycles, with cyclin A being degraded in advance of cyclin B. Cyclin destruction normally occurs during a 5-min window in mitosis, but in the monopolar mitosis that occurs after parthenogenetic activation of clam oocytes, or when colchicine is added to fertilized eggs about to enter first mitosis, the destruction of cyclin B is strongly delayed, whereas proteolysis of cyclin A is maintained in an activated state for the duration of metaphase arrest. Under either of these abnormal conditions, inhibition of protein synthesis causes a premature return to interphase that correlates with the time when cyclin B disappears.     

The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells

Recent evidence shows that the COOH-terminal CaaX motif of lamins is necessary to target newly synthesized proteins to the nuclear envelope membranes. Isoprenylation at the CaaX-cysteine has been taken to explain the different fates of A- and B-type lamins during cell division. A-type lamins, which loose their isoprenylation shortly after incorporation into the lamina structure, become freely soluble upon mitotic nuclear envelope breakdown. Somatic B-type lamins, in contrast, are permanently isoprenylated and, although depolymerized during mitosis, remain associated with remnants of nuclear envelope membranes. However, Xenopus lamin B3, the major B-type lamin of amphibian oocytes and eggs, becomes soluble after nuclear envelope breakdown in meiotic metaphase. Here we show that Xenopus lamin B3 is permanently isoprenylated and carboxyl methylated in oocytes (interphase) and eggs (meiotic metaphase). When transfected into mouse L cells Xenopus lamin B3 is integrated into the host lamina and responds to cell cycle signals in a normal fashion. Notably, the ectopically expressed Xenopus lamin does not form heterooligomers with the endogenous lamins as revealed by a coprecipitation experiment with mitotic lamins. In contrast to the situation in amphibian eggs, a significant portion of lamin B3 remains associated with membranes during mitosis. We conclude from these data that the CaaX motif-mediated modifications, although necessary, are not sufficient for a stable association of lamins with membranes and that additional factors are involved in lamin-membrane binding.     

Thr 3 phosphorylated histone H3 concentrates at centromeric chromatin at metaphase

Thr 3 was one of the newly characterized phosphorylation sites on histone H3. However, the functional significance of histone H3 Thr 3 phosphorylation during mitosis is unclear. In this study, SDS-PAGE and Western blotting analysis showed that histone H3 Thr 3 was phosphorylated specially during mitosis in MCF-10A and ECV-304 cells. Using indirect immunofluorescence labeling and laser confocal microscopy, we demonstrated that histone H3 Thr 3 phosphorylation occurred from prophase to anaphase and dephosphorylated completely in telophase. Remarkably, Thr 3 phosphorylated histone H3 mostly concentrated at centromeric chromatin at metaphase, which was distinct with Ser 10 phosphorylation aggregated at the telomere, but similar to that characteristic of Thr 11 phosphorylated H3 which is largely restricted to the centromeric chromatin. Using chromatin immunoprecipitation (ChIP) assay, we provided direct evidence that the Thr 3 phosphorylated H3 is associated with centromeric DNA at metaphase. These findings suggested that at metaphase Thr 3 phosphorylated histone H3 may also participate in kinetochore assembly to promote faithful chromosome segregation and serve as another recognition code for kinetochore proteins.