Cell cycle-dependent association of Rad51 with the nuclear matrix

Progression of the cells through the S phase of the cell cycle is connected with accumulation of stalled and collapsed replication forks that are repaired by homologous recombination. To investigate the temporal order of homologous recombination events during the S phase, HeLa cells synchronized at the G1/S phase boundary with mimosine were released to progress into the S phase and the phosphorylation of the histone variant H2AX, the appearance of Rad51 nuclear foci and the subcellular redistribution of Rad51 were followed. The results showed that there was gradual accumulation of double-strand breaks as judged by the increase in the phosphorylation of H2AX during the S phase. Rad51 nuclear foci did not appear until middle S phase, and this was accompanied by an increase in the chromatin- and nuclear matrix-bound Rad51 in the middle to late S phase. To determine the role of the intra S phase checkpoint in the S phase-dependent redistribution of Rad51 the cells were released in the S phase in the presence of the protein kinase inhibitors caffeine and wortmannin. The results suggest that the association of Rad51 with the nuclear matrix is regulated by activation of the intra S phase ATR-dependent checkpoint pathway.     

Cell cycle-dependent phosphorylation, nuclear localization, and activation of human condensin

Condensin, one of the most abundant components of mitotic chromosomes, is a conserved protein complex composed of two structural maintenance of chromosomes (SMC) subunits (SMC2- and SMC4-type) and three non-SMC subunits, and it plays an essential role in mitotic chromosome condensation. Purified condensin reconfigures DNA structure using energy provided by ATP hydrolysis. To know the regulation of condensin in somatic cells, the expression level, subcellular localization, and phosphorylation status of human condensin were examined during the cell cycle. The levels of condensin subunits were almost constant throughout the cell cycle, and the three non-SMC subunits were phosphorylated at specific sites in mitosis and dephosphorylated upon the completion of mitosis. Subcellular fractionation studies revealed that a proportion of condensin was tightly bound to mitotic chromosomes and that this form was phosphorylated at specific sites. Condensin purified from mitotic cells had much stronger supercoiling activity than that purified from interphase cells. These results suggest that condensin functions in somatic cells are regulated by phosphorylation in two ways during the cell cycle; the phosphorylation of specific sites correlates with the chromosomal targeting of condensin, and its biochemical activity is stimulated by phosphorylation.     

Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells

Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. Here, we have visualized the dynamics of five green fluorescent protein (GFP)-tagged mitotic kinesins (class 5, 6, 8, 13, and 14) in live Drosophila Schneider cell line (S2), after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNA interference (RNAi)-based rescue strategy. Class 8 (Klp67A) and class 14 (Ncd) kinesin are sequestered in an active form in the nucleus during interphase and engage their microtubule targets upon nuclear envelope breakdown (NEB). Relocalization of Klp67A to the cytoplasm using a nuclear export signal resulted in the disassembly of the interphase microtubule array, providing support for the hypothesis that this kinesin class possesses microtubule-destabilizing activity. The interactions of Kinesin-5 (Klp61F) and -6 (Pavarotti) with microtubules, on the other hand, are activated and inactivated by Cdc2 phosphorylation, respectively, as shown by examining localization after mutating Cdc2 consensus sites. The actions of microtubule-destabilizing kinesins (class 8 and 13 [Klp10A]) seem to be controlled by cell cycle-dependent changes in their localizations. Klp10A, concentrated on microtubule plus ends in interphase and prophase, relocalizes to centromeres and spindle poles upon NEB and remains at these sites throughout anaphase. Consistent with this localization, RNAi analysis showed that this kinesin contributes to chromosome-to-pole movement during anaphase A. Klp67A also becomes kinetochore associated upon NEB, but the majority of the population relocalizes to the central spindle by the timing of anaphase A onset, consistent with our RNAi result showing no effect of depleting this motor on anaphase A. These results reveal a diverse spectrum of regulatory mechanisms for controlling the localization and function of five mitotic kinesins at different stages of the cell cycle.     

Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics

The lamina-associated polypeptide (LAP) 2 family comprises up to six alternatively spliced proteins in mammalian cells and three isoforms in Xenopus. LAP2beta is a type II integral protein of the inner nuclear membrane, which binds to lamin B and the chromosomal protein BAF, and may link the nuclear membrane to the underlying lamina and provide docking sites for chromatin. LAP2alpha shares only the N-terminus with the other isoforms and contains a unique C-terminus. It is a nonmembrane protein associated with the nucleoskeleton and may help to organize higher order chromatin structure by interacting with A-lamins and chromosomes. Recent studies using mutant proteins have just begun to unravel functions of LAP2 isoforms during postmitotic nuclear reassembly. LAP2alpha associates with chromosomes via an alpha-specific domain at early stages of assembly, possibly providing a structural framework for chromosome reorganization. The subsequent interaction of both LAP2alpha and LAP2beta with the chromosomal BAF may stabilize chromatin structure and target membranes to the chromosomes. At later stages LAP2 may regulate the assembly of lamins. LAP2 isoforms have been found to share a homologous approximately 40 amino acid long region, the LEM domain, with nuclear membrane proteins MAN1 and emerin, which has been implicated in Emery-Dreifuss muscular dystrophy.     

Cell cycle-dependent nuclear location of the matricellular protein SPARC: association with the nuclear matrix.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that inhibits cellular adhesion and proliferation. In this study, we report the detection of SPARC in the interphase nuclei of embryonic chicken cells in vivo. Differential partitioning of SPARC was also noted in the cytoplasm of these cells during discrete stages of M-phase: cells in metaphase and anaphase exhibited strong cytoplasmic immunoreactivity, whereas cells in telophase were devoid of labeling. Immunocytochemical analysis of embryonic chicken cells in vitro likewise showed the presence of SPARC in the nucleus. Furthermore, elution of soluble proteins and DNA from these cells indicated that SPARC might be a component of the nuclear matrix. We subsequently examined cultured bovine aortic endothelial cells, which initially appeared to express SPARC only in the cytoplasm. However, after elution of soluble proteins and chromatin, we also detected SPARC in the nuclear matrix of these cells. Embryonic chicken cells incubated with recombinant SPARC were seen to take up the protein and to translocate it to the nucleus progressively over a period of 17 h. These observations provide new information about SPARC, generally recognized as a secreted glycoprotein that mediates interactions between cells and components of the extracellular matrix. The evidence presented in this study indicates that SPARC might subserve analogous functions in the nuclear matrix.     

Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells

To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.     

Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1alpha). The mitotic index and doubling time for LLCPK-1alpha were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1alpha cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1alpha and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1alpha cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.     

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.     

Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1)

Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1).     

Cell cycle-dependent nuclear localization of yeast RNase III is required for efficient cell division

Members of the double-stranded RNA-specific ribonuclease III (RNase III) family were shown to affect cell division and chromosome segregation, presumably through an RNA interference-dependent mechanism. Here, we show that in Saccharomyces cerevisiae, where the RNA interference machinery is not conserved, an orthologue of RNase III (Rnt1p) is required for progression of the cell cycle and nuclear division. The deletion of Rnt1p delayed cells in both G1 and G2/M phases of the cell cycle. Nuclear division and positioning at the bud neck were also impaired in Deltarnt1 cells. The cell cycle defects were restored by the expression of catalytically inactive Rnt1p, indicating that RNA cleavage is not essential for cell cycle progression. Rnt1p was found to exit from the nucleolus to the nucleoplasm in the G2/M phase, and perturbation of its localization pattern delayed the progression of cell division. A single mutation in the Rnt1p N-terminal domain prevented its accumulation in the nucleoplasm and slowed exit from mitosis without any detectable effects on RNA processing. Together, the data reveal a new role for a class II RNase III in the cell cycle and suggest that at least some members of the RNase III family possess catalysis-independent functions.     

Cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells studied by in vitro translation

A combination of methods was used to study the cell cycle-dependent expression of nuclear matrix proteins of Ehrlich ascites cells: Separation of asynchronous cells growing in vivo into fractions of G1-, S- and G2- phase cells by centrifugal elutriation with less than 10% cross-contamination. Isolation of poly(A+) RNA populations from total cytoplasmic RNA by affinity chromatography on messenger affinity paper (mAP). In vitro translation of poly(A+) RNA from asynchronous and phase synchronous cells. Immunoprecipitation of in vitro synthesized nuclear matrix proteins by a monoclonal antibody with anti-lamin specificity (PKB8) and by a polyspecific anti-nuclear matrix serum (AMS5) followed by analysis of immunoprecipitated materials on SDS-polyacrylamide gels. The results indicate that mRNAs for nuclear matrix-associated proteins including the lamins B and C are either exclusively or at least predominantly present in the cytoplasm of cells in S phase suggesting a high rate of in vivo synthesis of these proteins during S phase. This is consistent with an anticipated biological function of the nuclear matrix which is considered to organize parental and newly synthesized DNA in higher order structures.     

Cell cycle-dependent AgNOR analysis in invasive breast cancer

OBJECTIVE: To investigate to what extent analysis of silver-stained nucleolar organizer regions (AgNORs) is cell cycle dependent in breast cancer and to assess the prognostic value of an AgNOR analysis that takes into consideration the cell cycle status of tumor cells. STUDY DESIGN: In 97 cases of invasive breast carcinoma, morphometric AgNOR analysis was performed in tumor cells with immunohistochemical MIB-1 reactivity (NORcyc analysis) and in MIB-1-negative tumor cells (NORnon analysis). Additionally, conventional (NORconv) analysis without preceding MIB-1 staining was done. Findings were compared with the Nottingham prognostic index (NPI). RESULTS: In comparison to noncycling tumor cells, cycling ones exhibited significantly higher AgNOR numbers (mean values, 3.84 +/- 1.09 vs. 2.40 +/- 0.78 per nucleus), higher total AgNOR areas (5.95 +/- 3.17 vs. 5.62 +/- 3.05 micron 2, NS) and significantly lower mean AgNOR areas (2.08 +/- 1.14 vs. 2.93 +/- 1.69 micron 2). When related to NPI, correlation coefficients of NORnon analysis were higher than those of NORcyc analysis but lower than those of NORconv analysis. Among the different AgNOR parameters, total AgNOR area correlated best with NPI. CONCLUSION: Cell cycle status has a high impact on AgNOR analysis. However, the best prognostic information in breast cancer is derived from an AgNOR analysis that considers both cycling and noncycling tumor cells.     

Cell cycle-dependent proteolysis and ectopic overexpression of cyclin B1 in tobacco BY2 cells

Activation of cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Although cyclin gene expression has been extensively studied in plants, not much is known at the level of the protein stability and function. Here, we demonstrated by using the highly synchronizable tobacco BY2 cell culture, that endogenous cyclin B1 protein undergoes cell cycle-dependent proteolysis and is stabilized when the spindle checkpoint has been activated. Furthermore, we established transgenic tobacco BY2 cell cultures expressing under the control of an inducible promoter, cyclin B1 protein as well as its non-degradable form as fusion proteins with GFP and found that the ectopic expression of these proteins did not dramatically disturb the cell cycle progression. These results indicate that, to a certain extent, cell cycle exit is possible without cyclin B1 proteolysis.