Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A and TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAi-mediated silencing of each of the four genes led to loss of spindle assembly, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN-B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote.
Cytokinesis in Trypanosoma brucei, an early branching protozoan, occurs along its longitudinal axis uni-directionally from the anterior tip of the new flagellum attachment zone filament toward the cell's posterior end. However, the underlying mechanisms remain elusive. Here we report that cytokinesis in T. brucei is regulated by a concerted action of Polo-like kinase, Aurora B kinase, and a trypanosome-specific protein CIF1. Phosphorylation of CIF1 by Polo-like kinase targets it to the anterior tip of the new flagellum attachment zone filament, where it subsequently recruits Aurora B kinase to initiate cytokinesis. Consistent with its role, CIF1 depletion inhibits cytokinesis initiation from the anterior end of the cell, but, surprisingly, triggers cytokinesis initiation from the posterior end of the cell, suggesting the activation of an alternative cytokinesis from the opposite cell end. Our results reveal the mechanistic roles of CIF1 and Polo-like kinase in cytokinesis initiation and elucidate the mechanism underlying the recruitment of Aurora B kinase to the cytokinesis initiation site at late anaphase. These findings also delineate a signaling cascade controlling cytokinesis initiation from the anterior end of the cell and uncover a backup cytokinesis that is initiated from the posterior end of the cell when the typical anterior-to-posterior cytokinesis is compromised.cytokinesis | Polo-like kinase | Aurora B kinase | backup cytokinesis |
A one-step straightforward strategy has been developed to incorporate free imidazole molecules into a highly stable metal-organic framework (NENU-3, ([Cu(BTC)(HO)][HPWO])·Guest). The resulting material Im@(NENU-3) exhibits a very high proton conductivity of 1.82 × 10 S cm at 90% RH and 70 °C, which is significantly higher than 3.16 × 10 S cm for Im-Cu@(NENU-3a) synthesized through a two-step approach with mainly terminal bound imidazole molecules inside pores. Single crystal structure reveals that imidazole molecules in Im-Cu@(NENU-3a) isolate lattice water molecules and then block proton transport pathway, whereas high concentration of free imidazole molecules within Im@(NENU-3) significantly facilitate successive proton-hopping pathways through formation of hydrogen bonded networks.
Cyclins bind and activate cyclin-dependent kinases that regulate cell cycle progression in eukaryotes. Cell cycle control in Trypanosoma brucei was analyzed in the present study. Genes encoding four PHO80 cyclin homologues and three B-type cyclin homologues but no G 1 cyclin homologues were identified in this organism. Through knocking down expression of the seven cyclin genes with the RNA interference technique in the procyclic form of T. brucei, we demonstrated that one PHO80 homologue (CycE1/CYC2) and a B-type cyclin homologue (CycB2) are the essential cyclins regulating G 1 /S and G 2 /M transitions, respectively. This lack of overlapping cyclin function differs significantly from that observed in the other eukaryotes. Also, PHO80 cyclin is known for its involvement only in phosphate signaling in yeast with no known function in cell cycle control. Both observations thus suggest the presence of simple and novel cell cycle regulators in trypanosomes. T. brucei cells deficient in CycE1/CYC2 displayed a long slender morphology, whereas those lacking CycB2 assumed a fat stumpy form. These cells apparently still can undergo cytokinesis generating small numbers of anucleated daughter cells, each containing a single kinetoplast known as a zoid. Two different types of zoids were identified, the slender zoid derived from reduced CycE1/CYC2 expression and the stumpy zoid from CycB2 deficiency. This observation indicates an uncoupling between the kinetoplast and the nuclear cycle, resulting in cell division driven by kinetoplast segregation with neither a priori S phase nor mitosis in the trypanosome.
On-board fuel cell technology requires proton conducting materials with high conductivity not only at intermediate temperatures for work but also at room temperature and even at subzero temperature for startup when exposed to the colder climate. To develop such materials is still challenging because many promising candidates for the proton transport on the basis of extended microstructures of water molecules suffer from significant damage by heat at temperatures above 80 °C or by freeze below -5 °C. Here we show imidazole loaded tetrahedral polyimides with mesopores and good stability (Im@Td-PNDI 1 and Im@Td-PPI 2) exhibiting a high anhydrous proton conductivity over a wide temperature range from -40 to 90 °C. Among all anhydrous proton conductors, the conductivity of 2 is the highest at temperatures below 40 °C and comparable with the best materials, His@[Al(OH)(1,4-ndc)]n and [Zn3(H2PO4)6(H2O)3](Hbim), above 40 °C.
The chromosomal passenger protein aurora kinases have been implicated in regulating chromosome segregation and cell division. Three aurora kinase homologues were identified (TbAUK1, -2 and -3) in the Trypanosome Genomic Data Base, and their expressions in the procyclic form of Trypanosoma brucei were knocked down individually by using the RNA interference technique. Only a knockdown of TbAUK1 arrested the cells in G 2 /M phase with each cell showing an extended posterior end, two kinetoplasts, and an enlarged nucleus, apparently the result of an inhibited kinetoplast multiplication and a failed mitosis. There is no mitotic spindle structure in the TbAUK1-depleted cell. The two kinetoplasts moved apart from each other but stopped just before cytokinesis, suggesting that cytokinesis was blocked in its early phase. Overexpression of TbAUK1 in the cells resulted in little change in cell growth. By immunofluorescence, TbAUK1 was primarily localized to the nucleus in interphase and to the mitotic spindle during apparent metaphase and anaphase. Thus, differing from other eukaryotes, TbAUK1 has an apparent triple function in coupling mitosis and kinetoplast replication with cytokinesis in T. brucei. T. brucei polo-like kinase, previously identified as the initiator of cytokinesis without apparent involvement in mitosis in the trypanosome, was either depleted or overexpressed in the TbAUK1-deficient cells. A dominant TbAUK1-depleted phenotype was demonstrated in both cases, suggesting that TbAUK1 plays an essential role in cytokinesis that cannot be affected by changes in the level of T. brucei polo-like kinase. To our knowledge, this is the first time that the function of an aurora B-like kinase is a prerequisite for polo-like kinase action in initiating cytokinesis. TbAUK1 is also the first identified protein that couples both mitosis and kinetoplast replication with cytokinesis in the trypanosome.During cell cycle progression in eukaryotes, mitosis is a critical phase in ensuring that the two daughter cells inherit the same genetic background upon subsequent cell division. Although it represents the shortest phase in the metazoan cell cycle, mitosis is when the cell undergoes the most rapid and dramatic structural reorganizations. The interphase microtubule network de-polymerizes between the end of G 2 phase and the beginning of prophase (1). The centrosomes, which have duplicated during S phase, migrate around the nucleus and form a spindle, whereas chromatin condenses upon the entry into prophase. After spindle formation and nuclear envelope breakdown, the kinetochores capture the plus ends of microtubules and align the chromosomes at the metaphase plate. Two identical sets of chromosomes then begin to separate by decreasing the length of kinetochore fibers in anaphase A (2). Further separation of the chromosomes takes place by departing the spindle poles in anaphase B, which is followed by cytokinesis initiation and cell division.
Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei, depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.Trypanosoma brucei is an ancient unicellular eukaryote whose propagation involves a cyclic transmission between the mammalian host and the insect vector tsetse fly. A trypanosome cell contains a single flagellum, basal body, nucleus, mitochondrion, and the mitochondrial DNA complex known as the kinetoplast. Each of these organelles/structures is replicated and segregated into the two daughter cells in a wellsynchronized manner during cell division, implying a delicate coordination among these events (25). Other than the usual sequential G 1 , S, G 2 , and M phases, there is also an S phase for the kinetoplast completed prior to the nuclear S phase (45). The kinetoplast is replicated prior to mitosis and closely associated with the flagellum basal body, with its segregation dependent on the separation of replicated basal bodies, leading the cell through cytokinesis and eventual cell division in good coordination with nuclear division (30).Treatment of the procyclic form of T. brucei cells with okadaic acid prevented kinetoplast/basal body segregation and resulted in multinucleated cells, suggesting a normal progression of mitosis in cells defective in cytokinesis, which is controlled largely by the kinetoplast cycle (7). Treatment with aphidicolin or rhizoxin (29) or RNA interference (RNAi) silencing of the G 1 or mitotic cyclins (15, 24) or the cdc2-related kinases (CRKs) (37) in the procyclic-form cells resulted in inhibition of nuclear division but not kinetoplast/basal body segregation and subsequent cytokinesis, resulting in anucleate daughter cells designated the zoids. These results suggest that the checkpoint linking mitosis with cytokinesis may be weak or absent in the procyclic form of T. brucei and that kinetoplast/ basal body segregation alone is sufficient to drive some of t...
Trypanosoma brucei undergoes cytokinesis uni-directionally from the anterior tip of the new flagellum attachment zone (FAZ) toward the posterior end of the cell. We recently delineated a novel signaling pathway composed of polo-like kinase, cytokinesis initiation factor 1 (CIF1), and aurora B kinase that acts in concert at the new FAZ tip to regulate cytokinesis initiation. To identify new cytokinesis regulators, we carried out proximity-dependent biotin identification and identified many CIF1 binding partners and near neighbors. Here we report a novel CIF1-binding protein, named CIF2, and its mechanistic role in cytokinesis initiation. CIF2 interacts with CIF1 in vivo and co-localizes with CIF1 at the new FAZ tip during early cell cycle stages. RNAi of CIF2 inhibited the normal, anterior-toposterior cytokinesis but activated an alternative, posterior-toanterior cytokinesis. CIF2 depletion destabilized CIF1 and disrupted the localization of polo-like kinase and aurora B kinase to the new FAZ tip, thus revealing the mechanistic role of CIF2 in cytokinesis initiation. Surprisingly, overexpression of CIF2 also inhibited the normal, anterior-to-posterior cytokinesis and triggered the alternative, posterior-to-anterior cytokinesis, suggesting a tight control of CIF2 protein abundance. These results identified a new regulator in the cytokinesis regulatory pathway and reiterated that a backup cytokinesis pathway is activated by inhibiting the normal cytokinesis pathway.Trypanosoma brucei, an early divergent parasitic protozoan causing sleeping sickness in humans and nagana in cattle in sub-Saharan Africa, possesses a complex life cycle by alternating between the insect vector and the mammalian host. Within the insect midgut and the mammalian bloodstream, the parasite proliferates through binary fission along its longitudinal axis between the two flagella and their associated cytoskeletal structure termed flagellum attachment zone (FAZ) 2 (1). The cell division plane in a dividing trypanosome is determined by the length of the elongating new flagellum/FAZ, and the anterior tip of the new FAZ constitutes the site from which cytokinesis cleavage furrow ingression is initiated (2, 3). Before cytokinesis cleavage furrow initiation, invagination of cell body occurs between the two flagella, leading to the formation of the so-called division fold (4). Subsequently, the anterior tip of the new flagellum is released from the old flagellum due to the dissolution of the flagella connector (5), and cleavage furrow ingression begins from the anterior tip of the new FAZ and proceeds along the division fold toward the posterior end of the cell (6). At the very late stage of cytokinesis, the two daughter cells are connected at the posterior ends via a thread of membrane termed the cytoplasmic bridge (4), which is finally severed to generate two uni-flagellate daughter cells. Although the morphological events of cytokinesis in T. brucei have been well described (4), the mechanisms underlying this unusual mode of cytokinesis and the reg...
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