ABSTRACT
Microtubules constitute a vital part of the cytoskeleton in eukaryotes by mediating cell morphogenesis, cell motility, cell division, and intracellular transport. The cytoskeleton of the parasite Trypanosoma brucei contains an array of subpellicular microtubules with their plus-ends positioned toward the posterior cell tip, where extensive microtubule growth and cytoskeleton remodeling take place during early cell cycle stages. However, the control mechanism underlying microtubule dynamics at the posterior cell tip remains elusive. Here, we report that the S-phase cyclin-dependent kinase-cyclin complex CRK2-CYC13 in T. brucei regulates microtubule dynamics by phosphorylating ß-tubulin on multiple evolutionarily conserved serine and threonine residues to inhibit its incorporation into cytoskeletal microtubules and promote its degradation in the cytosol. Consequently, knockdown of CRK2 or CYC13 causes excessive microtubule extension and loss of microtubule convergence at the posterior cell tip, leading to cytoskeleton elongation and branching. These findings uncover a control mechanism for cytoskeletal microtubule dynamics by which CRK2 phosphorylates ß-tubulin and fine-tunes cellular ß-tubulin protein abundance to restrict excess microtubule extension for the maintenance of cytoskeleton architecture.
Subject(s)
Trypanosoma brucei brucei , Tubulin , Tubulin/metabolism , Trypanosoma brucei brucei/metabolism , Microtubules/metabolism , Cytoskeleton/metabolism , MorphogenesisABSTRACT
Perovskite nanocrystals (NCs) have emerged as a promising building block for the fabrication of optic-/optoelectronic-/electronic devices owing to their superior characteristics, such as high absorption coefficient, rapid ion mobilities, and tunable energy levels. However, their low structural stability and poor surface passivation have restricted their application to next-generation devices. Herein, a drug delivery system (DDS)-inspired post-treatment strategy is reported for improving their structural stability by doping of Ag into CsPbBr3 (CPB) perovskite NCs; delivery to damaged sites can promote their structural recovery slowly and uniformly, averting the permanent loss of their intrinsic characteristics. Ag NCs are designed through surface-chemistry tuning and structural engineering to enable their circulation in CPB NC dispersions, followed by their delivery to the CPB NC surface, defect-site recovery, and defect prevention. The perovskite-structure healing process through the DDS-type process (with Ag NCs as the drug) is analyzed by a combination of theoretical calculations (with density functional theory) and experimental analyses. The proposed DDS-inspired healing strategy significantly enhances the optical properties and stability of perovskite NCs, enabling the fabrication of white light-emitting diodes.
ABSTRACT
The human parasite Trypanosoma brucei contains a motile flagellum that determines the plane of cell division, controls cell morphology, and mediates cell-cell communication. During the cell cycle, inheritance of the newly formed flagellum requires its correct positioning toward the posterior of the cell, which depends on the faithful segregation of multiple flagellum-associated cytoskeletal structures including the basal body, the flagellar pocket collar, the flagellum attachment zone, and the hook complex. A specialized group of four microtubules termed the microtubule quartet (MtQ) originates from the basal body and runs through the flagellar pocket collar and the hook complex to extend, along the flagellum attachment zone, toward the anterior of the cell. However, the physiological function of the MtQ is poorly understood, and few MtQ-associated proteins have been identified and functionally characterized. We report here that an MtQ-localized protein named NHL1 interacts with the microtubule-binding protein TbSpef1 and depends on TbSpef1 for its localization to the MtQ. We show that RNAi-mediated knockdown of NHL1 impairs the segregation of flagellum-associated cytoskeletal structures, resulting in mispositioning of the new flagellum. Furthermore, knockdown of NHL1 also causes misplacement of the cell division plane in dividing trypanosome cells, halts cleavage furrow ingression, and inhibits completion of cytokinesis. These findings uncover a crucial role for the MtQ-associated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei.
Subject(s)
Trypanosoma brucei brucei , Basal Bodies/metabolism , Chromosome Segregation , Flagella/metabolism , Humans , Microtubules/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolismABSTRACT
BACKGROUND: Faithful DNA replication is essential to maintain genomic stability in all living organisms, and the regulatory pathway for DNA replication initiation is conserved from yeast to humans. The evolutionarily ancient human parasite Trypanosoma brucei, however, lacks many of the conserved DNA replication factors and may employ unusual mechanisms for DNA replication. Neither the S-phase cyclin-dependent kinase (CDK) nor the regulatory pathway governing DNA replication has been previously identified in T. brucei. RESULTS: Here we report that CRK2 (Cdc2-related kinase 2) complexes with CYC13 (Cyclin13) and functions as an S-phase CDK to promote DNA replication in T. brucei. We further show that CRK2 phosphorylates Mcm3, a subunit of the Mcm2-7 sub-complex of the Cdc45-Mcm2-7-GINS complex, and demonstrate that Mcm3 phosphorylation by CRK2 facilitates interaction with Sld5, a subunit of the GINS sub-complex of the Cdc45-Mcm2-7-GINS complex. CONCLUSIONS: These results identify the CRK2-CYC13 complex as an S-phase regulator in T. brucei and reveal its role in regulating DNA replication through promoting the assembly of the Cdc45-Mcm2-7-GINS complex.
Subject(s)
CDC2 Protein Kinase/genetics , Cyclins/genetics , DNA Replication , Protozoan Proteins/genetics , S-Phase Kinase-Associated Proteins/genetics , Trypanosoma brucei brucei/genetics , CDC2 Protein Kinase/metabolism , Cyclins/metabolism , Protozoan Proteins/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Trypanosoma brucei brucei/metabolismABSTRACT
Faithful chromosome segregation depends on correct spindle microtubule-kinetochore attachment and requires certain spindle-associated proteins (SAPs) involved in regulating spindle dynamics and chromosome segregation. Little is known about the spindle-associated proteome in the early divergent Trypanosoma brucei and its roles in chromosome segregation. Here we report the identification of a cohort of divergent SAPs through localization-based screening and proximity-dependent biotin identification. We identified seven new SAPs and seventeen new nucleolar proteins that associate with the spindle, and demonstrated that the kinetochore protein KKIP4 also associates with the spindle. These SAPs localize to distinct subdomains of the spindle during mitosis, and all but one localize to nucleus during interphase and post-mitotic phases. Functional analyses of three nucleus- and spindle-associated proteins (NuSAPs) revealed distinct functions in chromosome segregation. NuSAP1 is a kinetoplastid-specific protein required for equal chromosome segregation and for maintaining the stability of the kinetochore proteins KKIP1 and KKT1. NuSAP2 is a highly divergent ASE1/PRC1/MAP65 homolog playing an essential role in promoting the G2/M transition. NuSAP3 is a kinetoplastid-specific Kif13-1-binding protein maintaining Kif13-1 protein stability and regulating the G2/M transition. Together, our work suggests that chromosome segregation in T. brucei requires a cohort of kinetoplastid-specific and divergent SAPs with distinct functions.
Subject(s)
Chromosome Segregation , Kinetochores/metabolism , Microtubule-Associated Proteins/metabolism , Protozoan Proteins/metabolism , Spindle Apparatus/metabolism , Trypanosoma brucei brucei/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , In Situ Hybridization, Fluorescence , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Mitosis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protozoan Proteins/genetics , RNA Interference , Trypanosoma brucei brucei/cytology , Trypanosoma brucei brucei/geneticsABSTRACT
A naturally occurring stilbene, resveratrol, shows promising effects in the treatment of malignant pleural mesothelioma (MPM) both as a single agent and in combination with chemotherapeutic drugs. To discover new anticancer agents targeting MPM, stilbene-targeted isolation was performed on the roots of Polygonum multiflorum Thunb., an herbal medicine rich in stilbene compounds. In this study, seven stilbene glycosides (1-7) were isolated, along with four non-stilbenes (8-11), of which compounds 4 and 9-11 have not previously been isolated from this species. Stiquinoside A (1) is a previously undescribed stilbene glycoside, and its structure was elucidated as (E)-2,3,5,4'-tetrahydroxystilbene 2-O-ß-d-quinovopyranoside based on 1D and 2D-NMR, HR-ESI-MS, and acid hydrolysis experiments. Compounds 1, 4, 6, and 8 significantly inhibit the growth of MPM cancer cells H2452. These results demonstrate the potential utility of stilbenes in new strategies for the treatment of MPM.
Subject(s)
Antineoplastic Agents, Phytogenic , Fallopia multiflora , Mesothelioma, Malignant , Plant Roots , Stilbenes , Humans , Stilbenes/pharmacology , Stilbenes/isolation & purification , Plant Roots/chemistry , Molecular Structure , Cell Line, Tumor , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/isolation & purification , Mesothelioma, Malignant/drug therapy , Fallopia multiflora/chemistry , Phytochemicals/pharmacology , Phytochemicals/isolation & purification , Glycosides/pharmacology , Glycosides/isolation & purification , Mesothelioma/drug therapy , Lung Neoplasms/drug therapy , ChinaABSTRACT
Cu2Se nanoparticles were synthesized using the standard Schlenk line and glove box techniques, with the hot-injection method. The X-ray diffraction (XRD) analysis showed that the initial nanoparticles were formed in a stoichiometric Cu2Se phase with a cubic structure. When the nanoparticles are exposed to air, the diffraction peaks shift to higher angles. This suggests that the nanoparticles are changed to a nonstoichiometric Cu2-deltaSe phase with copper vacancies. The mean size of the nanoparticles was about 7 nm. The magnetic results show that the initial nanoparticles were diamagnetic, and after 1-week air exposure, they became paramagnetic. This dramatic change from diamagnetic to paramagnetic can be explained by the oxidation of Cu+ into Cu2+ at the nanoparticle surface. In addition, the superparamagnetic properties were observed to have a blocking temperature of 150 K. The coercive field decreased as the temperature approached the blocking temperature, and eventually vanished above the blocking temperature.
ABSTRACT
Cytokinesis in Trypanosoma brucei occurs unidirectionally from the anterior toward the posterior through mechanisms distinct from those of its human host and is controlled by a signaling pathway comprising evolutionarily conserved and trypanosome-specific regulatory proteins. The mechanistic roles and the functional interplay of these cytokinesis regulators remain poorly understood. Here, we investigate the requirement of the structural motifs in the trypanosome-specific cytokinesis regulator CIF3 for the initiation of cytokinesis, the interaction with other cytokinesis regulators, and the recruitment of CIF3-interacting proteins to the cytokinesis initiation site. We demonstrate that the internal and C-terminal coiled-coil motifs, but not the N-terminal coiled-coil motif, of CIF3 play essential roles in cytokinesis and interact with distinct cytokinesis regulators. CIF3 interacts with TbPLK, CIF1, CIF4, and FPRC through the N-terminal and C-terminal coiled-coil motifs and with KAT80 through all three coiled-coil motifs. The C-terminal coiled-coil motif of CIF3 is required for the localization of CIF3 and all of its interacting proteins, and additionally, the internal coiled-coil motif of CIF3 is required for KAT80 localization. Conversely, all the CIF3-interacting proteins are required to maintain CIF3 at the cytokinesis initiation site at different cell cycle stages. These results demonstrate that CIF3 cooperates with multiple interacting partner proteins to promote cytokinesis in T. brucei. IMPORTANCE Cytokinesis is the final stage of cell division and is regulated by a signaling pathway conserved from yeast to humans. Cytokinesis in Trypanosoma brucei, an early-branching protozoan parasite causing human sleeping sickness, is regulated by mechanisms that are distinct from those of its human host, employing a number of trypanosome-specific regulatory proteins to cooperate with evolutionarily conserved regulators. The functional interplay of these cytokinesis regulators is still poorly understood. In this work, we investigated the structural requirement of the trypanosome-specific cytokinesis regulator CIF3 for the initiation of cytokinesis, the interaction with other cytokinesis regulatory proteins, and the recruitment of CIF3-interacting proteins. We demonstrated that different structural motifs of CIF3 played distinct roles in cytokinesis, interacted with distinct cytokinesis regulatory proteins, and were required for the recruitment of distinct cytokinesis regulatory proteins. These findings provided novel insights into the cooperative roles of cytokinesis regulators in promoting cytokinesis in T. brucei.
Subject(s)
Trypanosoma brucei brucei , Cell Division , Cytokinesis , Humans , Protein Domains , Protozoan Proteins/metabolism , Transcription Factors/metabolism , Trypanosoma brucei brucei/metabolismABSTRACT
Cytokinesis in eukaryotes is regulated by a Polo-like kinase-mediated and Aurora B kinase-mediated signalling pathway that promotes the assembly of the actomyosin contractile ring, a cytokinesis machinery conserved across evolution from yeast to humans. Trypanosoma brucei, an early divergent parasitic protozoan, employs an actomyosin-independent mechanism for its unusual cytokinesis that is controlled by a regulatory pathway comprising the Polo-like kinase TbPLK, the Aurora B kinase TbAUK1 and multiple trypanosomatid-specific regulators. However, whether any of these trypanosomatid-specific regulators function as substrates of TbPLK and/or TbAUK1 and how they cooperate with TbPLK and TbAUK1 to promote cytokinesis remain unknown. Here, we demonstrate that TbPLK and TbAUK1 phosphorylate the cytokinesis regulators CIF1 and CIF2 on multiple sites within their intrinsically disordered regions. We further show that TbPLK localization depends on its interaction with CIF1 from S/G2 phases, that TbPLK maintains CIF1 and CIF2 localization from G2 phase until early mitosis, and that TbAUK1 maintains CIF1 and CIF2 localization from late mitosis. Finally, we demonstrate that the cytokinesis regulators CIF4 and FPRC are not substrates of TbPLK and TbAUK1, and that they function upstream of TbPLK and TbAUK1 in the cytokinesis regulatory pathway. Together, these results provide insights into the functional interplay and the order of actions between the two protein kinases and the trypanosomatid-specific cytokinesis regulators in T. brucei.
Subject(s)
Trypanosoma brucei brucei , Actomyosin/metabolism , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Cytokinesis/physiology , Humans , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolismABSTRACT
All-solid-state batteries (ASSBs) that employ anode-less electrodes have drawn attention from across the battery community because they offer competitive energy densities and a markedly improved cycle life. Nevertheless, the composite matrices of anode-less electrodes impose a substantial barrier for lithium-ion diffusion and inhibit operation at room temperature. To overcome this drawback, here, the conversion reaction of metal fluorides is exploited because metallic nanodomains formed during this reaction induce an alloying reaction with lithium ions for uniform and sustainable lithium (de)plating. Lithium fluoride (LiF), another product of the conversion reaction, prevents the agglomeration of the metallic nanodomains and also protects the electrode from fatal lithium dendrite growth. A systematic analysis identifies silver (I) fluoride (AgF) as the most suitable metal fluoride because the silver nanodomains can accommodate the solid-solution mechanism with a low nucleation overpotential. AgF-based full cells attain reliable cycling at 25 °C even with an exceptionally high areal capacity of 9.7 mAh cm-2 (areal loading of LiNi0.8 Co0.1 Mn0.1 O2 = 50 mg cm-2 ). These results offer useful insights into designing materials for anode-less electrodes for sulfide-based ASSBs.
ABSTRACT
To obtain high catalytic properties, finely modulating the electronic structure and active sites of catalysts is important. Herein, we report the design and economical synthesis of Pd@Pt core-shell nanoparticles for high productivity in the direct synthesis of hydrogen peroxide. Pd@Pt core-shell nanoparticles with a partially covered Pt shell on a Pd cube were synthesized using a simple direct seed-mediated growth method. The synthesized Pd@Pt core-shell nanoparticles were composed of high index faceted Pt on the corners and edges, while the Pd-Pt alloy was located on the terrace area of the Pd cubes. Because of the high-indexed Pt and Pd-Pt alloy sites, the synthesized concave Pd@Pt7 nanoparticles exhibited both high H2 conversion and H2O2 selectivity compared with Pd cubes.
ABSTRACT
The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote. IMPORTANCE: The basal body in the early-branching protozoan Trypanosoma brucei nucleates flagellum assembly and also regulates organelle segregation, cell morphogenesis, and cell division. However, the molecular composition and the assembly process of the basal body remain poorly understood. Here, we identify 14 conserved basal body proteins and 25 trypanosome-specific basal body proteins via bioinformatics, localization-based screening, and proximity-dependent biotin identification. We further localized these proteins to distinct subdomains of the basal body by using fluorescence microscopy and superresolution microscopy, discovered novel regulators of basal body duplication and separation, and uncovered new functions of conserved basal body proteins in basal body duplication and separation. This work lays the foundation for dissecting the mechanisms underlying basal body biogenesis and inheritance in T. brucei.
Subject(s)
Basal Bodies/chemistry , Basal Bodies/metabolism , Organelle Biogenesis , Proteome/analysis , Protozoan Proteins/analysis , Trypanosoma brucei brucei/physiology , Axoneme/metabolism , Flagella/metabolism , Locomotion , Trypanosoma brucei brucei/metabolismABSTRACT
Ni0.5Cu0.3Zn0.2Fe2O4 thin films with thickness ranging from 25 nm to 500 nm were grown on Si substrate using pulsed laser deposition technique and their structural and magnetic properties were investigated. From the atomic force microscopy (AFM) analysis, it is observed that the film roughness (Ra) depends strongly on the thickness of the fabricated film. The magnetizations of the thin films were found to decrease when the film thickness increases. The thinner films showed a larger magnetization than the thick films. All the films showed a blocking temperature indicating their superparamagnetic behavior.
ABSTRACT
UNLABELLED: The hetero-trimeric PP2A serine/threonine phosphatases containing the regulatory subunit B56, and in particular B56γ, can function as tumor suppressors. In response to DNA damage, the B56γ subunit complexes with the PP2A AC core (B56γ-PP2A) and binds p53. This event promotes PP2A-mediated dephosphorylation of p53 at Thr55, which induces expression of p21, and the subsequent inhibition of cell proliferation and transformation. In addition to dephosphorylation of p53, B56γ-PP2A also inhibits cell proliferation and transformation by a second, as yet unknown, p53-independent mechanism. Here, we interrogated a panel of B56γ mutations found in human cancer samples and cell lines and showed that these mutations lost B56γ tumor-suppressive activity by two distinct mechanisms: one is by disrupting interactions with the PP2A AC core and the other with B56γ-PP2A substrates (p53 and unknown proteins). For the first mechanism, due to the absence of the C catalytic subunit in the complex, the mutants are unable to mediate dephosphorylation of any substrate and thus failed to promote both the p53-dependent and -independent tumor-suppressive functions of B56γ-PP2A. For the second mechanism, the mutants lacked specific substrate interactions and thus partially lost tumor-suppressive function, i.e., either the p53-dependent or p53-independent contingent upon which substrate binding was affected. Overall, these data provide new insight into the mechanisms of tumor suppression by B56γ. IMPLICATIONS: This study further indicates the importance of B56γ-PP2A in tumorigenesis.
Subject(s)
Carcinogenesis , Protein Phosphatase 2/chemistry , Protein Phosphatase 2/genetics , Cell Line, Tumor , Cell Transformation, Neoplastic , HCT116 Cells , Humans , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Phosphorylation , Protein Phosphatase 2/metabolism , Protein Structure, Tertiary , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
One-dimensional ferromagnetic iron dendritic wire array film is prepared by facile electrodeposition. The space hindrance effect caused by neighbouring crystals resists the free growth directions parallel to the substrate, which is considered as a possible growth mechanism of one-dimensional morphology. Dendritic iron wire can be transformed into α-Fe(2)O(3) without destroying the dendritic morphology by thermal oxidation.