A kinesin-13 family kinesin in Trypanosoma brucei regulates cytokinesis and cytoskeleton morphogenesis by promoting microtubule bundling.

The early branching eukaryote Trypanosoma brucei divides uni-directionally along the longitudinal cell axis from the cell anterior toward the cell posterior, and the cleavage furrow ingresses along the cell division plane between the new and the old flagella of a dividing bi-flagellated cell. Regulation of cytokinesis in T. brucei involves actomyosin-independent machineries and trypanosome-specific signaling pathways, but the molecular mechanisms underlying cell division plane positioning remain poorly understood. Here we report a kinesin-13 family protein, KIN13-5, that functions downstream of FPRC in the cytokinesis regulatory pathway and determines cell division plane placement. KIN13-5 localizes to multiple cytoskeletal structures, interacts with FPRC, and depends on FPRC for localization to the site of cytokinesis initiation. Knockdown of KIN13-5 causes loss of microtubule bundling at both ends of the cell division plane, leading to mis-placement of the cleavage furrow and unequal cytokinesis, and at the posterior cell tip, causing the formation of a blunt posterior. In vitro biochemical assays demonstrate that KIN13-5 bundles microtubules, providing mechanistic insights into the role of KIN13-5 in cytokinesis and posterior morphogenesis. Altogether, KIN13-5 promotes microtubule bundle formation to ensure cleavage furrow placement and to maintain posterior cytoskeleton morphology in T. brucei.

CRK2 controls cytoskeleton morphogenesis in Trypanosoma brucei by phosphorylating ß-tubulin to regulate microtubule dynamics.

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.

The microtubule quartet protein SNAP1 in Trypanosoma brucei facilitates flagellum and cell division plane positioning by promoting basal body segregation.

The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubule-binding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis.

Angular uniformity improvement of diffractive waveguide display based on region geometry optimization.

Augmented reality (AR) near-eye displays have significantly progressed due to advances in nanostructure fabrication. However, for diffractive waveguide AR displays requiring exit pupil expansion, the angular uniformity of each exit pupil position still needs to improve. In this paper, an angular uniformity improvement method based on region geometry optimization is proposed. This optimization method essentially introduces the interaction number of the light with the grating as one of the variables to manipulate the energy distribution. This distribution is obtained by the rigorous coupled wave analysis (RCWA) method and ray tracing process and is further optimized by a multi-objective genetic algorithm. A model is built, and the feasibility of the proposed method is verified. The diffractive waveguide system has a 10m m×10m m exit pupil size at the eye relief of 25 mm and a field of view (FOV) of 21∘×12∘. After the optimization, the overall optical efficiency of the central field and the angular uniformity at the center exit pupil position increased from 0.9% and 66% to 3.1% and 80%, respectively.

Assessment of osteopontin as an early nephrotoxicity indicator in human renal proximal tubule cells and its application in evaluating lanthanum-induced nephrotoxicity.

Nephrotoxicity is a common adverse effect induced by various chemicals, necessitating the development of reliable toxicity screening models for nephrotoxicity assessment. In this study, we assessed a group of nephrotoxicity indicators derived from different toxicity pathways, including conventional endpoints and kidney tubular injury biomarkers such as clusterin (CLU), kidney injury molecule-I (KIM-1), osteopontin (OPN), and neutrophil gelatinase-associated lipocalin (NGAL), using HK-2 and induced pluripotent stem cells (iPSCs)-derived renal proximal tubular epithelial-like cells (PTLs). Among the biomarkers tested, OPN emerged as the most discerning and precise marker. The predictive potential of OPN was tested using a panel of 10 nephrotoxic and 5 non-nephrotoxic compounds. The results demonstrated that combining OPN with the half-maximal inhibitory concentration (IC50) enhanced the diagnostic accuracy in both cellular models. Additionally, PTLs cells showed superior predictive efficacy for nephrotoxicity compared to HK-2 cells in this investigation. The two cellular models were utilized to evaluate the nephrotoxicity of lanthanum. The findings indicated that lanthanum possesses nephrotoxic properties; however, the degree of nephrotoxicity was relatively low, consistent with the outcomes of in vivo experiments.

[Ursolic acid improved demyelination and interstitial fluid drainage disorders in schizophrenia mice].

OBJECTIVE: To unveil the pathological changes associated with demyelination in schizophrenia (SZ) and its consequential impact on interstitial fluid (ISF) drainage, and to investigate the therapeutic efficacy of ursolic acid (UA) in treating demyelination and the ensuing abnormalities in ISF drainage in SZ. METHODS: Female C57BL/6J mice, aged 6-8 weeks and weighing (20±2) g, were randomly divided into three groups: control, SZ model, and UA treatment. The control group received intraperitoneal injection (ip) of physiological saline and intragastric administration (ig) of 1% carboxymethylcellulose sodium (CMC-Na). The SZ model group was subjected to ip injection of 2 mg/kg dizocilpine maleate (MK-801) and ig administration of 1% CMC-Na. The UA treatment group underwent ig administration of 25 mg/kg UA and ip injection of 2 mg/kg MK-801. The treatment group received UA pretreatment via ig administration for one week, followed by a two-week drug intervention for all the three groups. Behavioral assessments, including the open field test and prepulse inhibition experiment, were conducted post-modeling. Subsequently, changes in the ISF partition drainage were investigated through fluorescent tracer injection into specific brain regions. Immunofluorescence analysis was employed to examine alterations in aquaporin 4 (AQP4) polarity distribution in the brain and changes in protein expression. Myelin reflex imaging using Laser Scanning Confocal Microscopy (LSCM) was utilized to study modifications in myelin within the mouse brain. Quantitative data underwent one-way ANOVA, followed by TukeyHSD for post hoc pairwise comparisons between the groups. RESULTS: The open field test revealed a significantly longer total distance [(7 949.39±1 140.55) cm vs. (2 831.01±1 212.72) cm, P < 0.001] and increased central area duration [(88.43±22.06) s vs. (56.85±18.58) s, P=0.011] for the SZ model group compared with the controls. The UA treatment group exhibited signifi-cantly reduced total distance [(2 415.80±646.95) cm vs. (7 949.39±1 140.55) cm, P < 0.001] and increased central area duration [(54.78±11.66) s vs. (88.43±22.06) s, P=0.007] compared with the model group. Prepulse inhibition test results demonstrated a markedly lower inhibition rate of the startle reflex in the model group relative to the controls (P < 0.001 for both), with the treatment group displaying significant improvement (P < 0.001 for both). Myelin sheath analysis indicated significant demyelination in the model group, while UA treatment reversed this effect. Fluorescence tracing exhibited a significantly larger tracer diffusion area towards the rostral cortex and reflux area towards the caudal thalamus in the model group relative to the controls [(13.93±3.35) mm2 vs. (2.79±0.94) mm2, P < 0.001 for diffusion area; (2.48±0.38) mm2 vs. (0.05±0.12) mm2, P < 0.001 for reflux area], with significant impairment of drainage in brain regions. The treatment group demonstrated significantly reduced tracer diffusion and reflux areas [(7.93±2.48) mm2 vs. (13.93±3.35) mm2, P < 0.001 for diffusion area; (0.50±0.30) mm2 vs. (2.48±0.38) mm2, P < 0.001 for reflux area]. Immunofluorescence staining revealed disrupted AQP4 polarity distribution and reduced AQP4 protein expression in the model group compared with the controls [(3 663.88±733.77) µm2 vs. (13 354.92±4 054.05) µm2, P < 0.001]. The treatment group exhibited restored AQP4 polarity distribution and elevated AQP4 protein expression [(11 104.68±3 200.04) µm2 vs. (3 663.88±733.77) µm2, P < 0.001]. CONCLUSION: UA intervention ameliorates behavioral performance in SZ mice, Thus alleviating hyperactivity and anxiety symptoms and restoring sensorimotor gating function. The underlying mechanism may involve the improvement of demyelination and ISF drainage dysregulation in SZ mice.

KLIF-associated cytoskeletal proteins in Trypanosoma brucei regulate cytokinesis by promoting cleavage furrow positioning and ingression.

Cytokinesis in the early divergent protozoan Trypanosoma brucei occurs from the anterior cell tip of the new-flagellum daughter toward the nascent posterior end of the old-flagellum daughter of a dividing biflagellated cell. The cleavage furrow ingresses unidirectionally along the preformed cell division fold and is regulated by an orphan kinesin named kinesin localized to the ingressing furrow (KLIF) that localizes to the leading edge of the ingressing furrow. Little is known about how furrow ingression is controlled by KLIF and whether KLIF interacts with and cooperates with other cytokinesis regulatory proteins to promote furrow ingression. Here, we investigated the roles of KLIF in cleavage furrow ingression and identified a cohort of KLIF-associated cytoskeletal proteins as essential cytokinesis regulators. By genetic complementation, we demonstrated the requirement of the kinesin motor activity, but not the putative tropomyosin domain, of KLIF in promoting furrow ingression. We further showed that depletion of KLIF impaired the resolution of the nascent posterior of the old-flagellar daughter cell, thereby stalking cleavage furrow ingression at late stages of cytokinesis. Through proximity biotinylation, we identified a subset of cytoskeleton-associated proteins (CAPs) as KLIF-proximal proteins, and functional characterization of these cytoskeletal proteins revealed the essential roles of CAP46 and CAP52 in positioning the cleavage furrow and the crucial roles of CAP42 and CAP50 in promoting cleavage furrow ingression. Together, these results identified multiple cytoskeletal proteins as cytokinesis regulators and uncovered their essential and distinct roles in cytokinesis.

A Spef1-interacting microtubule quartet protein in Trypanosoma brucei promotes flagellar inheritance by regulating basal body segregation.

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.

Fine mapping and candidate gene analysis of qFL-A12-5: a fiber length-related QTL introgressed from Gossypium barbadense into Gossypium hirsutum.

KEY MESSAGE: The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC4F3:5). A single segment substitution line (CSSL-106) screened from BC6F2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC7F2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length.

FAZ27 cooperates with FLAM3 and ClpGM6 to maintain cell morphology in Trypanosoma brucei.

The human parasite Trypanosoma brucei transitions from the trypomastigote form to the epimastigote form in the insect vector by repositioning its mitochondrial genome and flagellum-associated cytoskeleton. The molecular mechanisms underlying such changes in cell morphology remain elusive, but recent works demonstrated the involvement of three flagellar proteins, FLAM3, ClpGM6 and KIN-E, in this process by controlling the elongation of the flagellum attachment zone (FAZ). In this report, we identified a FAZ flagellum domain-localizing protein named FAZ27 and characterized its role in cell morphogenesis. Depletion of FAZ27 in the trypomastigote form caused major morphological changes and repositioning of the mitochondrial genome and flagellum-associated cytoskeleton, generating epimastigote-like cells. Furthermore, proximity biotinylation and co-immunoprecipitation identified FLAM3 and ClpGM6 as FAZ27-interacting proteins, and analyses of their functional interplay revealed an interdependency for assembly into the FAZ flagellum domain. Finally, we showed that assembly of FAZ27 occurred proximally, identical to the assembly pattern of other FAZ sub-domain proteins. Taken together, these results demonstrate a crucial role for the FAZ flagellum domain in controlling cell morphogenesis and suggest a coordinated assembly of all the FAZ sub-domains at the proximal end of the FAZ.

QTL mapping and candidate gene prediction for fiber yield and quality traits in a high-generation cotton chromosome substitution line with Gossypium barbadense segments.

Gossypium provides the foremost natural fiber for supporting the rapid development of the textile industry. Quantitative trait locus (QTL) mapping of fiber yield and quality traits is, thus, of great significance for providing a foundation for the genetic improvement of key target traits in cotton production. In this study, a superior chromosome segment substitution line (CSSL), MBI8255, with high yield and premium fiber quality characteristics was cultivated from the BC5F3:5 lineage derived from G. barbadense Hai1 and G. hirsutum CCRI36, and was chosen to construct a segregation population containing 123 F2 individuals with CCRI36. A total of 71 polymorphic SSR (simple sequence repeat) markers were identified based on a previous high-density linkage map, and 17 QTLs distributed on five chromosomes were detected, of which 10 QTLs for cotton yield explained 0.26-15.41% of phenotypic variations, while 7 QTLs for fiber quality explained 0.84-9.38% of phenotypic variations, separately containing four and one stable QTLs detected from over two environments. Among three identified QTL clusters, only the Chr19 QTL cluster harbored two stable and one unstable QTL for three different traits, and hence this significant region, which included 1546 genes, was subjected to functional enrichment and transcriptome expression analyses, ultimately screening eight candidate genes relevant to fiber development. This study not only provides useful information for the further fine-mapping and functional verification of candidate genes, but also offers a solid foundation for revealing the molecular mechanisms of fiber formation.

Genome-wide association study reveals that the cupin domain protein OsCDP3.10 regulates seed vigour in rice.

Seed vigour is an imperative trait for the direct seeding of rice. In this study, we examined the genetic regulation of seedling percentage at the early germination using a genome-wide association study in rice. One major quantitative trait loci qSP3 for seedling percentage was identified, and the candidate gene was validated as qSP3, encoding a cupin domain protein OsCDP3.10 for the synthesis of 52 kDa globulin. Disruption of this gene in Oscdp3.10 mutants reduced the seed vigour, including the germination potential and seedling percentage, at the early germination in rice. The lacking accumulation of 52 kDa globulin was observed in the mature grains of the Oscdp3.10 mutants. The significantly lower amino acid contents were observed in the mature grains and the early germinating seeds of the Oscdp3.10 mutants compared with those of wild-type. Rice OsCDP3.10 regulated seed vigour mainly via modulating the amino acids e.g. Met, Glu, His, and Tyr that contribute to hydrogen peroxide (H2 O2 ) accumulation in the germinating seeds. These results provide important insights into the application of seed priming with the amino acids and the selection of OsCDP3.10 to improve seed vigour in rice.

Portable autostereoscopic display based on multi-directional backlight.

A multi-directional backlight autostereoscopic display system with high resolution, low crosstalk, and motion parallax is developed in this paper. The proposed multi-directional backlight system is based on the Bragg mismatched reconstruction of volume holographic optical element (VHOE), and includes a set of light sources which are uniformly arrayed along one direction. Each light source produces its corresponding directional lighting to follow the human eye position detected by an eye tracker. Two scenarios are presented to build the multi-directional backlight system. The prism-type backlight system which guides the incident beam with a prism is relatively simple and easy to implement. The waveguide-type one which employs a transflective film to expand the incident light beam within the waveguide and modulate the intensity of the incident beam, is relatively thin and is applicable to large-area display. Two prototypes are built and the effectiveness of the proposed autostereoscopic display system is verified by the experimental results.

Cadmium chloride-induced apoptosis of HK-2 cells via interfering with mitochondrial respiratory chain.

Cadmium could induce cell apoptosis, probably related to the dysfunction of the mitochondrial respiratory chain. The human renal proximal tubule (HK-2) was used to explore the mechanism of mitochondrial respiratory chain dysfunction during apoptosis induced by cadmium chloride (CdCl2). Cell viability was evaluated by cell proliferation assay and different concentrations of 60, 80 and 100 µM were selected to evaluate the mitochondrial toxicity of CdCl2 respectively. Under the CdCl2 treatment for 24 h, the mitochondrial reactive oxygen species (ROS) of HK-2 cells increased and the superoxide dismutase (SOD) activity was inhibited at the above three concentrations separately. Both ATP content and mitochondrial membrane potential decreased significantly at 100 µM concentration. The levels of procaspase-3 and Bcl-2 had fallen in a concentration-dependent manner and Bax was significantly increased at 60, 80 and 100 µM concentration compared with no CdCl2 treatment respectively, which activated the mitochondrial apoptosis pathway. N-acetyl-cysteine (NAC) could partially resist CdCl2-induced cell apoptosis, while myxothiazol (Myx) promoted the process. Mitochondria relative alterations manifested as inhibition of complex III and V. In addition, both the quantity of mitochondrial coenzyme Q-binding protein CoQ10 homolog B (CoQ10B) and cytochrome c (Cyt c) had decreased significantly. Taken together, CdCl2 induced HK-2 apoptosis due to the mitochondrial respiratory chain dysfunction by reducing the CoQ10B level, offering a novel evaluating indicator for the environmental toxicity of CdCl2.

The CRK2-CYC13 complex functions as an S-phase cyclin-dependent kinase to promote DNA replication in Trypanosoma brucei.

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.

Maintenance of hook complex integrity and centrin arm assembly facilitates flagellum inheritance in Trypanosoma brucei.

Inheritance of the newly assembled flagellum in the human parasite Trypanosoma brucei depends on the faithful duplication and segregation of multiple flagellum-associated cytoskeletal structures, including the hook complex and its associated centrin arm. The biological functions of this unique hook complex-centrin arm assembly remain poorly understood. Here, we report a hook complex-associated protein named BOH2 that plays an essential role in promoting flagellum inheritance. BOH2 localizes to the hooked part of the hook complex by bridging the hook complex, the centrin arm, and the flagellum attachment zone filament. Depletion of BOH2 caused the loss of the shank part of the hook complex and its associated protein TbSmee1, disrupted the assembly of the centrin arm and the recruitment of centrin arm-associated protein CAAP1, inhibited the assembly of the flagellum attachment zone, and caused flagellum mispositioning and detachment. These results demonstrate crucial roles of BOH2 in maintaining hook complex integrity and promoting centrin arm formation and suggest that proper assembly of the hook complex-centrin arm structure facilitates flagellum inheritance.

Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei.

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

BOH1 cooperates with Polo-like kinase to regulate flagellum inheritance and cytokinesis initiation in Trypanosoma brucei.

Trypanosoma brucei possesses a motile flagellum that determines cell morphology and the cell division plane. Inheritance of the newly assembled flagellum during the cell cycle is controlled by the Polo-like kinase homolog TbPLK, which also regulates cytokinesis initiation. How TbPLK is targeted to its subcellular locations remains poorly understood. Here we report the trypanosome-specific protein BOH1 that cooperates with TbPLK to regulate flagellum inheritance and cytokinesis initiation in the procyclic form of T. brucei BOH1 localizes to an unusual sub-domain in the flagellum-associated hook complex, bridging the hook complex, the centrin arm and the flagellum attachment zone. Depletion of BOH1 disrupts hook-complex morphology, inhibits centrin-arm elongation and abolishes flagellum attachment zone assembly, leading to flagellum mis-positioning and detachment. Further, BOH1 deficiency impairs the localization of TbPLK and the cytokinesis regulator CIF1 to the cytokinesis initiation site, providing a molecular mechanism for its role in cytokinesis initiation. These findings reveal the roles of BOH1 in maintaining hook-complex morphology and regulating flagellum inheritance, and establish BOH1 as an upstream regulator of the TbPLK-mediated cytokinesis regulatory pathway.

Cadmium induces renal inflammation by activating the NLRP3 inflammasome through ROS/MAPK/NF-κB pathway in vitro and in vivo.

Cadmium (Cd) has been reported to induce kidney damage by triggering oxidative stress and inflammation. The NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome has been implicated a role in the pathogenesis of inflammation. However, the connection between Cd and NLRP3 inflammasome in the development of renal inflammation remains unknown. In this study, in vitro experiments based on the telomerase-immortalized human renal proximal-tubule epithelial cell line (RPTEC/TERT1) were carried out. Results revealed that CdCl2 (2-8 µM) increased ROS production and activated NLRP3, thereby enhancing secretion of IL-1ß and IL-18 (P < 0.05). Knock-down of NLRP3 rescued the RPTEC/TERT1 cells from Cd-induced inflammatory damage. Cd activated the MAPK/NF-κB signaling pathway in RPTEC/TERT1 cells (P < 0.05). In addition, treatment with N-acetylcysteine (NAC) improved inflammation and blocked the upregulation of the MAPK/NF-κB signaling pathway. Pre-treatment with MAPK and NF-κB inhibitors also suppressed NLRP3 inflammasome activation (P < 0.05). Moreover, CdCl2 (25-00 mg/L) stimulated the MAPK/NF-κB signaling pathway, activated the NLRP3 inflammasome, and increased inflammatory response (P < 0.05) leading to renal injury in rats. Exposure to cadmium elevated serum levels of NLRP3 and IL-1ß in populations (P < 0.05). Further analysis found that serum NLRP3 and IL-1ß levels were positively correlated with urine cadmium (UCd) and urine N-acetyl-ß-D-glucosaminidase (UNAG). Overall, Cd induced renal inflammation through the ROS/MAPK/NF-κB signaling pathway by activating the NLRP3 inflammasome. Our research thus provides new insights into the molecular mechanism that NLRP3 contributes to Cd-induced kidney damage.

A kinetochore-based ATM/ATR-independent DNA damage checkpoint maintains genomic integrity in trypanosomes.

DNA damage-induced cell cycle checkpoints serve as surveillance mechanisms to maintain genomic stability, and are regulated by ATM/ATR-mediated signaling pathways that are conserved from yeast to humans. Trypanosoma brucei, an early divergent microbial eukaryote, lacks key components of the conventional DNA damage-induced G2/M cell cycle checkpoint and the spindle assembly checkpoint, and nothing is known about how T. brucei controls its cell cycle checkpoints. Here we discover a kinetochore-based, DNA damage-induced metaphase checkpoint in T. brucei. MMS-induced DNA damage triggers a metaphase arrest by modulating the abundance of the outer kinetochore protein KKIP5 in an Aurora B kinase- and kinetochore-dependent, but ATM/ATR-independent manner. Overexpression of KKIP5 arrests cells at metaphase through stabilizing the mitotic cyclin CYC6 and the cohesin subunit SCC1, mimicking DNA damage-induced metaphase arrest, whereas depletion of KKIP5 alleviates the DNA damage-induced metaphase arrest and causes chromosome mis-segregation and aneuploidy. These findings suggest that trypanosomes employ a novel DNA damage-induced metaphase checkpoint to maintain genomic integrity.