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.

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.

Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is one of the most prevalent metabolic syndromes worldwide. However, no approved pharmacological treatments are available for MAFLD. Chenpi, one kind of dried peel of citrus fruits, has traditionally been utilized as a medicinal herb for liver diseases. Didymin is a newly identified oral bioactive dietary flavonoid glycoside derived from Chenpi. In this study, we investigated the therapeutic potential of Didymin as an anti-MAFLD drug and elucidated its underlying mechanisms. METHODS: High-fat diet (HFD)-induced MAFLD mice and alpha mouse liver 12 (AML12) cells were utilized to evaluate the effects and mechanisms of Didymin in the treatment of MAFLD. Liver weight, serum biochemical parameters, and liver morphology were examined to demonstrate the therapeutic efficacy of Didymin in MAFLD treatment. RNA-seq analysis was performed to identify potential pathways that could be affected by Didymin. The impact of Didymin on Sirt1 was corroborated through western blot, molecular docking analysis, microscale thermophoresis (MST), and deacetylase activity assay. Then, a Sirt1 inhibitor (EX-527) was utilized to confirm that Didymin alleviates MAFLD via Sirt1. Western blot and additional assays were used to investigate the underlying mechanisms. RESULTS: Our results suggested that Didymin may possess therapeutic potential against MAFLD in vitro and in vivo. By promoting Sirt1 expression as well as directly binding to and activating Sirt1, Didymin triggers downstream pathways that enhance mitochondrial biogenesis and function while reducing apoptosis and enhancing lipophagy. CONCLUSIONS: These suggest that Didymin could be a promising medication for MAFLD treatment. Furthermore, its therapeutic effects are mediated by Sirt1.

Exosomes derived from mesenchymal stem cells attenuate diabetic kidney disease by inhibiting cell apoptosis and epithelial-to-mesenchymal transition via miR-424-5p.

Diabetic kidney disease (DKD) is well-acknowledged as one of the most common complications in diabetes mellitus. Recent studies have demonstrated the promising role of mesenchymal stem cell-derived exosomes (MSC-exos) as a cell-free treatment strategy for DKD. The present study sought to investigate the therapeutic potential and the underlying mechanisms of MSC-exos in DKD. The authentication of MSC-exos was validated by western blot, transmission electron microscope (TEM), and nanosight tracking analysis (NTA). Apoptosis was detected by western blot, TUNEL staining, and flow cytometry. Epithelial-to-mesenchymal transition (EMT) was evaluated by western blot and immunofluorescence. The relationship between miR-424-5p and Yes-associated protein 1 (YAP1) was revealed by dual luciferase reporter assay. We observed that MSC-exos could attenuate DKD by decreasing cell apoptosis and inhibiting epithelial-to-mesenchymal transition (EMT) in diabetic kidneys in db/db mice. Besides, we documented that MSC-exos could reverse high glucose-induced apoptosis and EMT in HK2 cells. Interestingly, miR-424-5p derived from MSC-exos could inhibit YAP1 activation in HK2 cells, resulting in alleviation of high glucose-induced cell apoptosis and EMT. Our study provides novel insights into MSC-exos-mediated protective effect in DKD. MSC-exos could inhibit high glucose-induced apoptosis and EMT through miR-424-5p targeting of YAP1.

cGAS-STING mediates cytoplasmic mitochondrial-DNA-induced inflammatory signal transduction during accelerated senescence of pancreatic ß-cells induced by metabolic stress.

Type 2 diabetes mellitus (T2DM) is an age-related disease characterized by impaired pancreatic ß cell function and insulin resistance. Recent studies have shown that the accumulation of senescent ß cells under metabolic stress conditions leads to the progression of T2DM, while senolysis can improve the prognosis. However, the specific mechanism of ß cell senescence is still unclear. In this study, we found that the increased load of senescence pancreatic ß cells in both older mice and obese mice induced by high-fat diet (HFD) (DIO mice) was accompanied by activation of the Cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway and using cGAS or STING small interfering RNA or STING inhibitor C176 to downregulate this pathway reduced the senescence-associated secretion profile (SASP) and senescence of Min6 cells treated with palmitic acid or hydrogen peroxide. C176 intervention in DIO mice also significantly reduced the inflammation and senescence of the islets, thereby protecting the function of pancreatic ß cell and glucose metabolism. Our study further revealed that mitochondrial DNA (mtDNA) leakage under metabolic stress conditions was critical for the activation of the cGAS-STING pathway, which can be reversed by the mtDNA depleting agent ethidium bromide. Consistently, mtDNA leakage was more severe in older mice and was accelerated by a chronic HFD. In conclusion, we demonstrate that cytoplasmic mtDNA activates the cGAS-STING pathway to mediate SASP during the accelerated senescence of pancreatic ß-cells induced by metabolic stress, and this process can be downregulated by the STING inhibitor C176.

Puerarin ameliorates metabolic dysfunction-associated fatty liver disease by inhibiting ferroptosis and inflammation.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is frequently linked to type 2 diabetes mellitus (T2DM), and both conditions exacerbate the progression of the other. However, there is currently no standardized treatment or drug for MAFLD. In this study, A MAFLD animal model through a high-fat diet (HFD) along with administration of streptozotocin (STZ), and palmitic acid (PA)-induced AML12 cells were treated by puerarin. The objective of this study was to assess the therapeutic effect of puerarin, a flavonoid substance that possesses various pharmacological properties, on MAFLD. The results showed that puerarin administration enhanced glucose tolerance and insulin sensitivity, while also mitigating liver dysfunction and hyperlipidemia in MAFLD mice. Moreover, puerarin attenuated oxidative stress levels and inflammation in the liver. Transmission electron microscopy and Western blot analysis indicated that puerarin inhibited ferroptosis in vivo. Further mechanistic investigations revealed that puerarin upregulated SIRT1 expression, increased nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels, and facilitated translocation into the nucleus. The protective effect of puerarin on PA-induced AML12 cells was diminished by the utilization of EX-527 (a SIRT1 inhibitor) and Nrf2 siRNA. Overall, the results demonstrate that puerarin ameliorates MAFLD by suppressing ferroptosis and inflammation via the SIRT1/Nrf2 signaling pathway. The results emphasize the possible medicinal application of puerarin for managing MAFLD.

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.

Melatonin prevents diabetes-associated cognitive dysfunction from microglia-mediated neuroinflammation by activating autophagy via TLR4/Akt/mTOR pathway.

Cognitive dysfunction often occurs in diabetes mellitus patients. This study aimed to investigate the efficacy of melatonin (MLT) in improving diabetes-associated cognitive decline and the underlying mechanism involved. Type 2 diabetic mice and palmitic acid (PA)-stimulated BV-2 cells were treated by MLT, and the potential mechanisms among MLT, cognition, and autophagy were explored. The results showed that type 2 diabetic mice showed obvious learning and memory impairments in the Morris water maze test compared with normal controls, which could be ameliorated by MLT treatment. Meanwhile, MLT administration significantly improved neuroinflammation and regulated microglial apoptosis. Furthermore, autophagy inhibitor 3-methyladenine (3-MA) increased the microglial inflammation and apoptosis, indicating that the treatment effect of MLT was mediated by autophagy. Lastly, MLT treatment significantly decreased the levels of toll-like receptors 4 (TLR4), phosphorylated-protein kinase B (Akt), and phosphorylated-mechanistic target of rapamycin (mTOR), indicating that blocking TLR4/Akt/mTOR pathway might be an underlying basis for the anti-inflammatory and anti-apoptosis effects of MLT. Collectively, our study suggested that MLT could improve learning and memory in type 2 diabetic mice by activating autophagy via the TLR4/Akt/mTOR pathway, thereby inhibiting neuroinflammation and microglial apoptosis.

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.

Mesenchymal stem cell-conditioned medium improved mitochondrial function and alleviated inflammation and apoptosis in non-alcoholic fatty liver disease by regulating SIRT1.

Non-alcoholic fatty liver disease (NAFLD), an emerging risk factor for diabetes, is now recognized as the most common liver disease worldwide. Mesenchymal stem cells (MSCs), a promising tool in regenerative medicine, release abundant molecules into the conditioned medium (CM). Increasing evidence showed that MSC-CM is beneficial for diabetes-associated NAFLD. However, the mechanism of how MSC-CM improves NAFLD remains uncertain. In this study, to determine the effects of MSC-CM on NAFLD, streptozotocin (STZ) and high-fat diet (HFD) induced T2DM mice model and palmitic acid (PA)-stimulated L-O2 cells were used and treated with MSC-CM. Our results demonstrated that MSC-CM improved insulin resistance in diabetic mice, amended the pathological structure of the liver, enhanced the liver's total antioxidant capacity and mitochondrial function, reduced inflammation and cell apoptosis. We further verified that SIRT1 played a key role in mediating the protective effect of MSC-CM. These findings provide novel evidence that MSC-CM has the potential to treat T2DM patients with NAFLD clinically.

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.

The trypanosome-specific proteins FPRC and CIF4 regulate cytokinesis initiation by recruiting CIF1 to the cytokinesis initiation site.

The evolutionarily early divergent human parasite Trypanosoma brucei proliferates through binary cell fission in both its tsetse fly vector and mammalian host. The parasite divides unidirectionally along the longitudinal cell axis from the anterior cell tip toward the posterior cell tip through a mechanism distinct from that in the cells of its human host. Initiation of cytokinesis in T. brucei is regulated by two evolutionarily conserved protein kinases, the Polo-like kinase TbPLK and the Aurora B kinase TbAUK1, and a cohort of trypanosome-specific proteins, including the three cytokinesis initiation factors CIF1, CIF2, and CIF3. Here, using RNAi, in situ epitope tagging of proteins, GST pulldown, and coimmunoprecipitation assays, and immunofluorescence and scanning electron microscopy analyses, we report the identification and functional characterization of two trypanosome-specific proteins, flagellum attachment zone tip-localizing protein required for cytokinesis (FPRC) and CIF4. We found that the two proteins colocalize to the distal tips of the new and the old flagellum attachment zones and are required for cytokinesis initiation. Knockdown of FPRC or CIF4 disrupted the localization of CIF1, suggesting that they function upstream of CIF1. Moreover, depletion of CIF4 abolished FPRC localization, indicating that CIF4 acts upstream of FPRC. Together, these results identify two new cytokinesis regulators in T. brucei and integrate them into the CIF1-mediated cytokinesis regulatory pathway. These findings highlight the existence of a cytokinesis pathway in T. brucei that is different from that of its mammalian host and therefore suggest that cytokinesis in T. brucei could potentially be exploited as a new drug target.

Open eyes and closed eyes elicit different temporal properties of brain functional networks.

The eyes are our windows to the brain. There are differences in brain activity between people who have their eyes closed (EC) and eyes open (EO). Previous studies focused on differences in brain functional properties between these eyes conditions based on an assumption that brain activity is a static phenomenon. However, the dynamic nature of the brain activity in different eyes conditions is still unclear. In this study, we collected resting-state fMRI data from 21 healthy subjects in the EC and EO conditions. Using a sliding time window approach and a k-means clustering algorithm, we calculated the temporal properties of dynamic functional connectivity (dFC) states in the eyes conditions. We also used graph theory to estimate the dynamic topological properties of functional networks in the two conditions. We detected two dFC states, a hyper-connected State 1 and a hypo-connected State 2. We showed the following results: (i) subjects in the EC condition stayed longer in the hyper-connected State 1 than those in the EO; (ii) subjects in the EO condition stayed longer in the hypo-connected State 2 than those in the EC; and (iii) the dFC state transformed into the other state more frequently during EC than during EO. We also found the variance of the characteristic path length was higher during EC than during EO in the hyper-connected State 1. These results indicate that brain activity may be more active and unstable during EC than during EO. Our findings may provide insights into the dynamic nature of the resting-state brain and could be a useful reference for future rs-fMRI studies.

Functional analyses of an axonemal inner-arm dynein complex in the bloodstream form of Trypanosoma brucei uncover its essential role in cytokinesis initiation.

The flagellated eukaryote Trypanosoma brucei alternates between the insect vector and the mammalian host and proliferates through an unusual mode of cell division. Cell division requires flagellum motility-generated forces, but flagellum motility exerts distinct effects between different life cycle forms. Motility is required for the final cell abscission of the procyclic form in the insect vector, but is necessary for the initiation of cell division of the bloodstream form in the mammalian host. The underlying mechanisms remain elusive. Here we carried out functional analyses of a flagellar axonemal inner-arm dynein complex in the bloodstream form and investigated its mechanistic role in cytokinesis initiation. We showed that the axonemal inner-arm dynein heavy chain TbIAD5-1 and TbCentrin3 form a complex, localize to the flagellum, and are required for viability in the bloodstream form. We further demonstrated the interdependence between TbIAD5-1 and TbCentrin3 for maintenance of protein stability. Finally, we showed that depletion of TbIAD5-1 and TbCentrin3 arrested cytokinesis initiation and disrupted the localization of multiple cytokinesis initiation regulators. These findings identified the essential role of an axonemal inner-arm dynein complex in cell division, and provided molecular insights into the flagellum motility-mediated cytokinesis initiation in the bloodstream form of T. brucei.

Faithful chromosome segregation in Trypanosoma brucei requires a cohort of divergent spindle-associated proteins with distinct functions.

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.

The CIF1 protein is a master orchestrator of trypanosome cytokinesis that recruits several cytokinesis regulators to the cytokinesis initiation site.

To proliferate, the parasitic protozoan Trypanosoma brucei undergoes binary fission in a unidirectional manner along the cell's longitudinal axis from the cell anterior toward the cell posterior. This unusual mode of cell division is controlled by a regulatory pathway composed of two evolutionarily conserved protein kinases, Polo-like kinase and Aurora B kinase, and three trypanosome-specific proteins, CIF1, CIF2, and CIF3, which act in concert at the cytokinesis initiation site located at the distal tip of the newly assembled flagellum attachment zone (FAZ). However, additional regulators that function in this cytokinesis signaling cascade remain to be identified and characterized. Using proximity biotinylation, co-immunofluorescence microscopy, and co-immunoprecipitation, we identified 52 CIF1-associated proteins and validated six CIF1-interacting proteins, including the putative protein phosphatase KPP1, the katanin p80 subunit KAT80, the cleavage furrow-localized proteins KLIF and FRW1, and the FAZ tip-localized proteins FAZ20 and FPRC. Further analyses of the functional interplay between CIF1 and its associated proteins revealed a requirement of CIF1 for localization of a set of CIF1-associated proteins, an interdependence between KPP1 and CIF1, and an essential role of katanin in the completion of cleavage furrow ingression. Together, these results suggest that CIF1 acts as a master regulator of cytokinesis in T. brucei by recruiting a cohort of cytokinesis regulatory proteins to the cytokinesis initiation site.

The trypanosome-specific protein CIF3 cooperates with the CIF1 protein to promote cytokinesis in Trypanosoma brucei.

Cytokinesis, the terminal step in cell division, in the protist human pathogen Trypanosoma brucei occurs along the longitudinal axis from the anterior tip of the new flagellum attachment zone (FAZ) toward the posterior cell tip. This process is regulated by a signaling cascade composed of the Polo-like kinase homolog TbPLK, the Aurora B kinase homolog TbAUK1, and the trypanosome-specific CIF1-CIF2 protein complex. However, the regulatory mechanism and the signaling pathway for this unusual mode of cytokinesis remain poorly understood. Here, we report another trypanosome-specific protein assembly, the CIF1-CIF3 complex, and its essential role in cytokinesis initiation. Through biochemical and genetic approaches, we demonstrate that CIF3 interacts with CIF1 in a TbPLK-dependent manner and maintains CIF1 localization at the new FAZ tip. Conversely, CIF1 maintains CIF3 stability at the new FAZ tip. We further show that TbPLK is required for CIF3 localization and that CIF3 is necessary for targeting TbAUK1 to the new FAZ tip during anaphase. These results suggest that two trypanosome-specific CIF1-containing protein complexes cooperate with the evolutionarily conserved Polo-like kinase and Aurora B kinase to promote cytokinesis in T. brucei.

Functional analyses of the CIF1-CIF2 complex in trypanosomes identify the structural motifs required for cytokinesis.

Cytokinesis in trypanosomes occurs uni-directionally along the longitudinal axis from the cell anterior towards the cell posterior and requires a trypanosome-specific CIF1-CIF2 protein complex. However, little is known about the contribution of the structural motifs in CIF1 and CIF2 to complex assembly and cytokinesis. Here, we demonstrate that the two zinc-finger motifs but not the coiled-coil motif in CIF1 are required for interaction with the EF-hand motifs in CIF2. We further show that localization of CIF1 depends on the coiled-coil motif and the first zinc-finger motif and that localization of CIF2 depends on the EF-hand motifs. Deletion of the coiled-coil motif and mutation of either zinc-finger motif in CIF1 disrupts cytokinesis. Furthermore, mutation of either zinc-finger motif in CIF1 mislocalizes CIF2 to the cytosol and destabilizes CIF2, whereas deletion of the coiled-coil motif in CIF1 spreads CIF2 over to the new flagellum attachment zone and stabilizes CIF2. Together, these results uncover the requirement of the coiled-coil and zinc-finger motifs for CIF1 function in cytokinesis and for CIF2 localization and stability, providing structural insights into the functional interplay between the two cytokinesis regulators.

Aldosterone induced up-expression of ICAM-1 and ET-1 in pancreatic islet endothelium may associate with progression of T2D.

Previous studies have demonstrated that excess aldosterone impairs glucose metabolism. However, the underlying mechanism is still misty. Aldosterone has been proved a risk factor of fibrosis and inflammation. And the histology of islets from patients with type 2 diabetes (T2D) also displays inflammation and fibrosis. But it is unclear whether aldosterone has direct impact on islet inflammation and fibrosis in T2D. Islet endothelium plays a significant role in the maintenance of islet beta cell function and has a close relationship with islet fibrosis and inflammation. Therefore, we focused on the effect of aldosterone on the islet endothelium. In this study, we utilized a diabetic db/db mouse model and examined serum aldosterone levels, islet macrophages infiltration, and islet fibrosis. After we confirmed that there was an increased expression of intercellular cell adhesion molecule-1 (ICAM-1) and endothelin-1 (ET-1) in islet of diabetic mice compared with wild type mice. We next determined that aldosterone increased expression of ICAM-1 and ET-1 in both mRNA and protein levels in islet endothelium in vitro. And then we tested the expression of mineralocorticoid receptor (MR) in islet endothelium in vitro and in vivo. Our results showed that aldosterone can up-regulate the expression levels of ICAM-1 and ET-1 through MR. These findings suggest excess aldosterone might participate in islet inflammation and fibrosis in T2D.

CRL4WDR1 Controls Polo-like Kinase Protein Abundance to Promote Bilobe Duplication, Basal Body Segregation and Flagellum Attachment in Trypanosoma brucei.

The Polo-like kinase homolog in Trypanosoma brucei, TbPLK, plays essential roles in basal body segregation, flagellum attachment and cytokinesis. The level of TbPLK protein is tightly controlled, but the underlying mechanism remains elusive. Here, we report a Cullin-RING ubiquitin ligase composed of Cullin4, the DNA damage-binding protein 1 homolog TbDDB1 and a WD40-repeat protein WDR1 that controls TbPLK abundance in the basal body and the bilobe. WDR1, through its C-terminal domain, interacts with the PEST motif in TbPLK and, through its N-terminal WD40 motif, binds to TbDDB1. Depletion of WDR1 inhibits bilobe duplication and basal body segregation, disrupts the assembly of the new flagellum attachment zone filament and detaches the new flagellum. Consistent with its role in TbPLK degradation, depletion of WDR1 causes excessive accumulation of TbPLK in the basal body and the bilobe, leading to continuous phosphorylation of TbCentrin2 in the bilobe at late cell cycle stages. Together, these results identify a novel WD40-repeat protein as a TbPLK receptor in the Cullin4-DDB1 ubiquitin ligase complex for degrading TbPLK in the basal body and the bilobe after the G1/S cell cycle transition, thereby promoting bilobe duplication, basal body separation and flagellum-cell body adhesion.