Han X et al. (2010) Nat Cell Biol "Meiotic kinetochores get pushed aside by a CLS act."

Srayko M, Han X

[Nat Cell Biol, 2010]

Kinetochores link microtubules to DNA and provide force critical for chromosome segregation in mitosis. New data show that kinetochores are not necessary for acentrosomal meiotic chromosome segregation in Caenorhabditis elegans. Instead, CLS-2 (CLASP) generates a mid-zone bundle of microtubules that are suggested to act in pushing the chromosomes apart.

Han X et al. (2009) Genetics "The role of protein phosphatase 4 in regulating microtubule severing in the Caenorhabditis elegans embryo."

Sugimoto A, Mains PE, Han X, Pintard L, Birmingham CL, Gomes JE

[Genetics, 2009]

MEI-1 is the catalytic subunit of the Caenorhabditis elegans "katanin" microtubule-severing complex, which is required for meiotic spindle formation. However, MEI-1 must be inactivated with precise kinetics after the completion of meiosis to allow formation of the first mitotic spindle. Recent work has demonstrated that post-meiotic MEI-1 undergoes ubiquitin-dependent degradation mediated by two independent pathways. Here we describe another level of MEI-1 regulation involving the protein phosphatase 4 (PP4) complex. The PP4 R1 regulatory subunit ppfr-1 was identified in an RNAi screen for suppressors of a mei-1(gf) allele that is refractory to post-meiotic degradation. RNAi to the PP4 catalytic subunit PPH-4.1 or to the alpha4 regulatory PPFR-4 also suppressed lethality of ectopic MEI-1. These results suggests that PP4(+) activates MEI-1, therefore loss of PP4 decreases ectopic MEI-1(gf) activity. PPH-4.1 and MEI-1 co-immunoprecipitate with one another, indicating that the PP4 complex likely regulates MEI-1 activity directly rather than through an intermediate. The ppfr-1 mutant has subtle meiotic defects indicating that PPFR-1 also regulates MEI-1 during meiosis. MBK-2 is the only kinase known to phosphorylate MEI-1, thereby triggering post-meiotic MEI-1 degradation. However, genetic interactions between PP4 and mbk-2 were not consistent with an antagonistic relationship between the phosphatase and kinase. Additionally, reducing PP4 in mei-1(gf) did not change the level or localization of post-meiotic MEI-1. Thus by making use of a genetic background where MEI-1 is ectopically expressed, we have uncovered a third mechanism of MEI-1 regulation, one based on phosphorylation but independent of degradation. The redundant regulatory pathways likely contribute in different ways to the rapid and precise post-meiotic inactivation of MEI-1 microtubule severing activity.

Han, X. et al. (2019) International Worm Meeting "Regulation of GLP-1/Notch signaling in C. elegans Germline Stem Cells by Protein Interactions."

Han, X., Zhang, D., Hansen, D.

[International Worm Meeting, 2019]

Reproductive fitness requires a balance between stem cell self-renewal and differentiation. In the C. elegans germline, GLP-1/Notch signaling controls this balance. In the distal region of the germline, high GLP-1/Notch levels promote proliferation. GLP-1 signaling decreases moving proximally, allowing the GLD-1/NOS-3 and GLD-2/GLD-3 redundant pathways to be gradually activated, directing cells to enter meiosis/differentiation. Therefore, a stem cell's decision to self-renew or differentiate is governed by the establishment of an opposing gradient of GLP-1 signaling and the GLD-1/GLD-2 pathways along the distal-proximal axis of the germline. Previously, GLP-1 has been shown to indirectly repress the translation of gld-1 and gld-3. However, recent data from our lab suggests that protein-protein interactions between GLP-1 and GLD-1/GLD2 could result in the repression of their normal regulatory roles. The possibility of a direct physical interaction reveals a new level of regulation that was previously unknown. We hypothesize that GLP-1 physically interacts with GLD-1/GLD-2 to rapidly establish opposing domains. To test this, we will determine the protein regions and amino acids required for protein interactions using a yeast two-hybrid system. Results obtained thus far indicate that GLP-1(intra) interacts with the N-terminal regions of GLD-1 and GLD-2. In GLD-1, this region contains a QUA1 domain that's involved in GLD-1 dimerization. In GLD-2, this region is a germline specific isoform. Future work will focus on how this protein interaction may negatively regulate GLP-1/Notch or the GLD-1/GLD-2 pathways. This research will contribute to a better understanding of the precise regulatory mechanisms governing stem cell proliferation.

Han X et al. (2021) Lab Chip "A polymer index-matched to water enables diverse applications in fluorescence microscopy."

Potarazu D, Dai Q, Shroff H, Moyle MW, O'Neill KM, Han X, Sun Y, Albrecht DR, Christensen R, Su Y, Probst R, Vishwasrao HD, Pommier Y, Giniger E, Morgan NY, White H, Xu S, Huang SY

[Lab Chip, 2021]

We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

Han, X et al. (2009) International Worm Meeting "The mechanisms of the microtubule-depolymerizing kinesin in regulating MT dynamics in the early C. elegans embryos."

Han, X, Srayko, M

[International Worm Meeting, 2009]

Microtubules (MTs) are one of the major cytoskeletal components and they are essential for fundamental cellular processes including mitosis, cytokinesis, and vesicle transport. The temporal and spatial regulation of MT dynamics is key to our understanding of their cellular functions. I am specifically interested in how MT growth is controlled at centrosomes, the epicenters for MT outgrowth in the cell. Recently, the protein KLP-7 (kinesin-like protein) was implicated in regulating MT outgrowth at the centrosome (Srayko, et al., 2005, Schlaitz et al., 2007). KLP-7 is a member of MT-depolymerizing proteins. When the KLP-7 protein is removed, twice the number of MTs grow out of the centrosome. Work from the vertebrate homologues suggests that KLP-7 is turned off via phosphorylation by Aurora A kinase (Zhang X. et al., 2008), and there is indirect evidence in C. elegans to support a mechanism whereby KLP-7 is negatively regulated via the Aurora A kinase (Schlaitz et al., 2007). The KLP-7 protein has 13 identifiable Aurora kinase sites, in regions similar to the vertebrate homologues. A major focus of my work is to determine how phosphorylation/dephosphorylation of KLP-7''s Aurora kinase sites affects its intracellular location and/or function with respect to MT outgrowth at the centrosome. We are currently performing a structure-function analysis to determine which Aurora sites are required for KLP-7''s intracellular location and its depolymerase activity at the centrosome. Results obtained thus far indicate that mutating one of the C-terminal Aurora sites from serine to glutamic acid (to mimic constitutive phosphorylation) interferes with KLP-7 localization at the centrosomes. Experiments are underway to measure MT outgrowth in embryos expressing different phosphorylated forms of KLP-7. In parallel, we are pursuing the identification and characterization of putative KLP-7-interacting proteins through co-immunoprecipitations and yeast 2-hybrid.

Han X et al. (2024) Sci Total Environ "Mitochondria UPR stimulation by pelargonidin-3-glucoside contributes to ameliorating lipid accumulation under copper exposure."

Bian C, Gao Y, Han X, Yan F, Chen X, Chen W

[Sci Total Environ, 2024]

Intensification of copper pollution in the environment has led to its excessive accumulation in humans, causing oxidative stress and lipid metabolism disorders. It is necessary to look for effective targets and safe methods to alleviate copper toxicity. Pelargonidin-3-glucoside (Pg3G) is a natural anthocyanin with metal ion chelating ability and multiple physiological activities. In this study, lipid accumulation was investigated under copper exposure in Caenorhabditis elegans which can be improved by Pg3G. Transcriptome analysis revealed that differentially expressed genes are enriched in lipid metabolism and protein folding/degradation. Pg3G activates mitochondrial unfold protein response (UPR^mt) to mitigate mitochondrial damage caused by copper and regulates the expression of genes involved in lipid absorption, transport, and synthesis, thereby reducing lipid levels in C. elegans. This improvement disappears in the ubl-5 knockout strain, indicating that ubl-5 is one target of Pg3G. Meanwhile, in HepG2 cells, Pg3G enhances the cellular antioxidant capacity by activating UPR^mt for maintaining mitochondrial homeostasis, followed by inhibition of excessive lipid accumulation. Overall, these results suggested that UPR^mt activation can regard as a strategy for mitigating lipid disorders induced by copper and Pg3G with excellent ability to resist oxidative stress specially targeted for ubl-5 has a promising application in controlling copper contamination.

Han X et al. (2015) PLoS One "The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans."

Sykes EM, Han X, Adames K, Srayko M

[PLoS One, 2015]

Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase.

Han, X. et al. (2011) International Worm Meeting "Mechanisms Involved in Regulating the Activity and Localization of a Microtubule-depolymerizing Kinesin in the One-cell Stage C. elegans Embryo."

Cheung, K., Srayko, M., Han, X.

[International Worm Meeting, 2011]

Microtubules are required for multiple cellular processes including mitosis, cytokinesis, and vesicle transportation. Factors that regulate microtubule dynamics in the cell help determine the final form and precise cellular role of many cytoskeletal structures. Among the known modulators of microtubule dynamics, we are specifically interested in the microtubule-depolymerizing kinesins of the kinesin-13 family, such as KLP-7 (CeMCAK). In C. elegans, KLP-7 locates to the kinetochore and the centrosome. It has been implicated in regulating microtubule outgrowth at the centrosome because loss of KLP-7 results in an increase in the number of centrosomal microtubules (Srayko et al., 2005, Schlaitz et al., 2007). Extensive work from other labs on the vertebrate homologues of KLP-7 indicates that they are negatively regulated through phosphorylation by the Aurora kinases (Andrews et al., 2004, Lan et al., 2004, Ohi et al., 2004, Schlaitz et al., 2007, Zhang et al., 2007 and 2008). In order to understand how KLP-7 is regulated in the cell, we tested the possibility that Aurora kinases are directly involved. 2D gel-electrophoresis revealed an alteration in the ratio of potential KLP-7 phosphorylation variants in lysates from either air-1(RNAi) (Aurora A-depleted) or air-2(RNAi) (Aurora B-depleted) embryos, compared to wild type. Furthermore, we found that both AIR-1 and AIR-2 kinases phosphorylate KLP-7 in vitro. We used a combination of mass spectrometry, similarity to data on the vertebrate homologues, and an Aurora kinase phospho-site prediction algorithm to identify potential in vivo phosphorylation sites within KLP-7 (Zhou et al., 2004). We are currently performing a structure-function analysis to determine which putative Aurora sites are required for KLP-7's intracellular location and/or its depolymerase activity at the centrosome. Results obtained thus far indicate that mutating one of the C-terminal Aurora sites from serine to glutamic acid (to mimic constitutive phosphorylation) or to alanine (to mimic non-phosphorylation) interferes with KLP-7 function but not its ability to target to centrosomes or kinetochores.

Han X et al. (2017) Ecotoxicol Environ Saf "Investigating the performance of three modified activated sludge processes treating municipal wastewater in organic pollutants removal and toxicity reduction."

Zhou MX, Zuo YT, Liu AL, Zhang J, Hu Y, Han X, Lu WQ, Chen M, Tang F

[Ecotoxicol Environ Saf, 2017]

This study investigated the treatment performance of three types of modified activated sludge processes, i.e., anoxic/oxic (A/O), anaerobic/anoxic/oxic (A2/O) and oxidation ditch process, in treating municipal wastewater by measuring physicochemical and spectroscopic parameters, and the toxicity of the influents and effluents collected from 8 full-scale municipal wastewater treatment plants (MWTPs). The relationships between spectroscopic and physicochemical parameters of the wastewater samples and the applicability of the nematode Caenorhabditis elegans (C. elegans) bioassays for the assessment of the toxic properties of municipal wastewater were also evaluated. The results indicated that the investigated MWTPs employing any of A/O, A2/O and oxidation ditch processes could effectively control the discharge of major wastewater pollutants including biochemical oxygen demand (BOD), chemical oxygen demand, nitrogen and phosphorus. The oxidation ditch process appeared to have the advantage of removing tyrosine-like substances and presented slightly better removal efficiency of tryptophan-like fluorescent (peak T) substances than the A/O and A2/O processes. Both ultraviolet absorbance at 254nm and peak T may be used to characterize the organic load of municipal wastewater, and peak T can be adopted as a gauge of the BOD removal efficacy of municipal wastewater treatment. Using C. elegans-based oxygen consumption rate assay for monitoring municipal wastewater toxicity deserves further investigations.

Han H-P et al. (1992) Worm Breeder's Gazette "Expression and Localization of the cha-1 and unc-17 Gene Products"

Rand JB, Duerr JS, Han H-P

[Worm Breeder's Gazette, 1992]

EXPRESSION AND LOCALIZATION OF THE cha-l AND unc-17 GENE PRODUCTS He-ping Han, Janet Duerr, and Jim Rand, Oklahoma Medlcal Research Foundation, Oklahoma Clty, OK 73104