Han, Xue et al. (2013) International Worm Meeting "Regulation of C. elegans MCAK by Aurora Kinase Phosphorylation."

Srayko, Martin, Han, Xue

[International Worm Meeting, 2013]

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 mitosis, C. elegans KLP-7 locates to the kinetochore and the centrosome, and it functions to regulate spindle checkpoint and microtubule outgrowth at the centrosome. During female meiosis, KLP-7 localizes to chromosomes, the chromosomal passenger complex region and the spindle poles in metaphase, and localizes to chromosomes in anaphase and telophase. KLP-7 depletion resulted in an excessive microtubule network near the meiotic spindle and the cortex. Extensive work on the vertebrate homologues of KLP-7 indicates that they are negatively regulated through phosphorylation by the Aurora kinases. In order to understand how KLP-7 is regulated in worm embryos, 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 phosphorylated KLP-7 in vitro. We used a combination of in vitro kinase assays and an Aurora kinase phospho-site prediction algorithm to identify potential in vivo phosphorylation sites within KLP-7. Using in vitro kinase assays, we found that AIR-1 and AIR-2 kinases phosphorylate N- and C-terminal domains of KLP-7. We are also 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 S546 at the C-terminus from serine to glutamic acid (to mimic constitutive phosphorylation) or alanine (to mimic non-phosphorylation) interferes with KLP-7 function but not its ability to target to centrosomes or kinetochores.

Han, Xue et al. (2021) International Worm Meeting "Regulation of GLP-1/Notch signaling in C. elegans Germline Stem Cells by Protein Interactions"

Zhang, Dan, Smit, Ryan, Han, Xue, Schedl, Tim, Hansen, Dave

[International Worm Meeting, 2021]

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, GLD-2/GLD-3 and SCFPROM-1 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/SCFPROM-1 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. We hypothesize that GLP-1 physically interacts with GLD-1/GLD-2 to rapidly establish opposing domains. To test this, we determined the protein regions 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. Current work focuses on how this protein interaction may negatively regulate GLP-1/Notch or the GLD-1/GLD-2 pathways. One approach is to analyze GLP-1 signaling in germlines with ectopic expression of the N termini of GLD-1/2 domains. While GLD-2N was able to be ubiquitously expressed, GLD-1N localization largely resembles endogenous GLD-1, suggesting a novel mechanism of controlling GLD-1 accumulation. More work is underway to express GLD-1N in the distal germline. This research will contribute to a better understanding of the precise regulatory mechanisms governing stem cell proliferation.

Xue Han et al. (2007) International Worm Meeting "Protein Phosphatase 4 in MEI-1 Regulation."

Paul Mains, Cheryl Birmingham, Xue Han, Asako Sugimoto

[International Worm Meeting, 2007]

mei-1 encodes the worm katanin microtubule-severing protein. It is required during meiosis to regulate the shape and dynamics of meiotic spindles(McNally et al.,1993) but must be degraded before mitosis. MEL-26 recruits MEI-1 to the E3 ubiquitin ligase complex for post-meiotic ubiquitin-mediated degradation. In a screen of the chromosome I RNAi library, we identified F16A11.3 as a suppressor of a mei-1(gf) allele that is refractory to MEL-26 mediated degradation. RNAi against F16A11.3 in a mei-1(gf) background increased hatching from 1% to 14%. Lethality of a mel-26(lf) allele was also suppressed while a mei-2(lf) mutant with limited meiotic rather than excess mitotic MEI-1 was not affected. These findings suggested that F16A11.3 regulated mei-1 only after meiosis. F16A11.3(ppfr-1) encodes the R1 regulatory subunit of Protein Phosphatase 4 (PP4). Protein phosphatase 4 is a member of the PPP family of protein serine/threonine phosphatases. Like ectopic MEI-1, it localizes to centrosomes in mammals and flies(Brewis et al., 1993). One catalytic subunit, pph-4.1 plays an essential role in spindle formation in both mitosis and sperm meiosis in C.elegans(Sumiyoshi et al., 2002). We are investigating the mechanism of ppfr-1 suppression of mei-1(gf). In my work, I demonstrate that pph-4.1 and another regulatory subunit, alpha 4, act in the same pathway as ppfr-1 to suppress ectopic mitotic MEI-1. Other PP4 subunits (PPH4.2 and R2) do not suppress ectopic MEI-1. I also test several models to investigate how ppfr-1 suppresses mei-1(gf). It is unlikely that reduction in PP4 activity results in general increase in microtubule stability, making them resistant to ectopic MEI-1 severing. Another model, which proposes loss of PP4 phosphatase results in higher levels of MEI-1 phosphorylation, leading to its degradation, also does not seem to be the case. Reciprocal co-immunoprecipitation experiments indicate PP4 may regulate MEI-1 activity via direct binding.

Xue Han et al. (2005) International Worm Meeting "Protein Phosphatase 4 in MEI-1 Degradation"

Paul E Mains, Cheryl L Birmingham, Xue Han

[International Worm Meeting, 2005]

mei-1 encodes the worm katanin microtubule-severing protein. It is required during meiosis to regulate the shape and dynamics of meiotic spindles but must be degraded before mitosis. MEL-26 recruits MEI-1 to the E3 ubiquitin ligase complex for post-meiotic ubiquitin mediated degradation. In a screen of the chromosome I RNAi library, we identified F16A11.3 as a suppressor of a mei-1 allele that is refractory to MEL-26 mediated degradation. When we performed RNAi against F16A11.3 in a mei-1(gf) allele, hatching rates increased from 1% to 14%. Lethality of a mel-26(lf) allele was also suppressed by the same RNAi while a mei-2(lf) mutant, with limited meiotic mei-1, was not affected. These findings suggested that F16A11.3 regulated mei-1 only after meiosis. F16A11.3 encodes the R1 regulatory subunit of Protein Phosphatase 4. Protein phosphatase 4 is a member of the PPP family of protein serine/threonine phosphatases, and it localizes to centrosomes (Brewis et al., 1993). One catalytic subunit encoded by gene pph-4.1 plays an essential role in spindle formation in both mitosis and sperm meiosis in C. elegans, while the other catalytic subunit encoded by gene pph-4.2 does not (Sumiyoshi et al., 2002). We are investigating the mechanism of F16A11.3 suppression of mei-1(gf). Previous works demonstrated that MBK-2 kinase is required for ubiquitin mediated MEI-1 degradation and so loss of the corresponding phosphatase could result in increased phosphorylated MEI-1, increasing degradation. Alternatively, the phosphorylation state of MEI-1 could influence its level of activity and/or subcellular localization. Experiments are underway to explore these possibilities. We will also determine which catalytic subunit acts in conjunction with the F16A11.3 encoded R1 regulatory subunit.

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

Gruber J et al. (2017) Cell "Microbiome and Longevity: Gut Microbes Send Signals to Host Mitochondria."

Kennedy BK, Gruber J

[Cell, 2017]

The microbiome has emerged as a major determinant of the functioning of host organisms, affecting both health and disease. Here, Han etal. use the workhorse of aging research, C.elegans, to identify specific mechanisms by which gut bacteria influence mitochondrial dynamics and aging, a first steptoward analogous manipulations to modulate human aging.

Greenwald IS et al. (1990) Cell "Cell fates in C. elegans: in medias ras."

Greenwald IS, Broach JR

[Cell, 1990]

ras proteins play a central role in controlling cell proliferation in a variety of organisms and also appear to mediate specific developmental processes in certain cells. Nonetheless, how ras protein activity is regulated and how ras protein activation yields its biological effect have not been clearly defined in any metazoan. Now, Han and Sternberg (1990a) have found that a ras protein is encoded by the C. elegans let-60 gene. Since powerful classical and molecular genetic methods are available for C. elegans, this finding offers great promise for identifying components of the ras pathway and defining the nature of their interactions in this organism. In addition, the extensive literature on the biochemistry and genetics of ras function in other organisms should greatly facilitate translating genetic analysis of let-60 (Beitel et al., 1990; Han and Sternberg, 1990b) into detailed molecular models of cell fate determination.

Davies B et al. (1995) International C. elegans Meeting "STRUCTURE-FUNCTION STUDIES OF THE CELL DEATH PROTEIN CED-3."

Shaham S, Davies B, Horvitz HR

[International C. elegans Meeting, 1995]

CED-3, a protein required for the proper execution of programmed cell death in C. elegans, is similar in sequence to mammalian interleukin-1b converting enzyme (ICE), a protein involved in the inflammatory response of the human immune system, and to mammalian NEDD-2/ICH-1, a protein present in the developing mouse brain. CED-3, ICE and NEDD-2/ICH-1 define a family of cysteine proteases (see abstract by Xue and Horvitz). To better understand these proteases and their involvement in programmed cell death, we have been conducting structure-function studies of CED-3.

Yeh Chan-Hsien et al. (2007) International Worm Meeting "Screen for TLK-1-interacting proteins."

Yi-Chun Wu, Yeh Chan-Hsien, I-Ju Lee

[International Worm Meeting, 2007]

Tousled-like kinases (TLKs) form a conserved serine/threonine kinase family in multi-cellular organisms and have been implicated in transcription, DNA replication and repair and chromosome assembly and segregation. The tlk-1(RNAi) mutants exhibited defects in chromosome condensation and segregation. Previous studies of C. elegans TLK-1 indicates that tlk-1 may act in chromosome assembly by phosphorylating Asf-1 (anti-silencing function protein) and contribute to chromosome segregation as a substrate and activator of the Aurora B kinase AIR-2 (Han et al, 2005). To identify more TLK-1-interacting proteins, we performed a yeast-two hybrid screen. In the screen we isolated DLC-1 (Dynein light chain). DLC-1 is localized to spindle fiber during mitosis and this localization pattern is disrupted in tlk-1(RNAi) embryos. We are currently performing experiments to verify the interaction between DLC-1 and TLK-1 in vivo and investigating if TLK-1 may phosphorylate DLC-1. Reference Han, Z., Riefler, G.M., Saam, J.R., Mango, S.E., and Schumacher, J.M. (2005) Curr. Biol., 15, 894-904.

Gomes, Jose-Eduardo et al. (2009) International Worm Meeting "The protein-phosphatase 4 subunit PPFR-1 is a potential activator of MEI-1/Katanin."

Gomes, Jose-Eduardo, Formstecher, Etienne, Mains, Paul E., Han, Xue, Pintard, Lionel, Richaudeau, Benedicte

[International Worm Meeting, 2009]

Upon fertilization the C. elegans oocyte resumes and completes meiosis, and twenty minutes latter the first embryonic mitosis takes place. Despite sharing the same cytoplasm, the meiotic and mitotic spindles are radically different: while the small asterless meiotic spindle assembles close to the cell cortex, the mitotic spindle is large, with robust asters, and is positioned in the center of the embryo. MEI-1/Katanin is a microtubule-severing protein essential for meiotic spindle formation; however its activity is incompatible with the assembly of a fully functional mitotic spindle. In wild-type embryos, MEI-1 is inactivated at the meiosis-to-mitosis transition allowing for mitotic spindle assembly. We have previously established that, after meiosis, a Cullin-3-based E3-ligase targets MEI-1 for ubiquitin-mediated proteasomal degradation. MEI-1 is recruited by the CUL-3-based complex through the adaptor protein MEL-26. In order to identify new components of the pathway, we conducted a Yeast Two-Hybrid screen using MEL-26 as bait. In this screen we identified PPFR-1, a regulatory subunit of the serine/threonine phosphatase PP4 complex. Loss-of-function of ppfr-1, through both RNA-interference or a putative null allele, suppresses the lethality of ectopic MEI-1 expression during mitosis, either due to mel-26 loss-of-function or a to mei-1 gain-of-function allele encoding a protein refractory to MEL-26 mediated degradation. This suppression suggests the PP4 phosphatase is an activator of MEI-1. In addition, the ppfr-1 putative null allele displays a partly penetrant meiosis failure phenotype, again consistent with PP4 being an activator of MEI-1. To test whether PP4 modifies MEI-1 protein in vivo, we performed 2D gels on embryonic extracts; we were able to show that MEI-1 is phosophorylated in vivo and that these modifications are dependent on PPFR-1. Our results also show that PPFR-1 regulates MEI-1 independently of protein degradation. We are currently testing whether PPFR-1 is targeted for degradation by the CUL-3MEL-26 ligase at the meiosis-to-mitosis transition.