Hu J et al. (2007) Mol Biol Cell "STAM and Hrs down-regulate ciliary TRP receptors."

Hu J, Barr MM, Wittekind SG

[Mol Biol Cell, 2007]

Monitoring Editor: Jean Gruenberg Cilia are endowed with membrane receptors, channels, and signaling components whose localization and function must be tightly controlled. In primary cilia of mammalian kidney epithelia and sensory cilia of C. elegans neurons, polycystin-1 (PC1) and transient receptor polycystin-2 channel (TRPP2 or PC2), function together as a mechanosensory receptor-channel complex. Despite the importance of the polycystins in sensory transduction, the mechanisms that regulate polycystin activity and localization, or ciliary membrane receptors in general, remain poorly understood. We demonstrate that signal transduction adaptor molecule STAM-1A interacts with C. elegans LOV-1 (PC1), and that STAM functions with hepatocyte growth factor regulated tyrosine kinase substrate (Hrs) on early endosomes to direct the LOV-1-PKD-2 complex for lysosomal degradation. In a stam-1 mutant, both LOV-1 and PKD-2 improperly accumulate at the ciliary base. Conversely, overexpression of STAM or Hrs promotes the removal of PKD-2 from cilia, culminating in sensory behavioral defects. These data reveal that the STAM-Hrs complex, which downregulates ligand-activated growth factor receptors from the cell surface of yeast and mammalian cells, also regulates the localization and signaling of a ciliary PC1 receptor-TRPP2 complex.

Hu J et al. (2019) PLoS Biol "Distinct roles of two myosins in C. elegans spermatid differentiation."

Hu J, Wang H, Liu Y, Wu M, Liu S, Li X, Wang X, Cheng S

[PLoS Biol, 2019]

During spermatogenesis, interconnected haploid spermatids segregate undesired cellular contents into residual bodies (RBs) before detaching from RBs. It is unclear how this differentiation process is controlled to produce individual spermatids or motile spermatozoa. Here, we developed a live imaging system to visualize and investigate this process in C. elegans. We found that non-muscle myosin 2 (NMY-2)/myosin II drives incomplete cytokinesis to generate connected haploid spermatids, which are then polarized to segregate undesired cellular contents into RBs under the control of myosin II and myosin VI. NMY-2/myosin II extends from the pseudo-cleavage furrow formed between two haploid spermatids to the spermatid poles, thus promoting RB expansion. In the meantime, defective spermatogenesis 15 (SPE-15)/myosin VI migrates from spermatids towards the expanding RB to promote spermatid budding. Loss of myosin II or myosin VI causes distinct cytoplasm segregation defects, while loss of both myosins completely blocks RB formation. We found that the final separation of spermatids from RBs is achieved through myosin VI-mediated cytokinesis, while myosin II is dispensable at this step. SPE-15/myosin VI and F-actin form a detergent-resistant actomyosin VI ring that undergoes continuous contraction to promote membrane constriction between spermatid and RB. We further identified that RGS-GAIP-interacting protein C terminus (GIPC)-1 and GIPC-2 cooperate with myosin VI to regulate contractile ring formation and spermatid release. Our study reveals distinct roles of myosin II and myosin VI in spermatid differentiation and uncovers a novel myosin VI-mediated cytokinesis process that controls spermatid release.

Hu J et al. (2006) Mol Biol Cell "Casein kinase II and calcineurin modulate TRPP function and ciliary localization."

Knobel KM, Barr MM, Hu J, Bae YK

[Mol Biol Cell, 2006]

Monitoring Editor: Martin Chalfie Cilia serve as sensory devices in a diversity of organisms and their defects contribute to many human diseases. In primary cilia of kidney cells, the transient receptor potential polycystin (TRPP) channels polycystin-1 (PC-1) and polycystin-2 (PC-2) act as a mechanosensitive channel, with defects resulting in autosomal dominant polycystic kidney disease. In sensory cilia of Caenorhabditis elegans male-specific neurons, the TRPPs LOV-1 and PKD-2 are required for mating behavior. The mechanisms regulating TRPP ciliary localization and function are largely unknown. We identified the regulatory subunit of the serine-theonine casein kinase II (CK2) as a binding partner of LOV-1 and human PC-1. CK2 and the calcineurin phosphatase TAX-6 modulate male mating behavior and PKD-2 ciliary localization. The phospho-defective mutant PKD-2(S534A) localizes to cilia while a phospho-mimetic PKD-2(S534D) mutant is largely absent from cilia. Calcineurin is required for PKD-2 ciliary localization, but is not essential for ciliary gene expression, ciliogenesis, or localization of cilium structural components. This unanticipated function of calcineurin may be important for regulating ciliary protein localization. A dynamic phosphorylation-dephosphorylation cycle may represent a mechanism for modulating TRPP activity, cellular sensation, and ciliary protein localization.

Hu J et al. (2005) Mol Biol Cell "ATP-2 interacts with the PLAT domain of LOV-1 and is involved in Caenorhabditis elegans polycystin signaling."

Hu J, Barr MM

[Mol Biol Cell, 2005]

Caenorhabditis elegans is a powerful model to study the molecular basis of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by mutations in the polycystic kidney disease (PKD)1 or PKD2 gene, encoding polycystin (PC)-1 or PC-2, respectively. The C. elegans polycystins LOV-1 and PKD-2 are required for male mating behaviors and are localized to sensory cilia. The function of the evolutionarily conserved polycystin/lipoxygenase/alpha-toxin (PLAT) domain found in all PC-1 family members remains an enigma. Here, we report that ATP-2, the beta subunit of the ATP synthase, physically associates with the LOV-1 PLAT domain and that this interaction is evolutionarily conserved. In addition to the expected mitochondria localization, ATP-2 and other ATP synthase components colocalize with LOV-1 and PKD-2 in cilia. Disrupting the function of the ATP synthase or overexpression of atp-2 results in a male mating behavior defect. We further show that atp-2, lov-1, and pkd-2 act in the same molecular pathway. We propose that the ciliary localized ATP synthase may play a previously unsuspected role in polycystin signaling.

Hu Z et al. (2015) J Neurosci "NLP-12 engages different UNC-13 proteins to potentiate tonic and evoked release."

Vashlishan-Murray AB, Kaplan JM, Hu Z

[J Neurosci, 2015]

A neuropeptide (NLP-12) and its receptor (CKR-2) potentiate tonic and evoked ACh release at Caenorhabditis elegans neuromuscular junctions. Increased evoked release is mediated by a presynaptic pathway (egl-30 Gq and egl-8 PLC) that produces DAG, and by DAG binding to short and long UNC-13 proteins. Potentiation of tonic ACh release persists in mutants deficient for egl-30 Gq and egl-8 PLC and requires DAG binding to UNC-13L (but not UNC-13S). Thus, NLP-12 adjusts tonic and evoked release by distinct mechanisms.

Hu Y et al. (2013) J Proteome Res "Expanding tandem mass spectral libraries of phosphorylated peptides: advances and applications."

Lam H, Hu Y

[J Proteome Res, 2013]

The identification of phosphorylated proteins remains a challenge in proteomics, partially due to the difficulty in assigning tandem mass (MS/MS) spectra to their originating peptide sequences with correct phosphosite localization. Because of its advantages in efficiency and sensitivity, spectral library searching is a promising alternative to conventional sequence database searching. Our work aims to construct the largest collision-induced dissociation (CID) MS/MS spectral libraries of phosphorylated peptides in human (Homo sapiens) and four model organisms (Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) to date, to facilitate phosphorylated peptide identification by spectral library searching. We employed state-of-the-art search methods to published data and applied two recently published phosphorylation site localization tools (PhosphoRS and PTMProphet) to ascertain the phosphorylation sites. To further increase the coverage of this library, we predicted "semi-empirical" spectra for peptides containing known phosphorylation sites from the corresponding template unphosphorylated peptide spectra. The performance of the spectral libraries built were evaluated and found to be superior to conventional database searching in terms of sensitivity. Updated spectral libraries of phosphorylated peptides are made freely available for use with the spectral search engine SpectraST. The work flow being developed will be used to continuously update the libraries when new data become available.

Hu M et al. (2019) J Vis Exp "Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract."

Miller MA, Legg S, Hu M

[J Vis Exp, 2019]

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Hu K et al. (2024) J Agric Food Chem "Pongamol Alleviates Neuroinflammation and Promotes Autophagy in Alzheimer's Disease by Regulating the Akt/mTOR Signaling Pathway."

Ren J, Wu X, Yao Y, Miao J, Zhu S, Chen G, Hu K, Xu J, Wu S, Zhang Y

[J Agric Food Chem, 2024]

Alzheimer's disease (AD), one of the neurodegenerative disorders, is highly correlated with the abnormal hyperphosphorylation of Tau and aggregation of &#x3b2;-amyloid (A&#x3b2;). Oxidative stress, neuroinflammation, and abnormal autophagy are key drivers of AD and how they contribute to neuropathology remains largely unknown. The flavonoid compound pongamol is reported to possess a variety of pharmacological activities, such as antioxidant, antibacterial, and anti-inflammatory. This study investigated the neuroprotective effect and its mechanisms of pongamol in lipopolysaccharide (LPS)-induced BV2 cells, d-galactose/sodium nitrite/aluminum chloride (d-gal/NaNO₂/AlCl₃)-induced AD mice, and <i>Caenorhabditis elegans</i> models. Our research revealed that pongamol reduced the release of inflammatory factors IL-1&#x3b2;, TNF-&#x3b1;, COX-2, and iNOS in LPS-induced BV2 cells. Pongamol also protected neurons and significantly restored memory function, inhibited Tau phosphorylation, downregulated A&#x3b2; aggregation, and increased oxidoreductase activity in the hippocampus of AD mice. In addition, pongamol reversed the nuclear transfer of NF-&#x3ba;B and increased the levels of Beclin 1 and LC3 II/LC3 I. Most importantly, the anti-inflammatory and promoter autophagy effects of pongamol may be related to the regulation of the Akt/mTOR signaling pathway. In summary, these results showed that pongamol has a potential neuroprotective effect, which greatly enriched the research on the pharmacological activity of pongamol for improving AD.

Hu E et al. (1990) J Biol Chem "Expression of wild-type and mutated forms of the catalytic (alpha) subunit of Caenorhabditis elegans casein kinase II in Escherichia coli."

Hu E, Rubin CS

[J Biol Chem, 1990]

A full-length Caenorhabditis elegans cDNA that encodes the alpha subunit of casein kinase II was inserted into the inducible bacterial expression vector pET3a to generate the plasmid pCK alpha. Escherichia coli DE21 lysozyme S that was transformed with pCK alpha expressed soluble, catalytically active casein kinase II alpha upon induction with isopropyl beta-D-thiogalactopyranoside. The expressed alpha subunit was purified to homogeneity with a 60% yield by chromatography on CM-Sephadex, P-11 phosphocellulose, and heparin-agarose. The Mr values estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 42,000) or calculated from hydrodynamic measurements (s20,w = 3.3 S, Stokes radius = 2.8 nm, Mr = 37,000) were similar, thereby indicating that the expressed enzyme is monomeric. The native holoenzyme and the expressed alpha subunit exhibited several similar properties including the utilization of both ATP and GTP as substrates and the susceptibility to inhibition of phosphotransferase activity by low concentrations of heparin. However, the kcat for E. coli-derived alpha was only 9% of the kcat for the native holoenzyme, and catalytic activity was not stimulated by polyamines. Recombinant casein kinase II alpha aggregates at low ionic strength, and the aggregation is partially reversible. A mutant alpha subunit in which Lys74 and Lys75 were substituted by glutamic acid residues was constructed by site-directed mutagenesis. The mutant enzyme was not inhibited by typically effective concentrations of heparin (e.g. IC50 = 0.3 micrograms/ml) because the affinity of modified recombinant casein kinase II Glu-74Glu-75 for heparin decreased approximately 70-fold. Thus, Lys74 and Lys75 are implicated in the heparin binding, inhibitory domain. The successful expression of casein kinase II alpha in E. coli will facilitate the analysis of the structural basis for functional domains in this enzyme.

Hu E et al. (1991) J Biol Chem "Casein kinase II from Caenorhabditis elegans. Cloning, characterization, and developmental regulation of the gene encoding the beta subunit."

Rubin CS, Hu E

[J Biol Chem, 1991]

Complementary DNAs encoding the beta subunit of casein kinase II (CKII beta) from the nematode Caenorhabditis elegans were cloned and sequenced. The predicted beta subunit polypeptide comprises 234 amino acid residues and has a Mr of 26,452. CKII beta is not homologous with other types of proteins. In synchronously developing C. elegans the abundance of the 1.3-kilobase mRNA for CKII beta varies in parallel with the level of mRNA encoding the catalytic subunit (alpha) of CKII. Thus, the developmental expression of CKII subunits is controlled coordinately and pretranslationally. CKII beta and CKII alpha mRNAs are enriched 5-10-fold in C. elegans embryos relative to their concentrations at several other stages of nematode development. A 3.8-kilobase pair segment of C. elegans DNA that contains the CKII beta gene and an extensive 5'-flanking region was cloned and sequenced. The CKII beta gene is divided into 6 exons by introns ranging from 49 to 533 base pairs in length. The first exon encodes 88 nucleotides of 5'-untranslated mRNA. Exon 2 (72 base pairs) contains the initiator Met codon and only 5 additional codons. Exons 3-6 encode 52, 63, 64, and 49 amino acid residues, respectively. The 5' terminus of CKII beta mRNA is modified post-transcriptionally by trans-splicing with a leader sequence of 22 nucleotides. The CKII beta gene was mapped to a position on C. elegans chromosome 2 that is in close proximity to the lin-11 gene.