Gao L et al. (2004) J Biol Chem "Isoforms of the polarity protein par6 have distinct functions."

Macara IG, Gao L

[J Biol Chem, 2004]

PAR-6 is essential for asymmetric division of the Caenorhabditis elegans zygote. It is also critical for cell polarization in many other contexts, throughout the Metazoa. The Par6 protein contains a PDZ domain and a partial CRIB (Cdc42/Rac interactive binding) domain, which mediate interactions with other polarity proteins such as Par3, Cdc42, Pals1 and Lgl. A family of mammalian Par6 isoforms (Par6A-D) has been described, but the significance of this diversification has been unclear. Here, we demonstrate that Par6 family members localize differently when expressed in MDCK epithelial cells and have distinct effects on tight junction (TJ) assembly. Par6B localizes to the cytosol and inhibits TJ formation, but Par6A co-localizes predominantly with the TJ marker ZO-1 at cell-cell contacts and does not affect junctions. These functional differences correlate with differences in Pals1 binding - Par6B interacts strongly with Pals1, whereas Par6A binds very weakly to Pals1 even in the presence of active Cdc42. Pals1 has a low affinity for the isolated CRIB+PDZ domain of Par6A, but analysis of chimeras showed that in addition, Pals1 binding is blocked by an inhibitory property of the N-terminus of Par6A. Unexpectedly, the localization of Par6A to cell-cell contacts is Cdc42-independent.

Gao L (2015) Opt Express "Extend the field of view of selective plan illumination microscopy by tiling the excitation light sheet."

Gao L

[Opt Express, 2015]

Selective Plane Illumination Microscopy (SPIM) is attractive for its ability to acquire 3D images with high 3D spatial resolution, good optical sectioning capability and high imaging speed. However, tradeoffs have to be made when a large field of view (FOV) is required, results in lower axial resolution or worse optical sectioning capability. Here, we present a novel method for 3D imaging by SPIM that is capable to maintain its high 3D spatial resolution and good optical sectioning capability within a large FOV. Instead of trying to generate a large and uniformly thick excitation light sheet, the method tiles a relative small light sheet quickly to multiple positions within the image plane by defocusing the excitation beam used to create the light sheet, and takes one additional image at each position, so that a large FOV can be imaged by repeating this process and stitching all images together. By implementing this method, light sheets with thin thickness and good excitation light confinement can be used for SPIM imaging with slightly compromised imaging speed. The method was investigated through both numerical simulation and experiments, and the imaging performance was demonstrated by imaging fluorescent particles embedded in agarose gel and live C. elegans embryos.

Gao L et al. (2022) Elife "Shank promotes action potential repolarization by recruiting BK channels to calcium microdomains."

Ardiel EL, Nurrish S, Gao L, Zhao J, Hall Q, Kaplan JM

[Elife, 2022]

Mutations altering the scaffolding protein Shank are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. Among its many binding partners, Shank directly binds CaV1 voltage activated calcium channels. Here we show that the <i>C. elegans</i> SHN-1/Shank promotes CaV1 coupling to calcium activated potassium channels. Mutations inactivating SHN-1, and those preventing SHN-1 binding to EGL-19/CaV1 all increase action potential durations in body muscles. Action potential repolarization is mediated by two classes of potassium channels: SHK-1/KCNA and SLO-1 and SLO-2 BK channels. BK channels are calcium-dependent, and their activation requires tight coupling to EGL-19/CaV1 channels. SHN-1's effects on AP duration are mediated by changes in BK channels. In <i>shn-1</i> mutants, SLO-2 currents and channel clustering are significantly decreased in both body muscles and neurons. Finally, increased and decreased <i>shn-1</i> gene copy number produce similar changes in AP width and SLO-2 current. Collectively, these results suggest that an important function of Shank is to promote microdomain coupling of BK with CaV1.

Gao L et al. (2012) Cell "Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens."

Poulton JS, Davidson MW, Higgins CD, Wu X, Shao L, Goldstein B, Betzig E, Peifer M, Gao L

[Cell, 2012]

Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate invivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

Gao L et al. (2024) Proc Natl Acad Sci U S A "Voltage-induced calcium release in Caenorhabditis elegans body muscles."

Gao L, Ardiel E, Kaplan JM, Nurrish S

[Proc Natl Acad Sci U S A, 2024]

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in <i>Caenorhabditis elegans</i> body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

Gao L et al. (2016) J Nat Prod "-Dihydroagarofuran-Type Sesquiterpenes from the Seeds of Celastrus monospermus and Their Lifespan-Extending Effects on the Nematode Caenorhabditis elegans."

Wu D, Ning R, Lan J, Zhang R, Gao L, Chen D, Zhao W

[J Nat Prod, 2016]

Seventeen -dihydroagarofuran-type sesquiterpenes were isolated from the seeds of Celastrus monospermus, and, among them, 15 (1-15) were identified as new natural products. Nine isolates were evaluated for their lifespan-extending effect using the standard model animal nematode Caenorhabditis elegans. As a result, all of the tested compounds prolonged the lifespan of C. elegans when compared to the blank control group (p &lt; 0.0001). Among them, celaspermin E (5) extended the average lifespan and maximum lifespan of C. elegans, with effects similar to those of rapamycin, a positive control that has been found experimentally to delay the aging process of yeasts, worms, fruit flies, and mice.

Olson, Andrew et al. (2017) International Worm Meeting "The Role of Post-Translational Modifications in the Regulation of Serotonin Signaling."

Olson, Andrew, Koelle, Michael, Kumar, Santosh

[International Worm Meeting, 2017]

C. elegans uses serotonin as a neurotransmitter to slow locomotion, and we have used this model system to discover that post-translational modifications appear to regulate serotonin signaling. Through large-scale genetic screens for mutants that fail to paralyze in response to exogenous serotonin, we found that C. elegans mutants for either of two subunits of the ELPC Elongator Protein Complex are defective for serotonin signaling. Conversely, transgenic animals overexpressing ELPC are hypersensitive to the effects of exogenous serotonin. ELPC is conserved from C. elegans to humans and functions as a cytoplasmic lysine acetylase to reversibly modify other proteins. This is the first time that ELPC or lysine acetylation has been implicated in regulating serotonin signaling. We used two-dimensional gel electrophoresis to show that in C. elegans lysates, Gao, a neural G protein encoded by the goa-1 gene through which serotonin signals to slow locomotion, exists as a complex series of species of differing charge. Acetylation eliminates the positive charge on a lysine side chain, and differential acetylation on several Lys could produce the complex pattern of Gao species we see. Our preliminary results suggest that the series of differentially charged Gao species in wild-type lysates shifts to a less complex and more positively charged set of Gao species in lysates of ELPC mutants, as would be predicted if Gao is acetylated by ELPC. We isolated Gao from both mouse brain and C. elegans lysates and analyzed the purified proteins for post-translational modifications using mass spectrometry. In both species, Gao is acetylated on several conserved Lys residues near the N-terminus. We note that the signaling defects in ELPC mutants are much more restricted than those of Gao null mutants. Both ELPC and Gao mutants are defective for response to exogenous serotonin, but Gao null mutants have additional defects not seen in ELPC mutants, including defects in response to exogenous dopamine as well as in many behavioral assays. Thus we hypothesize that ELPC may reversibly acetylate Gao to specifically regulate its ability to be activated by serotonin receptors, while not strongly affecting its ability mediate signaling by other receptors. We are using CRISPR-Cas9 technology to mutate the acetylated Lys residues of Gao to the similar but non-acetylatable amino acid arginine to determine if acetylation at specific positions is responsible for regulating serotonin signaling.

Hofler, Catherine et al. (2011) International Worm Meeting "AGS-3 alters C. elegans behavior after food deprivation via RIC-8 activation of the neural G protein Gao."

Hofler, Catherine, Koelle, Michael

[International Worm Meeting, 2011]

Like all animals, C. elegans modifies a number of behaviors upon food deprivation in order to seek food. These behavioral changes depend on neurotransmitters and peptides that typically signal through the major neural G protein Gao, suggesting that signaling through this G protein ultimately mediates responses to food deprivation. Proteins containing the G Protein Regulator (GPR) domain bind to Gao in vitro, but the biological functions of GPR proteins in neurons and the mechanism by which they might affect G protein signaling has remained unclear. We found that the conserved GPR domain protein AGS-3 activates Gao signaling in vivo to allow C. elegans to alter several behaviors after food deprivation. These behaviors include octanol avoidance, area-restricted search, and egg laying. AGS-3 protein in whole worm lysates undergoes a progressive change in its biochemical fractionation pattern upon food deprivation, demonstrating that food deprivation changes the physical state of the protein. Cell-specific rescue and cell-specific inactivation experiments show that AGS-3 and Gao act together in the ASH chemosensory neurons to allow food deprivation to modify response to octanol. Genetic epistasis experiments show that AGS-3 activation of Gao in the ASHs requires the guanine nucleotide exchange factor RIC-8. Conversely, RIC-8 function in the ASHs also requires AGS-3. Using purified recombinant proteins, we characterized interactions of the proteins consistent with the genetic epistasis results. AGS-3 forms a complex with the the inactive, GDP-bound form of Gao, and RIC-8 can act on this complex to promote nucleotide exchange and dissociation of all the proteins, generating active Gao-GTP. These results identify a biological role for AGS-3 in response to food deprivation and indicate the mechanism for its activation of Gao signaling in vivo.

Heather A Hess et al. (2004) East Coast Worm Meeting "RGS-7 Completes a Receptor-independent Heterotrimeric G Protein Signaling Cycle to Regulate Mitotic Spindle Positioning in C. elegans"

Michael R Koelle, Heather A Hess

[East Coast Worm Meeting, 2004]

Heterotrimeric G proteins promote astral microtubule forces on centrosomes to position mitotic spindles properly during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor-independent, and the active form of the G proteins remains unclear. We obtained mutants for all 13 C. elegans regulators of G protein signaling and found that one, RGS-7, decreases the speed and magnitude of centrosome movements. The effects of RGS-7 require two redundant Gao-related G proteins and their non-receptor activators. Using recombinant proteins, we found that the non-receptor activator RIC-8 stimulates GTP binding by Gao, and the RGS domain of RGS-7 stimulates GTP hydrolysis by Gao. These results demonstrate that the active species in the receptor-independent G protein cycle is GaoGTP, and that RGS-7 completes the cycle by driving Gao to its inactive, GDP-bound form.

Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Lambie EJ, Kimble JE

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755