Biology subjects

Gordon, M. T.

Publications and source records attributed to Gordon, M. T..

5 recordsLinked to original sources

bioRxiv2026

A multifunctional polyketide synthase in nematodes produces divergent families of signaling molecules that control different developmental arrests

To improve their chances of reproductive success, nematodes not only must arrest their development in response to adverse growth conditions but also must quickly recover if conditions improve. A polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) hybrid assembly line that is expressed in the canal-associated neurons (CANs) of Caenorhabditis elegans promotes recovery from starvation-induced larval arrest. Here, we show that in the predatory nematode Pristionchus pacificus this assembly line produces a suite of secondary metabolites, including a family of hybrid polyketide-nonribosomal peptides known as the nemamides, the related nemaketides, and a family of ascarylose-modified polyketides named ascarenes. Depending on the starter unit that is loaded onto the PKS, the assembly line can produce dramatically different downstream products. Whereas the nemamides promote recovery from starvation-induced larval arrest, the ascarenes inhibit development of the dauer larval stage and promote recovery. This dichotomy suggests that the PKS-NRPS megasynthetase serves as a signaling hub in the CANs, controlling multiple developmental events. The PKS-NRPS assembly line is highly conserved across many nematode species, and identification of these chemical signals will help to elucidate the signaling pathways that control development in the worm and lead to novel anthelmintics.

biochemistry↗

bioRxiv2026

Lysosomal Expansion Compartments Mediate Zinc and Copper Homeostasis in Caenorhabditis elegans

Zinc is an essential transition metal that participates in many biological processes. In C. elegans, excess zinc is stored in lysosomes in intestinal cells; this process involves increasing the expression of the zinc transporter CDF-2 and remodeling of lysosomes characterized by an increase in the volume of the expansion compartment. To determine if this is a more general property, we investigated other metals. Here we report that lysosomes are remodeled in response to excess copper, manganese, and cadmium, with each metal causing an increase in the volume of the expansion compartment. Mutants with a reduced number of lysosomes were hypersensitive to growth retardation caused by excess copper and manganese, suggesting metal toxicity is prevented by metal sequestration in lysosomes. Using a novel method to analyze isolated lysosomes by X-ray Fluorescence Microscopy we demonstrated that zinc, copper and manganese are detectable in the lumen of lysosomes. To further analyze copper, we examined localization of CUA-1.1, a copper transporter that moves copper into the lumen of lysosomes. Like the zinc transporter CDF-2, CUA-1.1 localizes to both the acidified and expansion compartments in excess copper. These results indicate that the same intestinal lysosomes store zinc, copper and manganese. Lysosome remodeling characterized by an increase in volume of the expansion compartment is not specific to zinc but is a more general phenomenon during metal storage in lysosomes.

molecular biology↗

bioRxiv2023

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands

With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Forster resonance energy transfer (FRET), provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the [a]ngstrom scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose- dependent distance changes with an accuracy of [~]1.5 [A]. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 [A] to 20 [A]. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe2+ and Ru2+ to produce novel tmFRET acceptors to expand the working distance to as long as 50 [A], while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions. STATEMENT OF SIGNIFICANCEIn this work, we expand the working distance of transition metal ion Forster Resonance Energy Transfer (tmFRET) to allow the measurement of donor-acceptor distances from 10 [A] to 50 [A]. We develop new cysteine-reactive bipyridyl- and phenanthroline-based ligands for Cu2+, Fe2+, and Ru2+ and examine their ability to resolve small ligand-dependent distance changes in maltose binding protein. We extend these studies using pulsed electron paramagnetic resonance spectroscopy to demonstrate the high accuracy of tmFRET for studying protein allostery.

biophysics↗

bioRxiv2021

Single Molecule Studies Reveal Regulatory Interactions between Master Kinases PDK1, AKT1 and PKCalpha

Leukocyte migration is controlled by a leading edge chemosensory pathway that generates the regulatory lipid PIP3, a growth signal, thereby driving leading edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1/PKB and PKC are key components of a plasma membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, while PKC is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKC and other AGC kinases by phosphorylation at key sites. PDK1 is known to form PDK1:AKT1 and PDK1:PKC heterodimers stabilized by a PIF interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here we present the first, to our knowledge, single molecule studies of full length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full length PKC. The findings show that membrane-bound PDK1:AKT1 and PDK1:PKC heterodimers form under physiological conditions, and are stabilized by PIF interaction. PKC exhibits 8-fold higher PDK1 affinity than AKT1, thus PKC competitively displaces AKT1 from PDK1:AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1:AKT1 heterodimer, while PKC acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide new insights into molecular and regulatory interactions of the three master kinases on their target membrane, and suggest that the recently described tumor suppressor activity of PKC may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis. STATEMENT OF SIGNIFICANCEThis work investigates three master kinases that play central roles in guiding white blood cell migration to sites of infection, inflammation or tissue damage. More broadly, the same kinases help regulate production of a cell growth signal, and may trigger cancer when dysregulated. Using powerful single molecule methods, the work detects and analyzes the interactions between the three purified kinases on their target membrane surface. The findings reveal functionally important differences between pairwise binding affinities of different binding partners. Additional studies reveal that the highest affinity kinase can disrupt and inhibit the activated complex formed by association of the other two kinases. Such inhibition is proposed to help prevent cancer by limiting growth signal production by the activated complex.

biophysics↗

bioRxiv2021

Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan

Hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between megasynthases. In the nematode Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides in animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional stand-alone enzymes that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes in the biosynthetic pathway and discover a novel mechanism of trafficking intermediates between a PKS and an NRPS. Specifically, we show that the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.

biochemistry↗