Biology subjects

Collins, R. F.

Publications and source records attributed to Collins, R. F..

6 recordsLinked to original sources

bioRxiv2026

Acyl amides derived from a bacterial symbiont control development of its nematode host

Symbiotic bacteria frequently regulate host development through small-molecule signals. In the entomopathogenic Steinernema nematode - Xenorhabdus bacterium symbiosis, which is widely used in biological pest control, the signals that trigger nematode development have remained unknown. Using a phenotypic screen of Xenorhabdus mutants, we identified bacterial acyl amides, which we named stripteamides, as signals required for efficient recovery of Steinernema infective juveniles. Stripteamides are synthesized from phenylethylamine and activated fatty acid substrates by StaS, a previously uncharacterized amide synthase. Mechanistic and structural analyses indicate that StaS evolved from the initiation module of a nonribosomal peptide synthetase (NRPS) and was repurposed to generate signaling metabolites for host development. Stripteamide signaling is conserved across Xenorhabdus species and operates in different Steinernema hosts. These findings reveal a new function for widespread bacterial acyl amides that previously have been shown as cytotoxic and affecting bacterial quorum sensing, highlighting the context dependence of microbial natural product language.

microbiology↗

bioRxiv2025

Insights into the linker domain in ABCB1/P-glycoprotein

ABCB1/P-glycoprotein(P-gp) is one of the best-known ABC transporters and its involvement in the clearance of chemotherapy agents in cancer cells can lead to multidrug resistance. The N- and C-terminal halves of P-gp are joined by a flexible linker region. Linker regions in ABC transporters often play pivotal roles (i.e. CFTR), however, there is no consensus on the importance of the role of the linker, the complete 3D-structure of which has not yet been determined. Prior studies on linker length showed major effects on P-gp function. In this work, we combined microsecond simulations ({approx}50{micro}s) on mouse and human P-glycoprotein, cryo-EM and other computational methods to trace a profile of the linker region. Different experimentally verified conditions involving the linker region were simulated, analysed and compared. Attachment of the linker to the membrane was spontaneously and occasionally encountered via a recurrent series of amino-acids, despite random starting conformations, suggesting that the lipid bilayer might be involved in linker regulation. As carried out in previous experiments, shortening the linker could potentially disconnect the ATPase activity from the substrate transport by interfering with one ATP site while introducing a short partially-structured linker shows remarkable stability in accelerated MD. Finally, we investigated the linker region by positioning it in specific quadrants of the 3D space, with simulations suggesting 1) a possible influence on protein flexibility, 2) selective tightening and opening of the two halves of P-gp, and a 3) possible stabilization of the inward-facing/occluded-state. The interaction between the linker and ATPs was investigated using protein-protein docking, simulations and a re-refined cryo-EM map where medium-low resolution and weak Coulomb density was found between the nucleotide binding domains.

biochemistry↗

bioRxiv2024

Adenomatous polyposis coli (APC) regulates internalization and signaling of the chemorepellent receptor, Roundabout (ROBO) 1

The SLIT-ROBO signaling pathway regulates axon guidance and cell migration, and ROBO1 is a receptor for SLIT ligands. ROBO1 undergoes constitutive endocytosis which is enhanced upon SLIT2 binding, but the molecular mechanisms and functional consequences of this process are not well understood. Using pharmacologic inhibitors and molecular techniques, we found that clathrin-mediated endocytosis is necessary for SLIT2-induced inhibition of cell spreading. To explore the underlying mechanisms, we performed BioID to identify ROBO1-interacting proteins whose association with the cytoplasmic domain of ROBO1 is differentially regulated by SLIT2. We discovered that adenomatous polyposis coli (APC), a multifunctional tumor suppressor, constitutively interacts with ROBO1 but dissociates upon binding of SLIT2 and that this dissociation is necessary for clathrin-mediated endocytosis of ROBO1 and subsequent effects on cell morphology. These findings provide new insights into the functional mechanisms by which SLIT2 binding to ROBO1 effects changes in actin cytoskeletal architecture.

cell biology↗

bioRxiv2022

Septin-Mediated RhoA Activation Engages the Exocyst Complex to Recruit the Cilium Transition Zone

Septins are filamentous GTPases that play important, but poorly characterized roles in ciliogenesis. Here, we show that SEPTIN9 regulates RhoA signaling at the base of cilia by binding and activating the RhoA guanine nucleotide exchange factor, ARHGEF18. GTP-RhoA is known to activate the membrane targeting exocyst complex, and suppression of SEPTIN9 causes disruption of ciliogenesis and mislocalization of an exocyst subunit, SEC8. Using basal body-targeted proteins we show that upregulating RhoA signaling at the cilium can rescue ciliary defects and mislocalization of SEC8 caused by global SEPTIN9 depletion. Moreover, we demonstrate that transition zone components, RPGRIP1L and TCTN2 fail to accumulate at the transition zone in cells lacking SEPTIN9 or depleted of the exocyst complex. Thus, SEPTIN9 regulates the recruitment of transition zone proteins on Golgi-derived vesicles by activating the exocyst via RhoA to allow the formation of primary cilia.

cell biology↗

bioRxiv2021

Structure of ABCB1/P-glycoprotein bound to the CFTR potentiator ivacaftor.

ABCB1 (P-glycoprotein) is an ATP binding cassette transporter that is involved in the clearance of xenobiotics and it affects the disposition of many drugs in the body. Here we have studied ABCB1 in the drug-bound and drug-free states, simultaneously, using high contrast cryo-electron microscopy imaging and a Volta phase plate. The binding of the potent CFTR potentiator, ivacaftor, at a site in the central aqueous cavity is mediated by transmembrane -helices 3,6,10,11 & 12. Binding is associated with a wider separation of the two halves of the transporter in the inward-facing state. Induced-fit changes the nucleotide binding domains in a way that may explain their increased affinity for ATP when drug is bound. Comparison of ivacaftor-bound structures of CFTR and ABCB1 suggests common features in the binding modes.

biochemistry↗

bioRxiv2018

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9A resolution

P-glycoprotein (ABCB1) is a ATP-binding cassette transporter that plays an important role in the removal of drugs and xenobiotic compounds from the cell. It is also associated with multi-drug resistance in cancer. Here we report novel features of the cryo-EM-derived structure of P-glycoprotein in the post-hydrolytic state: The cytosolic nucleotide-binding domains (NBDs) are separated despite ADP remaining bound to the NBDs. Gaps in the TMDs that connect to the inner hydrophilic cavity are back-filled by detergent head-groups from the annular detergent micelle and are close to two regions predicted to delineate two pseudo-symmetry-related drug-binding sites. In this conformation, the (newly-resolved) N-terminal extension, NBD-TMD linker region and gap-filling detergents all appear to impede NBD dimerisation. We propose a model for the mechanism of action of the exporter where ATP will be bound to the protein for most of the time, consistent with the high physiological ATP concentrations in vivo.

biochemistry↗