Biology subjects

Goodman, K. M.

Publications and source records attributed to Goodman, K. M..

3 recordsLinked to original sources

bioRxiv2021

How clustered protocadherin binding specificity is tuned for neuronal self/non-self-recognition

The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self/non-self- discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans-homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self/non-self-discrimination.

biophysics↗

bioRxiv2020

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFR co-receptors recognise and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2-CLD3. Comparison with a cryo-EM structure of a ligand-engaged zebrafish RETECD-GDNF-GFR1 complex indicates conformational changes within a clade-specific CLD3 loop adjacent to co-receptor. Our observations indicate RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFR co-receptors, while its rigid arm recognises a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualise linear arrays of RETECD-GDNF-GFR1 suggesting a conserved contact stabilises higher-order species. Our study reveals ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands. HighlightsO_LICrystal structure of zebrafish RET cadherin-like module reveals conformational flexibility at the calcium-dependent CLD2-CLD3 interface C_LIO_LIComparison of X-ray and cryo-EM structures indicate conformational differences between unliganded and liganded RET involving a clade-specific CLD3 loop C_LIO_LIStrict spatial separation of RETECD C-termini is imposed by each cysteine-rich domain interaction with GFL dimer C_LIO_LIDifferences in co-receptor engagement and higher-order ligand-bound RET complexes indicate potentially divergent signalling mechanisms C_LI

biophysics↗

bioRxiv2019

Calcium-induced calcium release in noradrenergic neurons of the locus coeruleus

The locus coeruleus (LC) is a nucleus within the brainstem that consists of norepinephrine-releasing neurons. It is involved in broad processes including autonomic regulation, and cognitive and emotional functions such as arousal, attention and anxiety. Understanding the mechanisms that control the excitability of LC neurons is important because they innervate widespread regions of the central nervous system. One of the key regulators is the cytosolic calcium concentration ([Ca2+]c), the increases in which can be amplified by calcium-induced calcium release (CICR) from the intracellular calcium stores. Although the electrical activities of LC neurons are regulated by changes in [Ca2+]c, the extent of CICR involvement in this regulation has remained unclear. Here we show that CICR hyperpolarizes acutely dissociated LC neurons of the rat brain and demonstrate the pathway whereby it does this. When CICR was activated by extracellular application of 10 mM caffeine, LC neurons were hyperpolarized in the current-clamp mode of the patch-clamp recording, and the majority of neurons showed an outward current in the voltage-clamp mode. This outward current was accompanied by an increase in membrane conductance, and its reversal potential was close to the K+ equilibrium potential, indicating that it is mediated by the opening of K+ channels. The outward current was generated in the absence of extracellular calcium and was blocked when the calcium stores were inhibited by applying ryanodine. Pharmacological experiments indicated that the outward current was mediated by Ca2+-activated K+ channels of the non-small conductance type. Finally, the application of caffeine led to an increase in the [Ca2+]c in these neurons, as visualized by fluorescence microscopy. These findings delineate a mechanism whereby CICR suppresses the electrical activity of LC neurons, and indicate that it could play a dynamic role in modulating the LC-mediated noradrenergic tone in the brain.

neuroscience↗