Vinculin (Vcn) and its splice variant metavinculin (MVcn) are cell adhesion proteins that regulate cell morphology, adhesion, and motility. They function as scaffold proteins that anchor membrane receptors to filamentous actin (F-actin) at focal adhesions and cell-cell junctions. MVcn bears an extra 68 amino acid insert in the tail domain and is selectively expressed in cardiac and smooth muscle cells at substoichiometric levels relative to Vcn. Mutations in the MVcn tail domain (MVt) promote cardiomyopathy, yet how these mutations alter ligand interactions to promote defects in force transduction and reduced blood flow is unclear. One difference between Vcn and MVcn lies in the ability to reorganize F-actin, with MVcn negatively regulating Vcn-mediated F-actin bundling. Vcn associates with phosphatidylinositol 4,5-bisphosphate (PIP2) through its tail domain (Vt) to drive recruitment, activation, and focal adhesion turnover. However, it remains unclear whether MVcn specifically associates with PIP2-containing membranes and how such interactions might influence its functional interplay with Vcn in tissues where both isoforms coexist. To evaluate the interaction of MVt and MVt cardiomyopathy mutants with PIP2 membranes in comparison with Vt, we conducted mutagenesis, phospholipid-association assays, and computational modeling. We found that MVt shows reduced association for PIP2-containing liposomes relative to Vt due to sequence differences within the insert region. Moreover, mutations in MVt that promote cardiomyopathies do not affect PIP2-dependent lipid association. These findings suggest that MVcn differs from Vcn in driving PIP2-mediated membrane association and sheds light on the coordinate role of Vcn and MVcn in membrane association as well as MVcn cardiomyopathy defects.

The E3 ubiquitin ligase TRIM67 is enriched in the central nervous system and is required for proper neuronal development. Previously we demonstrated TRIM67 coordinates with the closely related E3 ubiquitin ligase TRIM9 to regulate cytoskeletal dynamics downstream of the netrin-1 during axon guidance and axon branching in early neuronal morphogenesis. Interestingly, loss of Trim67 impacts cognitive flexibility in a spatial learning and memory task. Despite this behavioral phenotype, it was previously uninvestigated if TRIM67 was involved in synapse formation or function. Here we demonstrate TRIM67 localizes to the post-synaptic density (PSD) within dendritic spines. Furthermore, we show that loss of Trim67 significantly changes the PSD proteome, including changes in the regulation of the actin and microtubule cytoskeletons. Collectively, our data propose a synaptic role for TRIM67.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitination regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitination impacts VASP activity was unknown. Here we show that mimicking multi-monoubiquitination of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitinated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitinated VASP maintained the ability to bind and protect barbed ends from capping protein. Lastly, we demonstrate the introduction of recombinant multi-monoubiquitinated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitination controls VASP-mediated actin dynamics.

During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1-dependent axon guidance and branching. Here, we demonstrate that TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the postsynaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9. In addition, we document changes in the synaptic receptors associated with loss of Trim9. These defects converge on a loss of netrin-dependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose that TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.

The cerebellin family of proteins influences synapse formation and function. In this issue of PLOS Biology, Han and colleagues identify a new role for Cerebellin-1 in axon growth and guidance.

The development of a functional nervous system entails establishing connectivity between appropriate synaptic partners. During axonal pathfinding, the developing axon navigates through the extracellular environment, extending toward postsynaptic targets. In the early 1900s, Ramon y Cajal suggested that the growth cone, a specialized, dynamic, and cytoskeletal-rich structure at the tip of the extending axon, is guided by chemical cues in the extracellular environment. A century of work supports this hypothesis and introduced myriad guidance cues and receptors that promote a variety of growth cone behaviors including extension, pause, collapse, retraction, turning, and branching. Here, we highlight research from the last two years regarding pathways implicated in axon pathfinding.

Appropriate axon guidance is necessary to form accurate neuronal connections. Guidance cues stimulate reorganization of the cytoskeleton within the distal growth cone at the tip of the extending axon. Filopodia at the periphery of the growth cone have long been considered sensors for axon guidance cues, yet how they perceive and respond to extracellular cues remains ill-defined. Our previous work found that the filopodial actin polymerase VASP is regulated via TRIM9-dependent nondegradative ubiquitination, and that appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 antagonizes VASP ubiquitination by outcompeting the TRIM9:VASP interaction. This antagonistic role is required for netrin-1 dependent filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model that coordinated regulation of nondegradative VASP ubiquitination by a pair of ligases is a critical element of axon guidance.

Appropriate axon guidance is necessary to form accurate neuronal connections. Axon guidance cues that stimulate cytoskeletal reorganization within the growth cone direct axon navigation. Filopodia at the growth cone periphery have long been considered sensors for axon guidance cues, yet how they respond to extracellular cues remains ill defined. Our previous work found that the filopodial actin polymerase VASP and consequently filopodial stability are negatively regulated via nondegradative TRIM9-dependent ubiquitination. Appropriate VASP ubiquitination and deubiquitination are required for axon turning in response to the guidance cue netrin-1. Here we show that the TRIM9-related protein TRIM67 outcompetes TRIM9 for interacting with VASP and antagonizes TRIM9-dependent VASP ubiquitination. The surprising antagonistic roles of two closely related E3 ubiquitin ligases are required for netrin-1-dependent filopodial responses, axon turning and branching, and fiber tract formation. We suggest a novel model in which coordinated regulation of VASP ubiquitination by a pair of interfering ligases is a critical element of VASP dynamics, filopodial stability, and axon guidance.

Branched‐chain polyamines are found exclusively in thermophilic bacteria and Euryarchaeota and play essential roles in survival at high temperatures. In the present study, kinetic analyses of a branched‐chain polyamine synthase from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk‐BpsA) were conducted, showing that N4‐bis(aminopropyl)spermidine was produced by sequential additions of decarboxylated S‐adenosylmethionine (dcSAM) aminopropyl groups to spermidine, through bifunctional catalytic action. Tk‐BpsA catalyzed the aminopropylation of the linear‐chain polyamines spermidine, spermine, norspermidine, and the tertiary‐branched polyamines N4‐aminopropylspermidine and N4‐aminopropylnorspermidine, but not of short‐chain diamines, putrescine, and cadaverine, suggesting that Tk‐BpsA does not catalyze the aminopropylation of primary amino groups of diamines. X‐ray structural analyses of Tk‐BpsA in the presence or absence of the substrates spermidine and dcSAM revealed that a large, negatively charged cavity is responsible for the binding of branched‐chain substrates. The binding is different from that in the active site of linear polyamine spermidine/spermine synthases, and loop‐closures occur upon the binding of spermidine. Based on structural analyses, further kinetic studies were carried out for various mutants, revealing that Asp159, positioned between the reactive secondary amino group of the substrate polyamine and a sulfur atom of the product 5ʹ‐methylthioadenosine and in a Gly‐Asp‐Asp‐Asp motif, functions as a catalytic center, with reactions proceeding via a ping‐pong mechanism. Our study provides a novel aminopropyltransfer reaction mechanism, distinct from the SN2 displacement mechanism found in other known linear spermidine/spermine synthases.DatabaseAtomic coordinates and structure factors have been deposited in the Protein Data Bank with PDB codes 5XNF for apo‐Tk‐BpsA, 5XNH for the binary complex, and 5XNC for the ternary complex.

Host‐defense peptides (HDPs) feature evolution‐tested potency against life‐threatening pathogens. While piscidin 1 (p1) and piscidin 3 (p3) are homologous and potent fish HDPs, only p1 is strongly membranolytic. Here, we hypothesize that another mechanism imparts p3 strong potency. We demonstrate that the N‐termini of both peptides coordinate Cu2+ and p3‐Cu cleaves isolated DNA at a rate on par with free Cu2+ but significantly faster than p1‐Cu. On planktonic bacteria, p1 is more antimicrobial but only p3 features copper‐dependent DNA cleavage. On biofilms and persister cells, p3‐Cu is more active than p1‐Cu, commensurate with stronger peptide‐induced DNA damage. Molecular dynamics and NMR show that more DNA‐peptide interactions exist with p3 than p1, and the peptides adopt conformations simultaneously poised for metal‐ and DNA‐binding. These results generate several important conclusions. First, homologous HDPs cannot be assumed to have identical mechanisms since p1 and p3 eradicate bacteria through distinct relative contributions of membrane and DNA‐disruptive effects. Second, the nuclease and membrane activities of p1 and p3 show that naturally occurring HDPs can inflict not only physicochemical but also covalent damage. Third, strong nuclease activity is essential for biofilm and persister cell eradication, as shown by p3, the homolog more specific toward bacteria and more expressed in vascularized tissues. Fourth, p3 combines several physicochemical properties (e.g., Amino Terminal Copper and Nickel binding motif; numerous arginines; moderate hydrophobicity) that confer low membranolytic effects, robust copper‐scavenging capability, strong interactions with DNA, and fast nuclease activity. This new knowledge could help design novel therapeutics active against hard‐to‐treat persister cells and biofilms.

Lipopolysaccharide (LPS) is one of the most extensively studied pathogen-associated molecular patterns (PAMPs), as it composes 90% of the membrane in Gram-negative bacteria. Recognition of micellar aggregates of LPS by the immune system subsequently mounts an inflammatory response against infection. However, if this process is over exaggerated, high levels of cytokines may become detrimental, leading to organ shutdown and septic shock. Various cationic antimicrobial peptides (AMPs) have been shown to decrease the transcription and release of pro-inflammatory cytokines associated with LPS recognition. Piscidin is a cationic antimicrobial peptide first isolated in the mast cells of fish. In the presence of a lipid bilayer, it folds into an amphipathic α-helix structure, which facilitates peptide-lipid bilayer binding. Based on the studies of other cationic AMPs, it was hypothesized that piscidin-1 (P1) and piscidin-3 (P3) would fold into their α-helical state and bind strongly to LPS, breaking apart its large aggregates. Using isothermal calorimetry to explore the thermodynamics of binding, it was shown that both peptides bound strongly to LPS through an exothermic reaction. Furthermore, fluorescence-dequenching studies were carried out using Fluorescein isothiocyanate labeled LPS (FITC-LPS), demonstrating piscidin's ability to separate FITC-LPS aggregates. Finally, both peptides were induced by LPS to be approximately 97% helical from their native random coil state, as shown by circular dichroism. Based upon these results, it is clear that significant interactions occur between LPS and piscidin, warranting future exploration of the effect of piscidin on LPS recognition by immune cells.