Mutation of the ALS-/FTD-Associated RNA-Binding Protein FUS Affects Axonal Development.

Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.

Biosensor for Multimodal Characterization of an Essential ABC Transporter for Next-Generation Antibiotic Research.

As the threat of antibiotic resistance increases, there is a particular focus on developing antimicrobials against pathogenic bacteria whose multidrug resistance is especially entrenched and concerning. One such target for novel antimicrobials is the ATP-binding cassette (ABC) transporter MsbA that is present in the plasma membrane of Gram-negative pathogenic bacteria where it is fundamental to the survival of these bacteria. Supported lipid bilayers (SLBs) are useful in monitoring membrane protein structure and function since they can be integrated with a variety of optical, biochemical, and electrochemical techniques. Here, we form SLBs containing Escherichia coli MsbA and use atomic force microscopy (AFM) and structured illumination microscopy (SIM) as high-resolution microscopy techniques to study the integrity of the SLBs and incorporated MsbA proteins. We then integrate these SLBs on microelectrode arrays (MEA) based on the conducting polymer poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) using electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA proteins in response to ATP hydrolysis. These EIS measurements can be correlated with the biochemical detection of MsbA-ATPase activity. To show the potential of this SLB approach, we observe not only the activity of wild-type MsbA but also the activity of two previously characterized mutants along with quinoline-based MsbA inhibitor G907 to show that EIS systems can detect changes in ABC transporter activity. Our work combines a multitude of techniques to thoroughly investigate MsbA in lipid bilayers as well as the effects of potential inhibitors of this protein. We envisage that this platform will facilitate the development of next-generation antimicrobials that inhibit MsbA or other essential membrane transporters in microorganisms.

Tuning riboflavin derivatives for photodynamic inactivation of pathogens.

The development of effective pathogen reduction strategies is required due to the rise in antibiotic-resistant bacteria and zoonotic viral pandemics. Photodynamic inactivation (PDI) of bacteria and viruses is a potent reduction strategy that bypasses typical resistance mechanisms. Naturally occurring riboflavin has been widely used in PDI applications due to efficient light-induced reactive oxygen species (ROS) release. By rational design of its core structure to alter (photo)physical properties, we obtained derivatives capable of outperforming riboflavin's visible light-induced PDI against E. coli and a SARS-CoV-2 surrogate, revealing functional group dependency for each pathogen. Bacterial PDI was influenced mainly by guanidino substitution, whereas viral PDI increased through bromination of the flavin. These observations were related to enhanced uptake and ROS-specific nucleic acid cleavage mechanisms. Trends in the derivatives' toxicity towards human fibroblast cells were also investigated to assess viable therapeutic derivatives and help guide further design of PDI agents to combat pathogenic organisms.

Nano-vehicles give new lease of life to existing antimicrobials.

Antibiotic resistance has become one of the greatest challenges for modern medicine, and new approaches for the treatment of bacterial infections are urgently needed to avoid widespread vulnerability again to infections that have so far been easily treatable with existing drugs. Among the many approaches investigated to overcome this challenge is the use of engineered nanostructures for the precise and targeted delivery of existing antimicrobial agents in a fashion that will potentiate their effect. This idea leans on lessons learned from pioneering research in cancer, where the targeted delivery of anti-cancer drugs to mammalian cells has been a topic for some time. In particular, new research has demonstrated that nanomaterials can be functionalised with active antimicrobials and, in some cases, with targeting molecules that potentiate the efficiency of the antimicrobials. In this mini-review, we summarise results that demonstrate the potential for nanoparticles, dendrimers and DNA nanostructures for use in antimicrobial delivery. We consider material aspects of the delivery vehicles and ways in which they can be functionalised with antibiotics and antimicrobial peptides, and we review evidence for their efficacy to kill bacteria both in vitro and in vivo. We also discuss the advantages and limitations of these materials and highlight the benefits of DNA nanostructures specifically for their versatile potential in the present context.

Turning the Mre11/Rad50 DNA repair complex on its head: lessons from SMC protein hinges, dynamic coiled-coil movements and DNA loop-extrusion?

The bacterial SbcC/SbcD DNA repair proteins were identified over a quarter of a century ago. Following the subsequent identification of the homologous Mre11/Rad50 complex in the eukaryotes and archaea, it has become clear that this conserved chromosomal processing machinery is central to DNA repair pathways and the maintenance of genomic stability in all forms of life. A number of experimental studies have explored this intriguing genome surveillance machinery, yielding significant insights and providing conceptual advances towards our understanding of how this complex operates to mediate DNA repair. However, the inherent complexity and dynamic nature of this chromosome-manipulating machinery continue to obfuscate experimental interrogations, and details regarding the precise mechanisms that underpin the critical repair events remain unanswered. This review will summarize our current understanding of the dramatic structural changes that occur in Mre11/Rad50 complex to mediate chromosomal tethering and accomplish the associated DNA processing events. In addition, undetermined mechanistic aspects of the DNA enzymatic pathways driven by this vital yet enigmatic chromosomal surveillance and repair apparatus will be discussed. In particular, novel and putative models of DNA damage recognition will be considered and comparisons will be made between the modes of action of the Rad50 protein and other related ATPases of the overarching SMC superfamily.

Coordinated regulation of the ESCRT-III component CHMP4C by the chromosomal passenger complex and centralspindlin during cytokinesis.

The chromosomal passenger complex (CPC)-composed of Aurora B kinase, Borealin, Survivin and INCENP-surveys the fidelity of genome segregation throughout cell division. The CPC has been proposed to prevent polyploidy by controlling the final separation (known as abscission) of the two daughter cells via regulation of the ESCRT-III CHMP4C component. The molecular details are, however, still unclear. Using atomic force microscopy, we show that CHMP4C binds to and remodels membranes in vitro Borealin prevents the association of CHMP4C with membranes, whereas Aurora B interferes with CHMP4C's membrane remodelling activity. Moreover, we show that CHMP4C phosphorylation is not required for its assembly into spiral filaments at the abscission site and that two distinctly localized pools of phosphorylated CHMP4C exist during cytokinesis. We also characterized the CHMP4C interactome in telophase cells and show that the centralspindlin complex associates preferentially with unphosphorylated CHMP4C in cytokinesis. Our findings indicate that gradual dephosphorylation of CHMP4C triggers a 'relay' mechanism between the CPC and centralspindlin that regulates the timely distribution and activation of CHMP4C for the execution of abscission.

Demonstration of ligand decoration, and ligand-induced perturbation, of G-quadruplexes in a plasmid using atomic force microscopy.

G-Quadruplexes are nucleic acid secondary structures consisting of a planar arrangement of four guanine residues. Potential G-quadruplex-forming sequences are widely distributed throughout the genome. Significantly, they are present in telomeres and are enriched in gene promoters and first introns, raising the possibility that perturbation of G-quadruplex stability might have therapeutic potential, for example in the treatment of cancer. Ligands that interact selectively with G-quadruplexes include both proteins and small molecules, although the interactions between ligands and their G-quadruplex targets have been monitored using indirect methods. In addition, the G-quadruplex targets have often been short DNA fragments. Here, we have used atomic force microscopy imaging to examine directly at the single-molecule level the interaction of ligands with G-quadruplexes generated during transcription of a plasmid containing a G-rich insert. We show that the structures produced during transcription are decorated specifically by the single-chain antibody HF1 and by the nuclear protein PARP-1, both of which are known to recognize G-quadruplexes. Our results provide clear structural evidence of G-quadruplex formation in a transcription-dependent case and demonstrate directly how small-molecule stabilizers and destabilizers can manipulate these structures in a biochemically functional system.