High-resolution imaging of plasma membrane proteins
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Dact1 induces Dishevelled oligomerization to facilitate binding partner switch and signalosome formation during convergent extension
Convergent extension (CE) is a universal morphogenetic engine that promotes polarized tissue extension. In vertebrates, CE is regulated by non-canonical Wnt ligands signaling through “core” proteins of the planar cell polarity (PCP) pathway, including the cytoplasmic protein Dishevelled (Dvl), receptor Frizzled (Fz) and tetraspan protein Van gogh-like (Vangl). PCP was discovered in Drosophila to coordinate polarity in the plane of static epithelium, but does not regulate CE in flies. Existing evidence suggests that adopting PCP for CE might be a vertebrate-specific adaptation with incorporation of new regulators. Herein we use Xenopus to investigate Dact1, a chordate-specific protein. Dact1 induces Dvl to form oligomers that dissociate from Vangl, but stay attached with Fz as signalosome-like clusters and co-aggregate with Fz into protein patches upon non-canonical Wnt induction. Functionally, Dact1 antagonizes Vangl, and synergizes with wild-type Dvl but not its oligomerization-defective mutants. We propose that, by promoting Dvl oligomerization, Dact1 couples Dvl binding partner switch with signalosome-like cluster formation to initiate non-canonical Wnt signaling during vertebrate CE.
Ion channel traffic jams: the significance of trafficking deficiency in long QT syndrome
A well-balanced ion channel trafficking machinery is paramount for the normal electromechanical function of the heart. Ion channel variants and many drugs can alter the cardiac action potential and lead to arrhythmias by interfering with mechanisms like ion channel synthesis, trafficking, gating, permeation, and recycling. A case in point is the Long QT syndrome (LQTS), a highly arrhythmogenic disease characterized by an abnormally prolonged QT interval on ECG produced by variants and drugs that interfere with the action potential. Disruption of ion channel trafficking is one of the main sources of LQTS. We review some molecular pathways and mechanisms involved in cardiac ion channel trafficking. We highlight the importance of channelosomes and other macromolecular complexes in helping to maintain normal cardiac electrical function, and the defects that prolong the QT interval as a consequence of variants or the effect of drugs. We examine the concept of “interactome mapping” and illustrate by example the multiple protein–protein interactions an ion channel may undergo throughout its lifetime. We also comment on how mapping the interactomes of the different cardiac ion channels may help advance research into LQTS and other cardiac diseases. Finally, we discuss how using human induced pluripotent stem cell technology to model ion channel trafficking and its defects may help accelerate drug discovery toward preventing life-threatening arrhythmias. Advancements in understanding ion channel trafficking and channelosome complexities are needed to find novel therapeutic targets, predict drug interactions, and enhance the overall management and treatment of LQTS patients.
In vivo surface-enhanced Raman scattering techniques: nanoprobes, instrumentation, and applications
Surface-enhanced Raman scattering (SERS) has emerged as a powerful tool in various biomedical applications, including in vivo imaging, diagnostics, and therapy, largely due to the development of near-infrared (NIR) active SERS substrates. This review provides a comprehensive overview of SERS-based applications in vivo, focusing on key aspects such as the design considerations for SERS nanoprobes and advancements in instrumentation. Topics covered include the development of NIR SERS substrates, Raman label compounds (RLCs), protective coatings, and the conjugation of bioligands for targeted imaging and therapy. The review also discusses microscope-based configurations such as scanning, widefield imaging, and fiber-optic setups. Recent advances in using SERS nanoprobes for in vivo sensing, diagnostics, biomolecule screening, multiplex imaging, intraoperative guidance, and multifunctional cancer therapy are highlighted. The review concludes by addressing challenges in the clinical translation of SERS nanoprobes and outlines future directions, emphasizing opportunities for advancing biomedical research and clinical applications.
Vastly different energy landscapes of the membrane insertions of monomeric gasdermin D and A3
Gasdermin D and gasdermin A3 belong to the same family of pore-forming proteins and executors of pyroptosis, a form of programmed cell death. To unveil the process of their pore formation, we examine the energy landscapes upon insertion of the gasdermin D and A3 monomers into a lipid bilayer by extensive atomistic molecular dynamics simulations. We reveal a lower free energy barrier of membrane insertion for gasdermin D than for gasdermin A3 and a preference of gasdermin D for the membrane-inserted and of gasdermin A3 for the membrane-adsorbed state, suggesting that gasdermin D first inserts and then oligomerizes while gasdermin A3 oligomerizes and then inserts. Gasdermin D stabilizes itself in the membrane by forming more salt bridges and pulling phosphatidylethanolamine lipids and more water into the membrane. Gasdermin-lipid interactions support the pore formation. Our findings suggest that both the gasdermin species and the lipid composition modulate gasdermin pore formation.
Anionic lipids direct efficient microfluidic encapsulation of stable and functionally active proteins in lipid nanoparticles
Because proteins do not efficiently pass through the plasma membrane, protein therapeutics are limited to target ligands located at the cell surface or in serum. Lipid nanoparticles can facilitate delivery of polar molecules across a membrane. We hypothesized that because most proteins are amphoteric ionizable polycations, proteins would associate with anionic lipids, enabling microfluidic chip assembly of stable EP-LNPs (Encapsulated Proteins in Lipid NanoParticles). Here, by employing anionic lipids we were able to efficiently load proteins into EP-LNPs at protein:lipid w:w ratios of 1:20. Several proteins with diverse molecular weights and isoelectric points were encapsulated at efficiencies of 70 75%–90% and remained packaged for several months. Proteins packaged in EP-LNPs efficiently entered mammalian cells and fungal cells with cell walls. The proteins delivered intracellularly were functional. EP-LNPs technology should improve cellular delivery of medicinal antibodies, enzymes, peptide antimetabolites, and dominant negative proteins, opening new fields of protein therapeutics
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