Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of gross synapse density in the ventral neuropil region at a specific larval stage. This reduction in the gross synapse density coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruits a PX-domain-containing motor protein, Klp98A, accelerating their synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via Vps34-depedent regulation of directed axonal transport of endosomes.

Significant advances in microscopy methods for biomedical applications have been made in the past two decades, and these methods are now being actively pursued for virus research [...]

The GTPase Rab4, which is essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed transient contraction of gross synapse density in the ventral neuropil region at a specific larval stage. This reduction in the gross synapse density coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruits a PX-domain-containing motor protein, Klp98A, accelerating their synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via Vps34-depedent regulation of directed axonal transport of endosomes.

Sensory cilia have a complex bipartite architecture containing 9+0 connecting cilia at the inner segment and singlet microtubule-supported highly membranous outer segments essential for receptor display. The mechanisms underlying the formation of such highly branched morphology and its microtubule-rich ciliary cytoskeleton are unclear. Here, we show that individual olfactory cilium inside the large basiconic sensillum grows in episodic steps following several pulsatile influxes of tubulin in developing Drosophila antenna. Transient elevations of the microtubule end-binding protein EB1 precede the tubulin influx events. We also demonstrate that EB1 interacts explicitly with the cargo-binding tail domain of Drosophila KLP68D, a kinesin-2β orthologue. Loss of EB1 in olfactory neurons during the outer segment growth reduces the tubulin influx and affects cilia stability. Finally, we show that the EB1 and tubulin influxes into the distal outer segment of the olfactory cilia require kinesin-2. Altogether, our findings elucidate a role of active EB1 transport in promoting the growth and stability of long-lived metazoan cilia involved in sensory perception.

In this study we have applied high-spatial and temporal label-free imaging of individual live multidrug-resistant bacteria and bacteria-infected cells and animal tissue using two-photon fluorescence lifetime imaging microscopy (2p-FLIM). 2p-FLIM can identify and quantify fluorescence intensity and lifetimes among bacteria, infected cells, and tissues. We have implemented 2p-FLIM in combination with phasor plot analysis for quantifying molecular differences of NAD(P)H intensities and lifetimes for fast, sensitive, high-resolution, non-destructive imaging of live bacteria and bacteria-infected cells and tissues. We have further developed a coordinated workflow using 2p-FLIM for high-resolution temporal-spatial mapping of bacteria infected cells and tissues that can be performed for near real-time quantitation of NAD(P)H intensities and lifetimes for identifying changes in metabolism. 2p-FLIM may have broad applicability for characterizing microbial infection at the molecular, subcellular, cellular and tissue levels. The ability to quantitate and directly monitor changes in NAD(P)H metabolism in near real-time in bacteria cells and tissues during an infection, offers a potential mechanism for understanding microbial pathogenesis and evaluating therapeutic treatments that can be applied to multiple model systems. Overall, the application of this label-free imaging approach has the potential to address biomedical research needs and technical problems that occur broadly across multiple biological systems and diseases.

DNA‐templated silver nanoclusters promise subwavelength light control for quantum photonics, sensing, and bioimaging. Yet deterministic one‐per‐site growth has lacked explicit quantitative criteria. Here, we define quantitative thresholds that render site‐specific nucleation of single silver nanoclusters reliable on DNA origami. By systematically varying oligocytosine (OligoC) handle number and Ag + reduction conditions, we identify a cooperative rule‐of‐10 OligoC handles per site and an AgNO 3 loading window that together produce one nanocluster per site with nanometer registration. To enable tailored emitter permutations, we program one‐, two‐, and three‐site arrays and show that the same thresholds hold for both substrate‐bound and solution‐phase syntheses. Correlative atomic force microscopy and single‐molecule two‐photon fluorescence lifetime imaging microscopy verify single‐emitter behavior, bright two‐photon fluorescence, single‐step photobleaching, and ≈1.2 ns lifetimes. We expect that these explicit rules provide physical insight into DNA‐directed nucleation and open practical routes to addressable quantum emitters and scalable nanocluster arrays for integrated nanophotonics and single‐molecule sensing.

The envelope (Env) spike (S) trimers of known SARS-CoV-2 strains have evolved to display conformational fluctuations between structural states, which in turn impact host receptor binding efficiency, the mechanism of infection, and susceptibility to antibody binding. Previously, we reported evidence that virion trimers alternate between two end states: one functional and the other nonfunctional. These dynamics, coupled with the close positioning of virion trimers, may constrain the total number of reactive mAb or receptor target sites. Such stoichiometric limits are important considerations for the infection efficiency and susceptibility to various antibody-mediated antiviral mechanisms. Here, we address this question using single-molecule fluorescence detection (SMD) methods and a step-detection algorithm to quantify bound ligands as a function of their stepwise fluorescence photobleaching on a captured virion. Importantly, this approach does not involve genetic or chemical modifications of the virion spike. Single-molecule photobleaching traces provide valuable information regarding the stoichiometry of receptor and/or ligand binding and accessibility of the binding sites present on a single virion, which can prove to be an impetus for improved vaccine designs based on stabilizing common transition state structures in the S trimer that might better elicit more cross-reactive antibodies. We find that the expected three photobleaching steps are detected with soluble trimers of the Wuhan and Omicron variants when reacted with soluble receptors or half-mAbs; i.e., reflecting 3:1 binding stoichiometry. On virions, a range of receptor binding stoichiometries was detected in 37 °C reactions, with a maxima of 12:1 or 9:1 for the Wuhan or Omicron variants, respectively. The Wuhan strain favored higher stoichiometries. The stoichiometries of binding to labeled half-mAbs were also distributed but with less interstrain variance, showing a maxima of 5:1-6:1 mAb/virion. In either case, such values were far lower than the theoretical number of spike binding sites on a virion. Notably, increasing the reaction temperature to 42 °C decreased receptor binding stoichiometry. Overall, these data indicate that a substantial fraction of S trimers on virions are constrained from binding receptors or certain cognate antibodies, either constitutively and/or due to the liganding of neighboring trimers, while a smaller fraction remains capable of achieving or maintaining a functional configuration.

Bacterial TIR proteins identified in pathogenic, nonpathogenic, commensal, and archaea have identified NADase activity, raising new and exciting questions about how bacterial TIR domains function in immunity. To answer questions about the role of bacterial TIR NADase activity and the regulation of human Toll and IL-1R signaling, we have sought to characterize the molecular mechanism of a bacterial TIR protein with NADase activity. Accordingly, we have determined the structure of the Acinetobacter baumannii Toll-Interleukin-1 receptor resistance domain protein (AbTIR), confirmed its enzymatic function in NAD+ hydrolysis, and mapped its interaction with NAD+ using HDX-MS. Upon binding NAD+, AbTir-TIR exhibits unique EX1 kinetics and large cooperative conformational changes. As part of this molecular characterization, we recently carried out a Course-Based Undergraduate Research Experience (CURE), which identified residues critical for bacterial TIR domain NADase activity. Students used bioinformatics to identify residues they hypothesized would play a role in NADase function. The results from these student-led studies support observations that NAD+ binding induces conformational changes within the bacteria TIR domain, as identified by hydrogen-deuterium exchange mass spectrometry studies and cryogenic electron microscopy. Collectively, these studies provide new insight and understanding into the molecular mechanisms of NADase-active bacterial TIR proteins. HHMI Inclusive Excellence Grant to Towson University Microbial, Parasitic, and Fungal Immunology (MPF)

Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of synapses in the ventral neuropil region at a specific larval stage. This coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruit a PX-domain-containing motor protein, Klp98A, accelerating synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via insulin signaling and directed axonal transport of endosomes. ### Competing Interest Statement The authors have declared no competing interest.

Fluorescence spectroscopy serves as an ultrasensitive sophisticated tool where background noises which serve as a major impediment to the detection of the desired signals can be safely avoided for detections down to the single-molecule levels. One such way of bypassing background noise is plasmon-enhanced fluorescence (PEF), where the interactions of fluorophores at the surface of metals or plasmonic nanoparticles are probed. The underlying condition is a significant spectral overlap between the localized surface plasmon resonance (LSPR) of the nanoparticle and the absorption or emission spectra of the fluorophore. The rationale being the coupling of the excited state of the fluorophore with the localized surface plasmon leads to an augmented emission, owing to local field enhancement. It is manifested in enhanced quantum yields concurrent with a decrease in fluorescence lifetimes, owing to an increase in radiative rate constants. This improvement in detection provided by PEF allows a significant scope of expansion in the domain of weakly emitting fluorophores which otherwise would have remained unperceivable. The concept of coupling of weak emitters with plasmons can bypass the problems of photobleaching, opening up avenues of imaging with significantly higher sensitivity and improved resolution. Furthermore, amplification of the emission signal by the coupling of free electrons of the metal nanoparticles with the electrons of the fluorophore provides ample opportunities for achieving lower detection limits that are involved in biological imaging and molecular sensing. One avenue that has attracted significant attraction in the last few years is the fast, label-free detection of bio-analytes under physiological conditions using plasmonic nanoparticles for point-of-care analysis. This review focusses on the applications of plasmonic nanomaterials in the field of biosensing, imaging with a brief introduction on the different aspects of LSPR and fabrication techniques.

In all tailed phages, the packaging of the double-stranded genome into the head by a terminase motor complex is an essential step in virion formation. Despite extensive research, there are still major gaps in the understanding of this highly dynamic process and the mechanisms responsible for DNA translocation. Over the last fifteen years, single-molecule fluorescence technologies have been applied to study viral nucleic acid packaging using the robust and flexible T4 in vitro packaging system in conjunction with genetic, biochemical, and structural analyses. In this review, we discuss the novel findings from these studies, including that the T4 genome was determined to be packaged as an elongated loop via the colocalization of dye-labeled DNA termini above the portal structure. Packaging efficiency of the TerL motor was shown to be inherently linked to substrate structure, with packaging stalling at DNA branches. The latter led to the design of multiple experiments whose results all support a proposed torsional compression translocation model to explain substrate packaging. Evidence of substrate compression was derived from FRET and/or smFRET measurements of stalled versus resolvase released dye-labeled Y-DNAs and other dye-labeled substrates relative to motor components. Additionally, active in vivo T4 TerS fluorescent fusion proteins facilitated the application of advanced super-resolution optical microscopy toward the visualization of the initiation of packaging. The formation of twin TerS ring complexes, each expected to be ~15 nm in diameter, supports a double protein ring–DNA synapsis model for the control of packaging initiation, a model that may help explain the variety of ring structures reported among pac site phages. The examination of the dynamics of the T4 packaging motor at the single-molecule level in these studies demonstrates the value of state-of-the-art fluorescent tools for future studies of complex viral replication mechanisms.

Through applications of fluorescence correlation spectroscopy, our research has provided a unique view of HIV virion-antibody interactions. We developed a novel FRET-FCS based assay to identify how neutralizing and non-neutralizing epitopes are expressed on single virions. Most of our methods can be expanded for application to studies of in vitro primary infection systems. Recently, we developed a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging NADH metabolism of virally infected cells and tissue sections.

Like a photoreceptor cilium, the sensory cilia have a complex bipartite architecture containing 9+0 connecting cilium at the base and a singlet microtubule-supported, highly membranous outer segment, essential for the receptor display. How such diverse cilia morphology and underlying microtubule cytoskeleton develops remains unclear. Here we show that individual olfactory cilium, inside the large basiconic sensilla in developing Drosophila antenna, grows in episodic steps following several pulsatile influxes of tubulin. Each tubulin influx event is preceded by transient elevations of a microtubule-stabilising protein, the End-binding protein 1 (EB1). Additionally, EB1 is found to specifically interact with the tail domain of Drosophila KLP68D, an orthologue of the kinesin-2β motor subunit, in vitro . Finally, the loss of EB1 in olfactory neurons preceding the growth surges reduces the tubulin influx as well as arrests the olfactory cilia assembly and stability. These findings suggest a novel mechanism of bipartite cilia assembly. ### Competing Interest Statement The authors have declared no competing interest.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2.

Abstract We have developed label-free imaging methods to identify and characterize microbial infection of live cells and tissues using a novel application of Fluorescence lifetime (FLT) imaging with 2-photon excitation (2p-FLIM) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). 2p-FLIM is often used in cancer biology to image metabolically altered cells of affected tissues and tumors. Whereas MALDI-MSI is one of the growing number of spatial ‘omic methods, that is also label-free and allows mapping of individual metabolites at cellular and tissue scales. We have previously reported the application of 2p-FLIM to characterize HIV-1-infected cells and tissues. We now report the novel application and integration of 2p-FLIM with MALDI-MSI for characterizing pathogenic bacteria patient isolates and infected tissue. We observe increases in NAD(P)H intensity and unique distributions of fluorescence lifetimes (FLTs) for select pathogenic bacteria patient isolates, which may reflect differences in OxPhos metabolism. We have further characterized Pseudomonas-infected mouse lung tissues which exhibit unique NAD(P)H fluorescence, average FLTs and spatial mapping of individual NAD+-associated metabolites. A highly integrative, cross-platform, label-free, multi-omics approach involving 2p-FLIM and MALDI-MSI may provide a better understanding of microbial infection for developing and assessing novel antimicrobial and immune-modulatory interventions.

Rab4 GTPase organizes endosomal sorting essential for maintaining the balance between recycling and degradative pathways. Rab4 localizes to many cargos whose transport in neurons is critical for regulating neurotransmission and neuronal health. Furthermore, elevated Rab4 levels in the CNS are associated with synaptic atrophy and neurodegeneration in Drosophila and humans, respectively. However, how the transport of Rab4-associated vesicles is regulated in neurons remains unknown. Using in vivo time-lapse imaging of Drosophila larvae, we show that activation of insulin signaling via Dilp2 and dInR increases the anterograde velocity, run length, and flux of Rab4 vesicles in the axons. Molecularly, we show that activation of neuronal insulin signaling further activates Vps34, elevates the levels of PI(3)P on Rab4-associated vesicles, recruits Klp98A (a PI(3)P-binding kinesin-3 motor) and activates their anterograde transport. Together, these observations delineate the role of insulin signaling in regulating axonal transport and synaptic homeostasis.

Colloidal metamaterials are highly desired artificial materials that recapitulate the structure of simple molecules. They exhibit exceptional functionalities conferred by the organization of and specific interaction among constituent elements. Harvesting such exquisite attributes for potential applications necessitates establishing precise control over their structural configuration with high precision. Yet, creating molecule-like small clusters of colloidal metamaterials remains profoundly challenging, as a lack of regioselectively encoded surface chemical heterogeneity prevents specific recognition interactions. Herein, we report a new strategy by harnessing magnetic -bead-assisted DNA cluster transferring to create discretely DNA cluster-patched nanoparticles for the self-assembly of colloidal metamaterials. This strategy affords broad generalizability and scalability for robustly patching DNA clusters on nanoparticles unconstrained by geometrical, dimensional, and compositional complex-ities commonly encountered in colloidal materials at the nano-and microscale. We direct judiciously patched nanoparticles into a wide variety of nanoassemblies and present a case study demonstrating the distinct metamaterial properties in enhancing the spontaneous emission of diamond nanoparticles. This newly invented strategy is readily implementable and extendable to construct a palette of structurally sophisticated and functionality-explicit architecture, paving the way for nanoscale manipulation of colloidal material functionalities with wide-ranging applications for biological sensing, optical engineering, and catalytic chemistry.