MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement.

Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.

Polymer Dots with Enhanced Photostability, Quantum Yield, and Two-Photon Cross-Section using Structurally Constrained Deep-Blue Fluorophores.

Semiconducting polymer dots (Pdots) have emerged as versatile probes for bioanalysis and imaging at the single-particle level. Despite their utility in multiplexed analysis, deep blue Pdots remain rare due to their need for high-energy excitation and sensitivity to photobleaching. Here, we describe the design of deep blue fluorophores using structural constraints to improve resistance to photobleaching, two-photon absorption cross sections, and fluorescence quantum yields using the hexamethylazatriangulene motif. Scanning tunneling microscopy was used to characterize the electronic structure of these chromophores on the atomic scale as well as their intrinsic stability. The most promising fluorophore was functionalized with a polymerizable acrylate handle and used to give deep-blue fluorescent acrylic polymers with Mn > 18 kDa and D < 1.2. Nanoprecipitation with amphiphilic polystyrene-graft-(carboxylate-terminated poly(ethylene glycol)) gave water-soluble Pdots with blue fluorescence, quantum yields of 0.81, and molar absorption coefficients of (4 ± 2) × 108 M-1 cm-1. This high brightness facilitated single-particle visualization with dramatically improved signal-to-noise ratio and photobleaching resistance versus an unencapsulated dye. The Pdots were then conjugated with antibodies for immunolabeling of SK-BR3 human breast cancer cells, which were imaged using deep blue fluorescence in both one- and two-photon excitation modes.

Photoluminescent Nanoparticles for Chemical and Biological Analysis and Imaging.

Research related to the development and application of luminescent nanoparticles (LNPs) for chemical and biological analysis and imaging is flourishing. Novel materials and new applications continue to be reported after two decades of research. This review provides a comprehensive and heuristic overview of this field. It is targeted to both newcomers and experts who are interested in a critical assessment of LNP materials, their properties, strengths and weaknesses, and prospective applications. Numerous LNP materials are cataloged by fundamental descriptions of their chemical identities and physical morphology, quantitative photoluminescence (PL) properties, PL mechanisms, and surface chemistry. These materials include various semiconductor quantum dots, carbon nanotubes, graphene derivatives, carbon dots, nanodiamonds, luminescent metal nanoclusters, lanthanide-doped upconversion nanoparticles and downshifting nanoparticles, triplet-triplet annihilation nanoparticles, persistent-luminescence nanoparticles, conjugated polymer nanoparticles and semiconducting polymer dots, multi-nanoparticle assemblies, and doped and labeled nanoparticles, including but not limited to those based on polymers and silica. As an exercise in the critical assessment of LNP properties, these materials are ranked by several application-related functional criteria. Additional sections highlight recent examples of advances in chemical and biological analysis, point-of-care diagnostics, and cellular, tissue, and in vivo imaging and theranostics. These examples are drawn from the recent literature and organized by both LNP material and the particular properties that are leveraged to an advantage. Finally, a perspective on what comes next for the field is offered.

Near-Infrared-Emitting Boron-Difluoride-Curcuminoid-Based Polymers Exhibiting Thermally Activated Delayed Fluorescence as Biological Imaging Probes.

Near-infrared-emitting polymers were prepared using four boron-difluoride-curcuminoid-based monomers using ring-opening metathesis polymerization (ROMP). Well-defined polymers with molecular weights of ≈20 kDa and dispersities <1.07 were produced and exhibited near-infrared (NIR) emission in solution and in the solid state with photoluminescence quantum yields (ΦPL ) as high as 0.72 and 0.18, respectively. Time-resolved emission spectroscopy revealed thermally activated delayed fluorescence (TADF) in polymers containing highly planar dopants, whereas room-temperature phosphorescence dominated with twisted species. Density functional theory demonstrated that rotation about the donor-acceptor linker can give rise to TADF, even where none would be expected based on calculations using ground-state geometries. Incorporation of TADF-active materials into water-soluble polymer dots (Pdots) gave NIR-emissive nanoparticles, and conjugation of these Pdots with antibodies enabled immunofluorescent labeling of SK-BR3 human breast-cancer cells.

Centriole-less pericentriolar material serves as a microtubule organizing center at the base of C. elegans sensory cilia.

During mitosis in animal cells, the centrosome acts as a microtubule organizing center (MTOC) to assemble the mitotic spindle. MTOC function at the centrosome is driven by proteins within the pericentriolar material (PCM), however the molecular complexity of the PCM makes it difficult to differentiate the proteins required for MTOC activity from other centrosomal functions. We used the natural spatial separation of PCM proteins during mitotic exit to identify a minimal module of proteins required for centrosomal MTOC function in C. elegans. Using tissue-specific degradation, we show that SPD-5, the functional homolog of CDK5RAP2, is essential for embryonic mitosis, while SPD-2/CEP192 and PCMD-1, which are essential in the one-cell embryo, are dispensable. Surprisingly, although the centriole is known to be degraded in the ciliated sensory neurons in C. elegans,1-3 we find evidence for "centriole-less PCM" at the base of cilia and use this structure as a minimal testbed to dissect centrosomal MTOC function. Super-resolution imaging revealed that this PCM inserts inside the lumen of the ciliary axoneme and directly nucleates the assembly of dendritic microtubules toward the cell body. Tissue-specific degradation in ciliated sensory neurons revealed a role for SPD-5 and the conserved microtubule nucleator γ-TuRC, but not SPD-2 or PCMD-1, in MTOC function at centriole-less PCM. This MTOC function was in the absence of regulation by mitotic kinases, highlighting the intrinsic ability of these proteins to drive microtubule growth and organization and further supporting a model that SPD-5 is the primary driver of MTOC function at the PCM.

Affinity Immobilization of Semiconductor Quantum Dots and Metal Nanoparticles on Cellulose Paper Substrates.

Colloidal semiconductor quantum dots (QDs), metal nanoparticles, and cellulose paper are materials with numerous applications in bioanalysis and beyond. The functional properties of QDs and metal NPs are substantially different than those of cellulose, such that their integration with cellulose paper is potentially enabling for many applications. Here, we characterize and evaluate multiple chemistries that modify cellulose paper substrates for the affinity-based immobilization of QDs, gold nanoparticles (Au NPs), and platinum nanoparticles (Pt NPs). These chemistries include grafting of cellulose fibers with imidazole and dithiol groups, as well as the aminosilanization of cellulose fibers (both with and without subsequent grafting with dithiol groups). Cellulose modifications and nanoparticle immobilization are characterized by multiple techniques, including, but not limited to, X-ray photoelectron spectroscopy, scanning electron microscopy, and optical imaging, extinction, and fluorescence measurements. We demonstrate the on-paper immobilization of color-tuned mixtures of QDs, on-paper patterning of QDs by microcontact printing, and post-immobilization enhancement of energy transfer and model assays of protease activity. The robustness of QD photoluminescence is also evaluated between immobilization chemistries. Paper-immobilized Au NPs and Pt NPs are evaluated as potential substrates for SERS and as supported catalysts for a model decolorization reaction. Our cumulative results indicate that there may not be a one-size-fits-all immobilization chemistry. Instead, the immobilization chemistry should be tailored and optimized for the downstream application.

Fully Self-Assembled Silica Nanoparticle-Semiconductor Quantum Dot Supra-Nanoparticles and Immunoconjugates for Enhanced Cellular Imaging by Microscopy and Smartphone Camera.

There is a growing need for brighter luminescent materials to improve the detection and imaging of biomarkers. Relevant contexts include low-abundance biomarkers and technology-limited applications, where an example of the latter is the emerging use of smartphones and other nonoptimal but low-cost and portable devices for point-of-care diagnostics. One approach to achieving brighter luminescent materials is incorporating multiple copies of a luminescent material into a larger supra-nanoparticle (supra-NP) assembly. Here, we present a facile method for the preparation and immunoconjugation of supra-NP assemblies (SiO2@QDs) that comprised many quantum dots (QDs) around a central silica nanoparticle (SiO2 NP). The assembly was entirely driven by spontaneous affinity interactions between the constituent materials, which included imidazoline-functionalized silica nanoparticles, ligand-coated QDs, imidazole-functionalized dextran, and tetrameric antibody complexes (TACs). The physical and optical properties of the SiO2@QDs were characterized at both the ensemble and single-particle levels. Notably, the optical properties of the QDs were preserved upon assembly into supra-NPs, and single SiO2@QDs were approximately an order of magnitude brighter than single QDs and nonblinking. In proof-of-concept applications, including selective immunolabeling of breast cancer cells, the SiO2@QDs provided higher sensitivity and superior signal-to-background ratios whether using research-grade fluorescence microscopy or smartphone-based imaging. Overall, the SiO2@QDs are promising materials for enhanced bioanalysis and imaging.

Yellow fluorescent protein-based label-free tension sensors for monitoring integrin tension.

Using enhanced yellow fluorescent protein, we demonstrate the feasibility to use fluorescent proteins as a label-free tension sensor to monitor integrin tension.

Dextran-Functionalized Quantum Dot Immunoconjugates for Cellular Imaging.

Brightly luminescent semiconductor quantum dots (QDs) are ideal materials for cellular imaging and analysis because of their advantageous optical properties and surface area that supports multivalent conjugation of biomolecules. An important design consideration for effective use of these materials is a hydrophilic, biocompatible surface chemistry that provides colloidal stability and minimizes nonspecific interactions with biological molecules and systems. Dextran coatings are able to satisfy these criteria. Despite frequent use of dextran coatings with other nanomaterials (e.g., iron oxide nanoparticles), there has been little development and application of dextran coatings for QDs. In this chapter, we describe methods for the synthesis and characterization of a dextran ligand for QDs, including preparation of an immunoconjugate via tetrameric antibody complexes (TAC). The utility of these immunoconjugates is demonstrated through immunofluorescent labeling and imaging of overexpressed human epidermal growth factor receptor 2 (HER2) on the surface of SK-BR3 breast cancer cells.

Dextran-Functionalized Semiconductor Quantum Dot Bioconjugates for Bioanalysis and Imaging.

The prerequisites for maximizing the advantageous optical properties of colloidal semiconductor quantum dots (QDs) in biological applications are effective surface functionalization and bioconjugation strategies. Functionalization with dextran has been highly successful with some nanoparticle materials, but has had very limited application with QDs. Here, we report the preparation, characterization, and proof-of-concept applications of dextran-functionalized QDs. Multiple approaches to dextran ligands were evaluated, including performance with respect to colloidal stability across a range of pH, nonspecific binding with proteins and cells, and microinjection into cells and viability assays. Multiple bioconjugation strategies were demonstrated and applied, including covalent coupling to develop a simple pH sensor, binding of polyhistidine-tagged peptides to the QD for energy transfer-based proteolytic activity assays, and binding with tetrameric antibody complexes (TACs) to enable a sandwich immunoassay and cell immunolabeling and imaging. Our results show that dextran ligands are highly promising for the functionalization of QDs, and that the design of the ligands is tailorable to help optimally meet the requirements of applications.

Dextran Functionalization of Semiconducting Polymer Dots and Conjugation with Tetrameric Antibody Complexes for Bioanalysis and Imaging.

Brightly fluorescent semiconducting polymer dots (Pdots) are emerging as very useful probes for bioanalysis and imaging. Unfortunately, Pdot materials often suffer from limitations such as poor colloidal and physical stability, nonspecific adsorption, and relatively few reported surface chemistries and bioconjugate chemistries. To help address these limitations, we have developed dextran-functionalized Pdots (Dex-Pdots). This functionalization improves particle stability over a range of pH and at high ionic strength, hinders surface-induced unfolding, and enables the preparation of immunoconjugates via tetrameric antibody complexes (TAC). The utility of TAC-conjugated Dex-Pdots is demonstrated through a proof-of-concept fluorescence-linked immunosorbent assay (FLISA) for human erythropoietin (EPO), and through immunolabeling of human epidermal growth factor receptor 2 (HER2)-positive SK-BR3 breast cancer cells. The conjugates exhibited less nonspecific binding and greater specific binding than Pdots without dextran functionalization. Overall, dextran functionalization is a highly promising surface chemistry for biological applications of Pdots.

Supraparticle Assemblies of Magnetic Nanoparticles and Quantum Dots for Selective Cell Isolation and Counting on a Smartphone-Based Imaging Platform.

There are numerous diagnostic and therapeutic applications for the detection and enumeration of specific cell types. Flow cytometry is the gold standard technique for this purpose but is poorly suited to point-of-need assays. The ideal platform for these assays would combine the immunocytochemical capabilities of flow cytometry with low-cost, portable instrumentation, and a simple and rapid assay workflow. Here, we present a smartphone-based imaging platform (SIP) in tandem with magnetic-fluorescent suprananoparticle assemblies as a step toward these ideal criteria. The assemblies (MNP@QD) are magnetic iron oxide nanoparticles surrounded by a dense corona of many brightly luminescent semiconductor quantum dots (QDs), where both the assemblies and their immunoconjugates are prepared by self-assembly. As proof of concept, we show that the MNP@QD and SIP pairing is able to selectively isolate, fluorescently immunolabel, and count breast cancer cells that are positive for human epidermal growth factor receptor 2 (HER2). These results are an important foundation for future point-of-need diagnostics capable of multiplexed isolation, counting, and immunoprofiling of cells on a smartphone, enabled by the highly advantageous optical properties of QDs.

Polyacrylamide gel electrophoresis of semiconductor quantum dots and their bioconjugates: materials characterization and physical insights from spectrofluorimetric detection.

Colloidal semiconductor quantum dot (QD) nanocrystals have ideal fluorescence properties for bioanalysis and bioimaging, but these materials must be functionalized with an inorganic shell, organic ligand or polymer coating, and conjugated with biomolecules to be useful in such applications. Several different analytical techniques are used to characterize QDs and their multiple layers of functionalization. Here, we revisit poly(acrylamide) gel electrophoresis (PAGE), which has been scarcely used for the characterization of QDs and their bioconjugates in deference to the routine use of agarose gel electrophoresis. We implemented PAGE in a novel "stubby" capillary format with spectrofluorimetric detection, the combination of which enabled more rapid and more detailed characterization of QDs than was possible with both poly(acrylamide) and agarose slab gels. Correlations between the peak photoluminescence (PL) emission wavelength and electropherogram peaks, especially when combined with Ferguson analysis, provided new and significant insight into the key factors that determine the electrophoretic mobility of QDs, and helped to resolve heterogeneity and sub-populations in ensembles of QDs. The method was useful for characterization of the inorganic core/shell nanocrystals, their organic ligand and polymer coatings, and their final bioconjugates, the latter of which were in the form of peptide and protein conjugates. With further development and optimization, we anticipate that capillary PAGE with spectrofluorimetric detection will become a valuable addition to the toolbox of characterization techniques suitable for QDs, their bioconjugates, and other nanoparticle materials as well.

Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1.

Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a "cage-like" structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles.

Kinetochore-independent chromosome segregation driven by lateral microtubule bundles.

During cell division, chromosomes attach to spindle microtubules at sites called kinetochores, and force generated at the kinetochore-microtubule interface is the main driver of chromosome movement. Surprisingly, kinetochores are not required for chromosome segregation on acentrosomal spindles in Caenorhabditis elegans oocytes, but the mechanism driving chromosomes apart in their absence is not understood. In this study, we show that lateral microtubule-chromosome associations established during prometaphase remain intact during anaphase to facilitate separation, defining a novel form of kinetochore-independent segregation. Chromosome dynamics during congression and segregation are controlled by opposing forces; plus-end directed forces are mediated by a protein complex that forms a ring around the chromosome center and dynein on chromosome arms provides a minus-end force. At anaphase onset, ring removal shifts the balance between these forces, triggering poleward movement along lateral microtubule bundles. This represents an elegant strategy for controlling chromosomal movements during cell division distinct from the canonical kinetochore-driven mechanism.

The herpes simplex virus 1 latency-associated transcript promotes functional exhaustion of virus-specific CD8+ T cells in latently infected trigeminal ganglia: a novel immune evasion mechanism.

Following ocular herpes simplex virus 1 (HSV-1) infection of C57BL/6 mice, HSV-specific (HSV-gB(498-505) tetramer(+)) CD8(+) T cells are induced, selectively retained in latently infected trigeminal ganglia (TG), and appear to decrease HSV-1 reactivation. The HSV-1 latency-associated transcript (LAT) gene, the only viral gene that is abundantly transcribed during latency, increases reactivation. Previously we found that during latency with HSV-1 strain McKrae-derived viruses, more of the total TG resident CD8 T cells expressed markers of exhaustion with LAT(+) virus compared to LAT(-) virus. Here we extend these findings to HSV-1 strain 17syn+-derived LAT(+) and LAT(-) viruses and to a virus expressing just the first 20% of LAT. Thus, the previous findings were not an artifact of HSV-1 strain McKrae, and the LAT function involved mapped to the first 1.5 kb of LAT. Importantly, to our knowledge, we show here for the first time that during LAT(+) virus latency, most of the HSV-1-specific TG resident CD8 T cells were functionally exhausted, as judged by low cytotoxic function and decreased gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. This resulted in LAT(-) TG having more functional HSV-gB(498-505) tetramer(+) CD8(+) T cells compared to LAT(+) TG. In addition, LAT expression, in the absence of other HSV-1 gene products, appeared to be able to directly or indirectly upregulate both PD-L1 and major histocompatibility complex class I (MHC-I) on mouse neuroblastoma cells (Neuro2A). These findings may constitute a novel immune evasion mechanism whereby the HSV-1 LAT directly or indirectly promotes functional exhaustion (i.e., dysfunction) of HSV-specific CD8(+) T cells in latently infected TG, resulting in increased virus reactivation.