Development of the ectodermally derived Drosophila tracheal system is based on branch outgrowth and fusion that interconnect metamerically arranged tracheal subunits into a highly stereotyped three-dimensional tubular structure. Recent studies have revealed that this process involves a specialized cell type of mesodermal origin, termed bridge-cell. Single bridge-cells are located between adjacent tracheal subunits and serve as guiding posts for the outgrowing dorsal trunk branches. We show that bridge-cell-approaching tracheal cells form filopodia-like cell extensions, which attach to the bridge-cell surface and are essential for the tracheal subunit interconnection. The results of both dominant-negative and gain-of-function experiments suggest that the formation of cell extensions require Cdc42-mediated Drosophila fibroblast growth factor activity.

During embryonic development neurons differentiate and extend axons and dendrites that have to reach their appropriate targets. In Caenorhabditis elegans the AVG neuron is the first neuron to extend an axon during the establishment of the ventral nerve cord, the major longitudinal axon tract in the animal. In genetic screens we isolated alleles of plr-1, which caused polarity reversals of the AVG neuron as well as outgrowth and navigation defects of the AVG axon. In addition plr-1 mutants show outgrowth defects in several other classes of neurons as well as the posterior excretory canals. plr-1 is predicted to encode a transmembrane E3 ubiquitin ligase and is widely expressed in the animal including the AVG neuron and the excretory cell. plr-1 has recently been shown to negatively regulate Wnt signalling by removing Wnt receptors from the cell surface. We observed that mutations in a gene reducing Wnt signalling as well as mutations in unc-53/NAV2 and unc-73/Trio suppress the AVG polarity defects in plr-1 mutants, but not the defects seen in other cells. This places plr-1 in a Wnt regulation pathway, but also suggests that plr-1 has Wnt independent functions and interacts with unc-53 and unc-73 to control cell polarity.

The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Förster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5' extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhβ protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.

Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell-cycle progression. We then show that the actin filament length regulators CFL1, CAPZB and DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation.

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo-/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C m = 1.9 µF/cm(2). In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C m values of 3.7-4.0 µF/cm(2), which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.

Cell walls are at the front line of interactions between walled-organisms and their environment. They support cell expansion, ensure cell integrity and, for multicellular organisms such as plants, they provide cell adherence, support cell shape morphogenesis and mediate cell-cell communication. Wall-sensing, detecting perturbations in the wall and signaling the cell to respond accordingly, is crucial for growth and survival. In recent years, plant signaling research has suggested that a large family of receptor-like kinases (RLKs) could function as wall sensors partly because their extracellular domains show homology with malectin, a diglucose binding protein from the endoplasmic reticulum of animal cells. Studies of several malectin/malectin-like (M/ML) domain-containing RLKs (M/MLD-RLKs) from the model plant Arabidopsis thaliana have revealed an impressive array of biological roles, controlling growth, reproduction and stress responses, processes that in various ways rely on or affect the cell wall. Malectin homologous sequences are widespread across biological kingdoms, but plants have uniquely evolved a highly expanded family of proteins with ML domains embedded within various protein contexts. Here, we present an overview on proteins with malectin homologous sequences in different kingdoms, discuss the chromosomal organization of Arabidopsis M/MLD-RLKs and the phylogenetic relationship between these proteins from several model and crop species. We also discuss briefly the molecular networks that enable the diverse biological roles served by M/MLD-RLKs studied thus far.

Using a series of multiheme cytochromes, the metal-reducing bacterium Shewanella oneidensis MR-1 can perform extracellular electron transfer (EET) to respire redox-active surfaces, including minerals and electrodes outside the cell. While the role of multiheme cytochromes in transporting electrons across the cell wall is well established, these cytochromes were also recently found to facilitate long-distance (micrometer-scale) redox conduction along outer membranes and across multiple cells bridging electrodes. Recent studies proposed that long-distance conduction arises from the interplay of electron hopping and cytochrome diffusion, which allows collisions and electron exchange between cytochromes along membranes. However, the diffusive dynamics of the multiheme cytochromes have never been observed or quantified in vivo, making it difficult to assess their hypothesized contribution to the collision-exchange mechanism. Here, we use quantum dot labeling, total internal reflection fluorescence microscopy, and single-particle tracking to quantify the lateral diffusive dynamics of the outer membrane-associated decaheme cytochromes MtrC and OmcA, two key components of EET in S. oneidensis. We observe confined diffusion behavior for both quantum dot-labeled MtrC and OmcA along cell surfaces (diffusion coefficients DMtrC = 0.0192 ± 0.0018 µm2/s, DOmcA = 0.0125 ± 0.0024 µm2/s) and the membrane extensions thought to function as bacterial nanowires. We find that these dynamics can trace a path for electron transport via overlap of cytochrome trajectories, consistent with the long-distance conduction mechanism. The measured dynamics inform kinetic Monte Carlo simulations that combine direct electron hopping and redox molecule diffusion, revealing significant electron transport rates along cells and membrane nanowires.

In this study, a previously known high-affinity silica binding protein (SB) was genetically engineered to fuse with an integrin-binding peptide (RGD) to create a recombinant protein (SB-RGD). SB-RGD was successfully expressed in Escherichia coli and purified using silica beads through a simple and fast centrifugation method. A further functionality assay showed that SB-RGD bound to the silica surface with an extremely high affinity that required 2 M MgCl₂ for elution. Through a single-step incubation, the purified SB-RGD proteins were noncovalently coated onto an electrospun silica nanofiber (SNF) substrate to fabricate the SNF-SB-RGD substrate. SNF-SB-RGD was characterized by a combination of scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and immunostaining fluorescence microscopy. As PC12 cells were seeded onto the SNF-SB-RGD surface, significantly higher cell viability and longer neurite extensions were observed when compared to those on the control surfaces. These results indicated that SB-RGD could serve as a noncovalent coating biologic to support and promote neuron growth and differentiation on silica-based substrates for neuronal tissue engineering. It also provides proof of concept for the possibility to genetically engineer protein-based signaling molecules to noncovalently modify silica-based substrates as bioinspired material.

Live intracellular imaging is a valuable tool in modern diagnostics and pharmacology. Surface Enhanced Raman Spectroscopy (SERS) stands out as a non-destructive and multiplexed technique, but intracellular SERS imaging still suffers from interfering background from endogenous components. Here we show the assembly of small colloidal SERS probes with Raman signal in the cell-silent window of 1800-2900 cm-1 for biorthogonal intracellular SERS imaging of dopamine that was undistinguishable from the endogenous cell background. By linking colloidal silver nanoparticles with alkyne-dopamine adducts, clusters are formed by 2-6 nanoparticles spaced by tight interparticle gaps that exhibited high electric field enhancement and strong SERS signals of alkyne and dopamines. Due to the cell-silent signals of the alkyne, intracellular in-vitro Raman imaging shows that the dopamines on the internalized clusters remain distinguishable across the cytoplasm with good spatial resolution. Our method can be a general-purpose method for real-time imaging of biomolecules, such as proteins, peptides, DNA and drugs.

In this study, we first synthesized a slow-degrading silica nanofiber (SNF2) through an electrospun solution with an optimized tetraethyl orthosilicate (TEOS) to polyvinyl pyrrolidone (PVP) ratio. Then, laminin-modified SNF2, namely SNF2-AP-S-L, was obtained through a series of chemical reactions to attach the extracellular matrix protein, laminin, to its surface. The SNF2-AP-S-L substrate was characterized by a combination of scanning electron microscopy (SEM), Fourier transform-infrared (FTIR) spectroscopy, nitrogen adsorption/desorption isotherms, and contact angle measurements. The results of further functional assays show that this substrate is a biocompatible, bioactive and biodegradable scaffold with good structural integrity that persisted beyond 18 days. Moreover, a synergistic effect of sustained structure support and prolonged biochemical stimulation for cell differentiation on SNF2-AP-S-L was found when neuron-like PC12 cells were seeded onto its surface. Specifically, neurite extensions on the covalently modified SNF2-AP-S-L were significantly longer than those observed on unmodified SNF and SNF subjected to physical adsorption of laminin. Together, these results indicate that the SNF2-AP-S-L substrate prepared in this study is a promising 3D biocompatible substrate capable of sustaining longer neuronal growth for tissue-engineering applications.

The olfactory epithelium (OE) is a neurosensory organ required for the sense of smell. Turbinates, bony projections from the nasal cavity wall, increase the surface area within the nasal cavity lined by the OE. Here, we use engineered fibroblast growth factor 20 (Fgf20) knockin alleles to identify a population of OE progenitor cells that expand horizontally during development to populate all lineages of the mature OE. We show that these Fgf20-positive epithelium-spanning progenitor (FEP) cells are responsive to Wnt/β-Catenin signaling. Wnt signaling suppresses FEP cell differentiation into OE basal progenitors and their progeny and positively regulates Fgf20 expression. We further show that FGF20 signals to the underlying mesenchyme to regulate the growth of turbinates. These studies thus identify a population of OE progenitor cells that function to scale OE surface area with the underlying turbinates.

The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA.

"Surface-associated astrocytes" (SAAs) in posterior piriform cortex (PPC) are unique by virtue of a direct apposition to the cortical surface and large-caliber processes that descend into layer I. In this study additional unique and functionally relevant features of SAAs in PPC of the rat were identified by light and electron microscopy. Examination of sections cut parallel to the surface of PPC and stained for glial fibrillar acidic protein revealed that, in addition to descending processes, SAAs give rise to an extensive matrix of "superficial processes." Electron microscopy revealed that these superficial processes, together with cell bodies, form a continuous sheet at the surface of PPC with features in common with the glia limitans that is formed by endfeet in other cortical areas. These include a glia limiting membrane with basal lamina and similar associated organelles, including a striking array of mitochondria. Of particular interest, SAAs lack the domain organization observed in neocortex and hippocampus. Rather, superficial processes overlap extensively with gap junctions between their proximal regions as well as between cell bodies. Study of the descending processes revealed thin extensions, many of which appose synaptic profiles. We conclude that SAAs provide a potential substrate for bidirectional signaling and transport between brain and the pial arteries and cerebrospinal fluid in the subarachnoid space. We postulate that the spatially distributed character of SAAs in PPC reflects and supports the spatially distributed circuitry and sensory representation that are also unique features of this area.

Collective cell migration is not only important for development and tissue homeostasis but can also promote cancer metastasis. To migrate collectively, cells need to coordinate cellular extensions and retractions, adhesion sites dynamics, and forces generation and transmission. Nevertheless, the regulatory mechanisms coordinating these processes remain elusive. Using A431 carcinoma cells, we identify the kinase MAP4K4 as a central regulator of collective migration. We show that MAP4K4 inactivation blocks the migration of clusters, whereas its overexpression decreases cluster cohesion. MAP4K4 regulates protrusion and retraction dynamics, remodels the actomyosin cytoskeleton, and controls the stability of both cell-cell and cell-substrate adhesion. MAP4K4 promotes focal adhesion disassembly through the phosphorylation of the actin and plasma membrane crosslinker moesin but disassembles adherens junctions through a moesin-independent mechanism. By analyzing traction and intercellular forces, we found that MAP4K4 loss of function leads to a tensional disequilibrium throughout the cell cluster, increasing the traction forces and the tension loading at the cell-cell adhesions. Together, our results indicate that MAP4K4 activity is a key regulator of biomechanical forces at adhesion sites, promoting collective migration.

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5.

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses.

The splenic marginal zone is a unique microenvironment where resident immune cells are exposed to the open blood circulation. Even though it has an important role in responses against blood-borne antigens, lymphocyte migration in the marginal zone has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a two-photon microscopy procedure to study marginal zone and follicular B-cell movement in the live mouse spleen. We show that marginal zone B cells are highly motile and exhibit long membrane extensions. Marginal zone B cells shuttle between the marginal zone and follicles with at least one-fifth of the cells exchanging between compartments per hour, a behaviour that explains their ability to deliver antigens rapidly from the open blood circulation to the secluded follicles. Follicular B cells also transit from follicles to the marginal zone, but unlike marginal zone B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. Follicular B-cell egress via the marginal zone is sphingosine-1-phosphate receptor-1 (S1PR1)-dependent. This study shows that marginal zone B cells migrate continually between marginal zone and follicles and establishes the marginal zone as a site of S1PR1-dependent B-cell exit from follicles. The results also show how adhesive differences of similar cells critically influence their behaviour in the same microenvironment.

Adaptive T and B lymphocytes expand, respond, and persist across a multitude of separable cell differentiation states. Small compartments of these cells present defined cell surface phenotype, but express potentially divergent immune functions. Here, we use high resolution flow cytometry to provide direct access to rare lymphocyte subpopulations for evaluation of steady-state or reactive transcriptional programs. We sort and index single cells by phenotype in 384-well format for quantification of targeted gene amplification through RNA sequencing (single cell qtSEQ). For complete details on the use and execution of this profile, please refer to Dufaud et al. (2021).

In vascular plants, the epidermal surfaces of leaves and flower petals often display cells with wavy geometries forming intricate jigsaw puzzle patterns. The prevalence and diversity of these complex epidermal patterns, originating from simple polyhedral progenitor cells, suggest adaptive significance. However, despite multiple efforts to explain the evolutionary drivers behind these geometrical features, compelling validation remains elusive. Employing a multidisciplinary approach that integrates microscopic and macroscopic fracture experiments with computational fracture mechanics, we demonstrate that wavy epidermal cells toughen the plants' protective skin. Through a multi-scale framework, we demonstrate that this energy-efficient patterning mechanism is universally applicable for toughening biological and synthetic materials. Our findings reveal a tunable structural-mechanical strategy employed in the microscale design of plants to protect them from deleterious surface fissures while facilitating and strategically directing beneficial ones. These findings hold implications for targeted plant breeding aimed at enhancing resilience in fluctuating environmental conditions. From an engineering perspective, our work highlights the sophisticated design principles the plant kingdom offers to inspire metamaterials.