Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes.

The rhizosphere, the narrow zone of soil around plant roots, is a complex network of interactions between plants, bacteria, and a variety of other organisms. The absolute dependence on host-derived signals, or xenognosins, to regulate critical developmental checkpoints for host commitment in the obligate parasitic plants provides a window into the rhizosphere's chemical dynamics. These sessile intruders use H2O2 in a process known as semagenesis to chemically modify the mature root surfaces of proximal host plants and generate p-benzoquinones (BQs). The resulting redox-active signaling network regulates the spatial and temporal commitments necessary for host attachment. Recent evidence from non-parasites, including Arabidopsis thaliana, establishes that reactive oxygen species (ROS) production regulates similar redox circuits related to root recognition, broadening xenognosins' role beyond the parasites. Here we compare responses to the xenognosin dimethoxybenzoquinone (DMBQ) between the parasitic plant Striga asiatica and the non-parasitic A. thaliana. Exposure to DMBQ simulates the proximity of a mature root surface, stimulating an increase in cytoplasmic Ca2+ concentration in both plants, but leads to remarkably different phenotypic responses in the parasite and non-parasite. In S. asiatica, DMBQ induces development of the host attachment organ, the haustorium, and decreases ROS production at the root tip, while in A. thaliana, ROS production increases and further growth of the root tip is arrested. Obstruction of Ca2+ channels and the addition of antioxidants both lead to a decrease in the DMBQ response in both parasitic and non-parasitic plants. These results are consistent with Ca2+ regulating the activity of NADPH oxidases, which in turn sustain the autocatalytic production of ROS via an external quinone/hydroquinone redox cycle. Mechanistically, this chemistry is similar to black and white photography with the emerging dynamic reaction-diffusion network laying the foundation for the precise temporal and spatial control underlying rhizosphere architecture.

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer, although sub-optimal tumor specificity and side effects have hindered its clinical application. We introduced a new strategy named nanobody-targeted PDT in which photosensitizers are delivered to tumor cells by means of nanobodies. As efficacy of targeted PDT can be hampered by heterogeneity of target expression and/or moderate/low target expression levels, we explored the possibility of combined targeting of endothelial and cancer cells in vitro. We developed nanobodies binding to the mouse VEGFR2, which is overexpressed on tumor vasculature, and combined these with nanobodies specific for the cancer cell target EGFR. The nanobodies were conjugated to the photosensitizer IRDye700DX and specificity of the newly developed nanobodies was verified using several endothelial cell lines. The cytotoxicity of these conjugates was assessed in monocultures and in co-cultures with cancer cells, after illumination with an appropriate laser. The results show that the anti-VEGFR2 conjugates are specific and potent PDT agents. Nanobody-targeted PDT on co-culture of endothelial and cancer cells showed improved efficacy, when VEGFR2 and EGFR targeting nanobodies were applied simultaneously. Altogether, dual targeting of endothelial and cancer cells is a promising novel therapeutic strategy for more effective nanobody-targeted PDT.

A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT.

Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage. Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT. Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h compared to 1 h after administration. Discussion: Although differences were observed in distribution of the two NB-PS conjugates, both led to similar necrosis. Clearly, the response to PDT using NB-PS conjugates is the result of a complex mixture of tumor cell responses and vascular effects, which is likely to be necessary for a maximally effective treatment.

Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.

Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high-resolution two-photon microscopy in organotypic hippocampal slices from GAD65-GFP mice of both sexes to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons, we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilized boutons rapidly recruited synaptic vesicles, followed by accumulation of postsynaptic gephyrin and were functional after 24 h, as determined by electrophysiology and immunohistochemistry. Inhibitory boutons are only sensitive to Sema4D at a specific stage during synapse formation and sensitivity to Sema4D is regulated by network activity. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurs through rapid remodeling of the actin cytoskeleton. This could be mimicked by the actin-depolymerizing drug latrunculin B or by reducing ROCK activity. We discovered that the intracellular signaling cascade requires activation of the receptor tyrosine kinase MET, which is a well known autism risk factor. By using a viral approach to reduce MET levels specifically in inhibitory neurons, we found that their axons are no longer sensitive to Sema4D signaling. Together, our data yield important insights into the molecular pathway underlying activity-dependent Sema4D-induced synapse formation and reveal a novel role for presynaptic MET at inhibitory synapses.SIGNIFICANCE STATEMENT GABAergic synapses provide the main inhibitory control of neuronal activity in the brain. We wanted to unravel the sequence of molecular events that take place when formation of inhibitory synapses is triggered by a specific signaling molecule, Sema4D. We find that this signaling pathway depends on network activity and involves specific remodeling of the intracellular actin cytoskeleton. We also reveal a previously unknown role for MET at inhibitory synapses. Our study provides novel insights into the dynamic process of inhibitory synapse formation. As defects in GABAergic synapses have been implied in many brain disorders, and mutations in MET are strong risk factors for autism, our findings urge for a further investigation of the role of MET at inhibitory synapses.

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10-15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Ã… X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.

The ∼15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies®) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with (68)Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for (89)Zr immuno-positron emission tomography (PET).

Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4-4 Ã… resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications.

Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and tumor-selective treatment, using nanobodies (NBs) to deliver a photosensitizer (PS) specifically to cancer cells. Upon local light application, reactive oxygen species are formed and consequent cell death occurs. NB-PDT has preclinically shown evident success and we next aim to treat cats with oral squamous cell carcinoma (OSCC), which has very limited therapeutic options and is regarded as a natural model of human head and neck SCC. Immunohistochemistry of feline OSCC tissue confirmed that the epidermal growth factor receptor (EGFR) is a relevant target with expression in cancer cells and not in the surrounding stroma. Three feline OSCC cell lines were employed together with a well-characterized human cancer cell line (HeLa), all with similar EGFR expression, and a low EGFR-expressing human cell line (MCF7), mirroring the EGFR expression level in the surrounding mucosal stroma. NBA was identified as a NB binding human and feline EGFR with comparable high affinity. This NB was developed into NiBh, a NB-PS conjugate with high PS payload able to effectively kill feline OSCC and HeLa cell lines, after illumination. Importantly, the specificity of NB-PDT was confirmed in co-cultures where only the feline OSCC cells were killed while surrounding MCF7 cells were unaffected. Altogether, NiBh can be used for NB-PDT to treat feline OSCC and further advance NB-PDT towards the human clinic.

Vγ9Vδ2-T cells constitute the predominant subset of γδ-T cells in human peripheral blood and have been shown to play an important role in antimicrobial and antitumor immune responses. Several efforts have been initiated to exploit these cells for cancer immunotherapy, e.g. by using phosphoantigens, adoptive cell transfer, and by a bispecific monoclonal antibody based approach. Here, we report the generation of a novel set of Vγ9Vδ2-T cell specific VHH (or nanobody). VHH have several advantages compared to conventional antibodies related to their small size, stability, ease of generating multispecific molecules and low immunogenicity. With high specificity and affinity, the anti-Vγ9Vδ2-T cell receptor VHHs are shown to be useful for FACS, MACS and immunocytochemistry. In addition, some VHH were found to specifically activate Vγ9Vδ2-T cells. Besides being of possible immunotherapeutic value, these single domain antibodies will be of great value in the further study of this important immune effector cell subset.

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT.

The epidermal growth factor receptor (EGFR) is involved in various developmental processes, and alterations of its extracellular segment are associated with several types of cancers, in particular glioblastoma multiforme (GBM). The EGFR extracellular region is therefore a primary target for therapeutic agents, such as monoclonal antibodies and variable domains of heavy chain antibodies (VHH), also called nanobodies. Nanobodies have been previously shown to bind to EGFR, and to inhibit ligand-mediated EGFR activation.

Probes are essential to visualize proteins in their cellular environment, both using light microscopy as well as electron microscopy (EM). Correlated light microscopy and electron microscopy (CLEM) requires probes that can be imaged simultaneously by both optical and electron-dense signals. Existing combinatorial probes often have impaired efficiency, need ectopic expression as a fusion protein, or do not target endogenous proteins. Here, we present FLIPPER-bodies to label endogenous proteins for CLEM. Fluorescent Indicator and Peroxidase for Precipitation with EM Resolution (FLIPPER), the combination of a fluorescent protein and a peroxidase, is fused to a nanobody against a target of interest. The modular nature of these probes allows an easy exchange of components to change its target or color. A general FLIPPER-body targeting GFP highlights histone2B-GFP both in fluorescence and in EM. Similarly, endogenous EGF receptors and HER2 are visualized at nm-scale resolution in ultrastructural context. The small and flexible FLIPPER-body outperforms IgG-based immuno-labeling, likely by better reaching the epitopes. Given the modular domains and possibilities of nanobody generation for other targets, FLIPPER-bodies have high potential to become a universal tool to identify proteins in immuno-CLEM with increased sensitivity compared to current approaches.

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell-cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation.

Antibody-drug conjugates (ADCs) are currently used for the targeted delivery of drugs to diseased cells, but intracellular drug delivery and therefore efficacy may be suboptimal because of the large size, slow internalization and ineffective intracellular trafficking of the antibody. Using a phage display method selecting internalizing phages only, we developed internalizing single domain antibodies (sdAbs) with high binding affinity to rat PDGFRβ, a receptor involved in different types of diseases. We demonstrate that these constructs have different characteristics with respect to internalization rates but all traffic to lysosomes. To compare their efficacy in targeted drug delivery, we conjugated the sdAbs to a cytotoxic drug. The conjugates showed improved cytotoxicity correlating to their internalization speed. The efficacy of the conjugates was inhibited in the presence of vacuolin-1, an inhibitor of lysosomal maturation, suggesting lysosomal trafficking is needed for efficient drug release. In conclusion, sdAb constructs with different internalization rates can be designed against the same target, and sdAbs with a high internalization rate induce more cell killing than sdAbs with a lower internalization rate in vitro. Even though the overall efficacy should also be tested in vivo, sdAbs are particularly interesting formats to be explored to obtain different internalization rates.

Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide-mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.