Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses.

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.

Optical Manipulation of F-Actin with Photoswitchable Small Molecules.

Cell-permeable photoswitchable small molecules, termed optojasps, are introduced to optically control the dynamics of the actin cytoskeleton and cellular functions that depend on it. These light-dependent effectors were designed from the F-actin-stabilizing marine depsipeptide jasplakinolide by functionalizing them with azobenzene photoswitches. As demonstrated, optojasps can be employed to control cell viability, cell motility, and cytoskeletal signaling with the high spatial and temporal resolution that light affords. Optojasps can be expected to find applications in diverse areas of cell biological research. They may also provide a template for photopharmacology targeting the ubiquitous actin cytoskeleton with precision control in the micrometer range.

Micropatterning as a tool to identify regulatory triggers and kinetics of actin-mediated endothelial mechanosensing.

Developmental processes, such as angiogenesis, are associated with a constant remodeling of the actin cytoskeleton in response to different mechanical stimuli. The mechanosensitive transcription factors MRTF-A (MKL1) and YAP (also known as YAP1) are important mediators of this challenging adaptation process. However, it is as yet unknown whether both pathways respond in an identical or in a divergent manner to a given microenvironmental guidance cue. Here, we use a micropatterning approach to dissect single aspects of cellular behavior in a spatiotemporally controllable setting. Using the exemplary process of angiogenesis, we show that cell-cell contacts and adhesive surface area are shared regulatory parameters of MRTF and YAP on rigid 2D surfaces. By analyzing MRTF and YAP under laminar flow conditions and during cell migration on dumbbell-shaped microstructures, we demonstrate that they exhibit different translocation kinetics. In conclusion, our work promotes the application of micropatterning techniques as a cell biological tool to study mechanosensitive signaling in the context of angiogenesis.

Chivosazole A Modulates Protein-Protein Interactions of Actin.

Actin is a protein of central importance for many cellular key processes. It is regulated by local interactions with a large number of actin binding proteins (ABPs). Various compounds are known to either increase or decrease the polymerization dynamics of actin. However, no actin binding compound has been developed for clinical applications yet because of selectivity issues. We provide a crystal structure of the natural product chivosazole A (ChivoA) bound to actin and show that-in addition to inhibiting nucleation, polymerization, and severing of F-actin filaments-it selectively modulates binding of ABPs to G-actin: Although unphysiological actin dimers are induced by ChivoA, interaction with gelsolin, profilin, cofilin, and thymosin-ß4 is inhibited. Moreover, ChivoA causes transcriptional effects differing from latrunculin B, an actin binder with a different binding site. Our data show that ChivoA and related compounds could serve as scaffolds for the development of actin binding molecules selectively targeting specific actin functions.

Transcriptional effects of actin-binding compounds: the cytoplasm sets the tone.

Actin has emerged as a versatile regulator of gene transcription. Cytoplasmatic actin regulates mechanosensitive-signaling pathways such as MRTF-SRF and Hippo-YAP/TAZ. In the nucleus, both polymerized and monomeric actin directly interfere with transcription-associated molecular machineries. Natural actin-binding compounds are frequently used tools to study actin-related processes in cell biology. However, their influence on transcriptional regulation and intranuclear actin polymerization is poorly understood to date. Here, we analyze the effects of two representative actin-binding compounds, Miuraenamide A (polymerizing properties) and Latrunculin B (depolymerizing properties), on transcriptional regulation in primary cells. We find that actin stabilizing and destabilizing compounds inversely shift nuclear actin levels without a direct influence on polymerization state and intranuclear aspects of transcriptional regulation. Furthermore, we identify Miuraenamide A as a potent inducer of G-actin-dependent SRF target gene expression. In contrast, the F-actin-regulated Hippo-YAP/TAZ axis remains largely unaffected by compound-induced actin aggregation. This is due to the inability of AMOTp130 to bind to the amorphous actin aggregates resulting from treatment with miuraenamide. We conclude that actin-binding compounds predominantly regulate transcription via their influence on cytoplasmatic G-actin levels, while transcriptional processes relying on intranuclear actin polymerization or functional F-actin networks are not targeted by these compounds at tolerable doses.

Short-term study on in-use stability of opened bevacizumab biosimilar PF-06439535 vials.

OBJECTIVES: Aggregation is one of the key critical points limiting the stability of monoclonal antibodies in solution. The present study aimed to investigate the in-use stability of a residual monoclonal antibody solution after withdrawal of most of the filling volume of PF-06439535 (bevacizumab biosimilar), addressing the physical and chemical stability with respect to aggregation and fragmentation. METHODS: The stability of residual PF-06439535 solution (25 mg/mL) after withdrawal of 80% (12.8 mL) filling volume with a 20G needle was monitored over a light-protected storage period of 8 days at 2-8°C and 25°C with measurement time points at D0 (start of storage), D2, D4, and D8 (2, 4, and 8 days of storage after start, respectively). Unopened vials stored under the same conditions served as control. For this purpose, the analytical results from size exclusion chromatography, dynamic light scattering, and micro-flow imaging obtained after the individual measurement time points up to 8 days were compared with those obtained at D0 and with those obtained for unopened vials stored under the same conditions. RESULTS: No aggregation or ongoing fragmentation due to partial withdrawal of filling volume could be observed in the residual PF-06439535 solution. Moreover, no changes in the particle size distribution at D8 compared with the D0 values were identified upon storage at either 2-8°C or 25°C (both opened and unopened vials). The total concentration of particles ≥10 µm of all samples was <100 particles/mL. In addition, no variations in the pH values or in the visual appearance were detected over the whole study period in all samples at all storage conditions. CONCLUSIONS: Consequently, residual PF-06439535 solution (25 mg/mL) in opened vials may be regarded as stable when stored light-protected over a period of 8 days in the refrigerator (2-8°C) or at 25°C.

Spatio-selective activation of nuclear translocation of YAP with light directs invasion of cancer cell spheroids.

The mechanical properties of the extracellular matrix strongly influence tumor progression and invasion. Yes-associated protein (YAP) has been shown to be a key regulator of this process translating mechanical cues from the extracellular matrix into intracellular signals. Despite its apparent role in tumor progression and metastasis, it is not clear yet, whether YAP activation can actively trigger the onset of invasion. To address this question, we designed a photo-activatable YAP (optoYAP), which allows for spatiotemporal control of its activation. The activation mechanism of optoYAP is based on optically triggered nuclear translocation of the protein. Activation of optoYAP induces downstream signaling for several hours and leads to increased proliferation in two- and three-dimensional cultures. Applied to cancer spheroids, optoYAP activation induces invasion. Site-selective activation of optoYAP in cancer spheroids strikingly directs invasion into the activated direction. Thus, nuclear translocation of YAP may be enough to trigger the onset of invasion.

Gene editing and synthetically accessible inhibitors reveal role for TPC2 in HCC cell proliferation and tumor growth.

The role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of cancer cells in vitro, affects their energy metabolism, and successfully abrogates tumor growth in vivo. Concurrently, we have developed simplified analogs of the alkaloid tetrandrine as potent TPC2 inhibitors by screening a library of synthesized benzyltetrahydroisoquinoline derivatives. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against cancer cells and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing a TPC2 inhibitor with antitumor efficacy in mice. Hence, our study unveils TPC2 as valid target for cancer therapy and provides easily accessible tetrandrine analogs as a promising option for effective pharmacological interference.

Inducible microRNA-200c decreases motility of breast cancer cells and reduces filamin A.

Cancer progression and metastases are frequently related to changes of cell motility. Amongst others, the microRNA-200c (miR-200c) was shown to maintain the epithelial state of cells and to hamper migration. Here, we describe two miR-200c inducible breast cancer cell lines, derived from miR-200c knock-out MCF7 cells as well as from the miR-200c-negative MDA-MB-231 cells and report on the emerging phenotypic effects after miR-200s induction. The induction of miR-200c expression seems to effect a rapid reduction of cell motility, as determined by 1D microlane migration assays. Sustained expression of miR200c leads to a changed morphology and reveals a novel mechanism by which miR-200c interferes with cytoskeletal components. We find that filamin A expression is attenuated by miRNA-200c induced downregulation of the transcription factors c-Jun and MRTF/SRF. This potentially novel pathway that is independent of the prominent ZEB axis could lead to a broader understanding of the role that miR200c plays in cancer metastasis.

Actin stabilizing compounds show specific biological effects due to their binding mode.

Actin binding compounds are widely used tools in cell biology. We compare the biological and biochemical effects of miuraenamide A and jasplakinolide, a structurally related prototypic actin stabilizer. Though both compounds have similar effects on cytoskeletal morphology and proliferation, they affect migration and transcription in a distinctive manner, as shown by a transcriptome approach in endothelial cells. In vitro, miuraenamide A acts as an actin nucleating, F-actin polymerizing and stabilizing compound, just like described for jasplakinolide. However, in contrast to jasplakinolide, miuraenamide A competes with cofilin, but not gelsolin or Arp2/3 for binding to F-actin. We propose a binding mode of miuraenamide A, explaining both its similarities and its differences to jasplakinolide. Molecular dynamics simulations suggest that the bromophenol group of miurenamide A interacts with residues Tyr133, Tyr143, and Phe352 of actin. This shifts the D-loop of the neighboring actin, creating tighter packing of the monomers, and occluding the binding site of cofilin. Since relatively small changes in the molecular structure give rise to this selectivity, actin binding compounds surprisingly are promising scaffolds for creating actin binders with specific functionality instead of just "stabilizers".

Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy.

Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.

Nucleotide-Dependent Dimer Association and Dissociation of the Chaperone Hsp90.

Hsp90 is an essential molecular chaperone, which has to be in a dimeric form for its correct function. While the affinity of the dimer has previously been measured, little is known about how it associates and dissociates and the factors that influence this. We perform an in-depth single molecule characterization of the C-terminal association and dissociation of Hsp90. We find more than one dissociation rate, indicating that the dimer has a stable and an unstable state. Furthermore, we find that the stability of the C-terminal association is dependent on the presence of ATP, despite the C-terminal dimerization interface being distal to the catalytic site.

Contractility as a global regulator of cellular morphology, velocity, and directionality in low-adhesive fibrillary micro-environments.

Recent reports demonstrated that migration in fibrillary environments can be mimicked by spatial confinement achieved with micro-patterning [1]. Here we investigated whether a model system based on linearly structured surfaces allows to draw conclusions about migration of endothelial cells (ECs) in fibrillary 3D environments. We found that ECs on 3 µm wide tracks (termed as 1D) migrate less efficient in comparison to ECs on broader tracks in regard to velocity and directional persistence. The frequent changes of direction in ECs on narrow tracks are accompanied by pronounced cell rounding and membrane blebbing, while cells migrating with an elongated morphology display a single lamellipodium. This behavior is contractility-dependent as both modes can be provoked by manipulating activity of myosin II (blebbistatin or calyculin A, respectively). The comparison between 1D and 3D migrating cells revealed a striking similarity in actin architecture and in switching between two morphologies. ECs move more directed but slower upon inhibition of contractility in 1D and 3D, in contrast to 2D cell culture. We conclude that micro-patterning can be used to study morphological switches in a controlled manner with a prognostic value for 3D environments. Moreover, we identified blebbing as a new aspect of EC migration.