Compartmentalization of androgen receptors at endogenous genes in living cells.

A wide range of nuclear proteins are involved in the spatio-temporal organization of the genome through diverse biological processes such as gene transcription and DNA replication. Upon stimulation by testosterone and translocation to the nucleus, multiple androgen receptors (ARs) accumulate in microscopically discernable foci which are irregularly distributed in the nucleus. Here, we investigated the formation and physical nature of these foci, by combining novel fluorescent labeling techniques to visualize a defined chromatin locus of AR-regulated genes-PTPRN2 or BANP-simultaneously with either AR foci or individual AR molecules. Quantitative colocalization analysis showed evidence of AR foci formation induced by R1881 at both PTPRN2 and BANP loci. Furthermore, single-particle tracking (SPT) revealed three distinct subdiffusive fractional Brownian motion (fBm) states: immobilized ARs were observed near the labeled genes likely as a consequence of DNA-binding, while the intermediate confined state showed a similar spatial behavior but with larger displacements, suggesting compartmentalization by liquid-liquid phase separation (LLPS), while freely mobile ARs were diffusing in the nuclear environment. All together, we show for the first time in living cells the presence of AR-regulated genes in AR foci.

Correlative light and electron microscopy reveals fork-shaped structures at actin entry sites of focal adhesions.

Focal adhesions (FAs) are the main cellular structures to link the intracellular cytoskeleton to the extracellular matrix. FAs mediate cell adhesion, are important for cell migration and are involved in many (patho)-physiological processes. Here we examined FAs and their associated actin fibres using correlative fluorescence and scanning electron microscopy (SEM). We used fluorescence images of cells expressing paxillin-GFP to define the boundaries of FA complexes in SEM images, without using SEM contrast enhancing stains. We observed that SEM contrast was increased around the actin fibre entry site in 98% of FAs, indicating increases in protein density and possibly also phosphorylation levels in this area. In nearly three quarters of the FAs, these nanostructures had a fork shape, with the actin forming the stem and the high-contrast FA areas the fork. In conclusion, the combination of fluorescent and electron microscopy allowed accurate localisation of a highly abundant, novel fork structure at the FA-actin interface.

DNA binding alters ARv7 dimer interactions.

Androgen receptor (AR) splice variants are proposed to be a potential driver of lethal castration-resistant prostate cancer. AR splice variant 7 (ARv7) is the most commonly observed isoform and strongly correlates with resistance to second-generation anti-androgens. Despite this clinical evidence, the interplay between ARv7 and the highly expressed full-length AR (ARfl) remains unclear. In this work, we show that ARfl/ARv7 heterodimers readily form in the nucleus via an intermolecular N/C interaction that brings the four termini of the proteins in close proximity. Combining fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that these heterodimers undergo conformational changes following DNA binding, indicating dynamic nuclear receptor interaction. Although transcriptionally active, ARv7 can only form short-term interactions with DNA at highly accessible high-occupancy ARfl binding sites. Dimerization with ARfl does not affect ARv7 binding dynamics, suggesting that DNA binding occupancy is determined by the individual protein monomers and not the homodimer or heterodimer complex. Overall, these biophysical studies reveal detailed properties of ARv7 dynamics as both a homodimer or heterodimer with ARfl.

Growth factor dependent changes in nanoscale architecture of focal adhesions.

Focal adhesions (FAs) are flat elongated structures that mediate cell migration and link the cytoskeleton to the extracellular matrix. Along the vertical axis FAs were shown to be composed of three layers. We used structured illumination microscopy to examine the longitudinal distribution of four hallmark FA proteins, which we also used as markers for these layers. At the FA ends pointing towards the adherent membrane edge (heads), bottom layer protein paxillin protruded, while at the opposite ends (tails) intermediate layer protein vinculin and top layer proteins zyxin and VASP extended further. At the tail tips, only intermediate layer protein vinculin protruded. Importantly, head and tail compositions were altered during HGF-induced scattering with paxillin heads being shorter and zyxin tails longer. Additionally, FAs at protruding or retracting membrane edges had longer paxillin heads than FAs at static edges. These data suggest that redistribution of FA-proteins with respect to each other along FAs is involved in cell movement.

Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells.

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation.

Uptake and subcellular distribution of radiolabeled polymersomes for radiotherapy.

Polymersomes have the potential to be applied in targeted alpha radionuclide therapy, while in addition preventing release of recoiling daughter isotopes. In this study, we investigated the cellular uptake, post uptake processing and intracellular localization of polymersomes. Methods: High-content microscopy was used to validate polymersome uptake kinetics. Confocal (live cell) microscopy was used to elucidate the uptake mechanism and DNA damage induction. Intracellular distribution of polymersomes in 3-D was determined using super-resolution microscopy. Results: We found that altering polymersome size and concentration affects the initial uptake and overall uptake capacity; uptake efficiency and eventual plateau levels varied between cell lines; and mitotic cells show increased uptake. Intracellular polymersomes were transported along microtubules in a fast and dynamic manner. Endocytic uptake of polymersomes was evidenced through co-localization with endocytic pathway components. Finally, we show the intracellular distribution of polymersomes in 3-D and DNA damage inducing capabilities of 213Bi labeled polymersomes. Conclusion: Polymersome size and concentration affect the uptake efficiency, which also varies for different cell types. In addition, we present advanced assays to investigate uptake characteristics in detail, a necessity for optimization of nano-carriers. Moreover, by elucidating the uptake mechanism, as well as uptake extent and geometrical distribution of radiolabeled polymersomes we provide insight on how to improve polymersome design.

Halogen-substituted anthranilic acid derivatives provide a novel chemical platform for androgen receptor antagonists.

Androgen receptor (AR) antagonists are used for hormone therapy of prostate cancer (PCa). However resistance to the treatment occurs eventually. One possible reason is the occurrence of AR mutations that prevent inhibition of AR-mediated transactivation by antagonists. To offer in future more options to inhibit AR signaling, novel chemical lead structures for new AR antagonists would be beneficial. Here we analyzed structure-activity relationships of a battery of 36 non-steroidal structural variants of methyl anthranilate including 23 synthesized compounds. We identified structural requirements that lead to more potent AR antagonists. Specific compounds inhibit the transactivation of wild-type AR as well as AR mutants that render treatment resistance to hydroxyflutamide, bicalutamide and the second-generation AR antagonist enzalutamide. This suggests a distinct mode of inhibiting the AR compared to the clinically used compounds. Competition assays suggest binding of these compounds to the AR ligand binding domain and inhibit PCa cell proliferation. Moreover, active compounds induce cellular senescence despite inhibition of AR-mediated transactivation indicating a transactivation-independent AR-pathway. In line with this, fluorescence resonance after photobleaching (FRAP) - assays reveal higher mobility of the AR in the cell nuclei. Mechanistically, fluorescence resonance energy transfer (FRET) - assays indicate that the amino-carboxy (N/C)-interaction of the AR is not affected, which is in contrast to known AR-antagonists. This suggests a mechanistically novel mode of AR-antagonism. Together, these findings indicate the identification of a novel chemical platform as a new lead structure that extends the diversity of known AR antagonists and possesses a distinct mode of antagonizing AR-function.

Staphylococcal Protein A Is a Key Factor in Neutrophil Extracellular Traps Formation.

Staphylococcus aureus are strong inducers of neutrophil extracellular traps (NETs), a defense mechanism of neutrophils against pathogens. Our aim was to explore the role of Protein A in S. aureus-induced NETosis. We determined the Protein A production of four different S. aureus strains and found a direct relationship between the degree of NETosis induction and Protein A production: strains producing higher concentrations of Protein A evoke significantly more NETs. A S. aureus strain in which Protein A as well as a second binding protein for immunoglobulins (Sbi) have been knocked-out (ΔSpA ΔSbi) induced significantly less NETosis than the wild-type strain. NETosis induction by this knockout strain can be rescued by the addition of purified Protein A. Dead S. aureus did not induce NETosis. In conclusion, Protein A is a determinant for NETosis induction by S. aureus.

In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review.

BACKGROUND: Multiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing. METHODS: In vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging. RESULTS: PMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis. CONCLUSION: Our systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events.

Novel Nor-Homo- and Spiro-Oxetan- Steroids Target the Human Androgen Receptor and Act as Antiandrogens.

The prostate adenocarcinoma is the cancer with the highest incidence for men in Western countries. Targeting the androgen receptor (AR) by antagonists is used as hormone therapy for prostate cancer (PCa), however, eventually therapy resistance occurs in most patients. In most of these cancer the AR signaling is active and thus AR remains an important drug target. Since many years we are characterizing novel chemical structural platforms to provide a broader possibility for compounds that bind to and act as AR antagonists. Here, we describe the chemical synthesis of a battery of novel steroidal derivatives as nor-homo-, spiro-oxolan- and spiro-oxetan- steroids. They modulate the transcriptional activity of the human AR. As AR antagonists, the spiro-oxetan- steroid derivatives seem to be the most potent steroid derivatives. They inhibit the transcriptional activity of both wild-type AR as well as the AR mutant T877A. In line with this, these compounds bind to the human AR and inhibit the proliferation of the human androgen-dependent growing PCa cell line LNCaP. Interestingly, the castration-resistant AR expressing human PC3-AR cells are also growth inhibited. On mechanistic level, fluorescence resonance energy transfer (FRET) assays with living cells indicate that the androgen-induced N/C terminal interaction of the AR is inhibited by the investigated compounds. Using fluorescence recovery after photobleaching (FRAP) assays in living cells suggest a higher mobility of the AR in the cell nuclei in the presence of spiro-oxetan- steroidal antagonists. Together, these findings suggest that spiro-oxetan- steroids are very useful as a chemical platform for novel AR antagonists.

Reversible aggregation of PABPN1 pre-inclusion structures.

Increased aggregation of misfolded proteins is associated with aging, and characterizes a number of neurodegenerative disorders caused by homopolymeric amino acid expansion mutations. PABPN1 is an aggregation-prone nuclear protein. Natural aggregation of wild-type (WT) PABPN1 is not known to be disease-associated, but alanine-expanded PABPN1 (expPABPN1) accumulates in insoluble intranuclear inclusions in muscle of patients with oculopharyngeal muscular dystrophy (OPMD). We applied microscopic image quantification to study PABPN1 aggregation process in living cells. We identified transitional pre-inclusion foci and demonstrate that these structures significantly differ between WT- and expPABPN1-expressing cells, while inclusions of these proteins are indistinguishable. In addition to the immobile PABPN1 in inclusions, in the nucleoplasm of expPABPN1 expressing cells we also found a fraction of immobile proteins, representing pre-aggregated species. We found that pre-aggregated and pre-inclusion structures are reverted by a PABPN1 specific affinity binder while inclusion structures are not. Together our results demonstrate that the aggregation process of WT- and expPABPN1 differs in steps preceding inclusion formation, suggesting that pre-aggregated protein species could represent the cytotoxic structures.

Application of fluorescence microscopy for investigation of cellular distribution of dinuclear platinum anticancer drugs.

The dinuclear platinum complexes with aliphatic diamines [{cis-Pt(NH(3))(2)Cl}(2)(mu-H(2)N(CH(2))(6)NH(2))](NO(3))(2) (1,1/c,c) and [{trans-Pt(NH(3))(2)Cl}(2)(mu-H(2)N(CH(2))(4)NH(2))](NO(3))(2) (1,1/t,t), which are known to be highly active in vitro against several cancer cell lines, have been modified with a fluorogenic reporter (carboxyfluorescein diacetate, CFDA) and a hapten (dinitrophenyl, DNP). These labeled complexes have been designed for fluorescence microscopy investigation of cellular pathways of promising dinuclear platinum anticancer drugs and present the first example of labeling biologically active dinuclear platinum complexes with a fluorescent reporter. The modified compounds interact with a guanine model base similarly to the label-free parent complexes. The uptake of the complexes with a fluorescent label and the respective unlabeled complexes in the U2-OS human osteosarcoma cell line and its cisplatin-resistant derivative, U2-OS/Pt cell line has been investigated. Cellular processing of the CFDA- and DNP-modified dinuclear platinum complexes in U2-OS and U2-OS/Pt cells has been studied.

A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain.

Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721-725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719-721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.

Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2.

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.