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Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins (ƒdark) in the ensemble. To date, ƒdark has rarely been quantified, and different methods to determine fdark have not been compared. Here, we use and compare two different methods to determine the fdark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain fdark. Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine ƒdark values between 30 and 60% for the different VFPs. The high values for ƒdark of these commonly used VFPs highlight that fdark has to be accounted for in quantitative microscopy and spectroscopy.

Kinases play key roles in signaling networks that are activated by G-protein-coupled receptors (GPCRs). Kinase activities are generally inferred from cell lysates, hiding cell-to-cell variability. To study the dynamics and heterogeneity of ERK and Akt proteins, we employed high-content biosensor imaging with kinase translocation reporters. The kinases were activated with GPCR ligands. We observed ligand concentration-dependent response kinetics to histamine, α2-adrenergic and S1P receptor stimulation. By using G-protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single-cell level, especially for ERK. Cluster analysis of time series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

A novel bright near-infrared fluorescent protein inserted into a nanobody enables visualization of native proteins inside living cells and specific manipulation of cell function, including Boolean protein-based operators.

Arbuscular mycorrhizal fungi function as conduits for underground nutrient transport. While the fungal partner is dependent on the plant host for its carbon (C) needs, the amount of nutrients that the fungus allocates to hosts can vary with context. Because fungal allocation patterns to hosts can change over time, they have historically been difficult to quantify accurately. We developed a technique to tag rock phosphorus (P) apatite with fluorescent quantum-dot (QD) nanoparticles of three different colors, allowing us to study nutrient transfer in an in vitro fungal network formed between two host roots of different ages and different P demands over a 3-week period. Using confocal microscopy and raster image correlation spectroscopy, we could distinguish between P transfer from the hyphae to the roots and P retention in the hyphae. By tracking QD-apatite from its point of origin, we found that the P demands of the younger root influenced both: (1) how the fungus distributed nutrients among different root hosts and (2) the storage patterns in the fungus itself. Our work highlights that fungal trade strategies are highly dynamic over time to local conditions, and stresses the need for precise measurements of symbiotic nutrient transfer across both space and time.

The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings identify a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair., The response to DNA damage-stalled RNA polymerase II leads to the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. Here, the authors reveal the complex assembly mechanism of the TCR complex in human cells.

G-protein-coupled receptors (GPCRs) are seven transmembrane spanning receptors that regulate a wide array of intracellular signaling cascades in response to various stimuli. To do so, they couple to different heterotrimeric G proteins and adaptor proteins, including arrestins. Importantly, arrestins were shown to regulate GPCR signaling through G proteins, as well as promote G protein-independent signaling events. Several research groups have reported successful isolation of exclusively G protein-dependent and arrestin-dependent signaling downstream of GPCR activation using biased agonists or receptor mutants incapable of coupling to either arrestins or G proteins. In the latter category, the DRY mutant of the angiotensin II type 1 receptor was extensively used to characterize the functional selectivity downstream of AT1AR. In an attempt to understand histamine 1 receptor signaling, we characterized the signaling capacity of the H1R DRY mutant in a panel of dynamic, live cell biosensor assays, including arrestin recruitment, heterotrimeric G protein activation, Ca2+ signaling, protein kinase C activity, GTP binding of RhoA, and activation of ERK1/2. Here, we show that both H1R DRY mutant and the AT1AAR DRY mutant are capable of efficient activation of G protein-mediated signaling. Therefore, contrary to the common belief, they do not constitute suitable tools for the dissection of the arrestin-mediated, G protein-independent signaling downstream of these receptors.

The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins.

Genetically encoded fluorescent proteins (FPs) are highly utilized in cell biology research to study proteins of interest or signal processes using biosensors. To perform well in specific applications, these FPs require a multitude of tailored properties. It is for this reason that they need to be optimized by using mutagenesis. The optimization process through screening is often based solely on bacterial colony brightness, but multiple parameters ultimately determine the performance of an optimal FP. Instead of characterizing other properties after selection, we developed a multiparameter screening method based on four critical parametersscreened simultaneously: fluorescence lifetime, cellular brightness, maturation efficiency, and photostability. First, a high-throughput primary screen (based on fluorescence lifetime and cellular brightness using a mutated FP library) is performed in bacterial colonies. A secondary multiparameter screen based on all four parameters, using a novel bacterial-mammalian dual-expression vector enables expression of the best FP variants in mammalian cell lines. A newly developed automated multiparameter acquisition and cell-based analysis approach for 96-well plates further increased workflow efficiency. We used this protocol to yield the record-bright mScarlet, a fast-maturating mScarlet-I, and a photostable mScarlet-H. This protocol can also be applied to other FP classes or Förster resonance energy transfer (FRET)-based biosensors with minor adaptations. With an available mutant library of a template FP and a complete and tested laboratory setup, a single round of multiparameter screening (including the primary bacterial screen, secondary mammalian cell screen, sequencing, and data processing) can be performed within 2 weeks.

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a key role in calcium homeostasis, by sensing free calcium levels in blood and regulating parathyroid hormone secretion in response. The CaSR is highly expressed in parathyroid gland and kidney where its role is well characterised, but also in other tissues where its function remains to be determined. The CaSR can be activated by a variety of endogenous ligands, as well as by synthetic modulators such as Cinacalcet, used in the clinic to treat secondary hyperparathyroidism in patients with chronic kidney disease. The CaSR couples to multiple G proteins, in a tissue-specific manner, activating several signalling pathways and thus regulating diverse intracellular events. The multifaceted nature of this receptor makes it a valuable therapeutic target for calciotropic and non-calciotropic diseases. It is therefore essential to understand the complexity behind the pharmacology, trafficking, and signalling characteristics of this receptor. This review provides an overview of the latest knowledge about the CaSR and discusses future hot topics in this field.

The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS — a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin.

In this thesis, 3 topics regarding the vascular system are discussed. First, a vessel-on-a-chip method is used to study the process of leukocyte transendothelial migration (TEM) in full. In the commonly used methods, the leukocytes encounter plastic after extravasating from the vessels instead of soft tissue. In the vessel-on-a-chip however, these cells encounter a physiological extracellular matrix. Using this state-of-the-art method, we were able to show that different leukocytes employ different methods of migration, something that previously could not be studied in vitro. In addition, we used this system to characterize neutrophils of a patient lacking the ARPC1B gene. These neutrophils are defective, especially in their migratory behavior. Using the vessel-on-a-chip, we could distinguish between hampered extravasation and diminished subsequent migration. The next topic concerns sprouting angiogenesis. A bead-sprouting assay is optimized for confocal microscopy, allowing the study of the process in much more detail. This assay is then used to study the role of Rab5C during VEGFR2 internalization and subsequent sprouting of the endothelial cells. Lastly, the role of the RhoGEF Trio in endothelial cells is described. Trio is well known for its role during neural growth; however, our group has shown it is an important factor in endothelial cells as well. Especially regarding the barrier function between endothelial cells. We created a vascular-specific, inducible mouse model to study this role in vivo.

During the inflammatory response, the white blood cells must exit the blood vessels at the right site to migrate into the tissue. To do so, the blood vessel wall cells – endothelial cells – provide signals when an inflammation in the underlying tissue occurs. The circulating white blood cells recognize these signals by interacting with the endothelial cells of the blood vessel wall. As inflammation often occurs in the underlying tissue, white blood cells maneuver out of the blood vessel, without causing excessive leakage of other blood components. Therefore, a tightly regulated interplay occurs when the white blood cells squeeze themselves between and through the endothelial cells. This process is called transendothelial migration; the migration of white blood cells through (trans) the blood vessel wall (endothelial layer). This process is tightly regulated to make it highly efficient. In this thesis new insights in the process of transendothelial migration are discussed. We describe a phenomenon called transendothelial migration hotspots. These preferred diapedesis sites are marked by adhesion molecules but also by endothelial membrane protrusions that are used by neutrophils to exit the vasculature. Furthermore, we discuss the post-diapedesis phase, where neutrophils migrate around focal adhesions underneath the endothelium. These new insights might help us better understanding TEM but also reveal targets for therapeutic intervention in disease situations.

Under inflammatory conditions, leukocytes are directed to exit the vasculature and pass through the endothelial barrier. In this thesis, we focus on specific aspects of the multistep transendothelial migration (TEM) process. First, we give a literature overview of the changes in endothelial actin dynamics upon inflammation and during leukocyte TEM that facilitate this process. Next, we performed a proteomics analysis of the ICAM-1 adhesome that is recruited at the docking site of the leukocyte, mapping the entire complex for the first time. We follow up on this screen by demonstrating the importance of CD44 for efficient cytotoxic T-lymphocyte (CTL) extravasation. Another follow-up study demonstrated that CTL form a transmigration synapse that is essential extravasation via the transcellular route. In the second part of this thesis, we generated a new leukocyte TEM assay by adapting blood-vessel-on-a-chip (BVOAC) technology. Current models to study leukocyte extravasation are based on endothelium cultured on stiff surfaces, which means TEM can only be studied until the point where leukocytes cross the endothelium. We demonstrate that BVOAC can be used to study the complete leukocyte TEM process from the vascular lumen into the perivascular tissue matrix with real-time and 3D microscopy. We applied this model to characterize migration dynamics induced by the endothelium in CTL and neutrophils. Finally, we show with our BVOAC model that neutrophils from a patient with a deficiency in the ARPC1B gene are impaired in both traversing the endothelium and migration into the tissue matrix.

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2–9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids., Currently, genetically encoded calcium indicators are not suitable for direct quantification. Here the authors engineer a fluorescence lifetime imaging calcium biosensor, Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS), and measure intracellular calcium concentrations in human-derived organoids.

Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores’ extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor’s (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)–SGFP2 and GerD–mScarlet-I, but there was no FRET between GerD–mScarlet-I and either GerRA–SGFP2 or GerRC–SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)–SGFP2 and GerD–mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)–SGFP2 and GerD–mScarlet-I, and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous.

Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein–protein interactions and is frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Förster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. The second messenger cyclic adenosine monophosphate (cAMP) is rapidly formed following activation of certain cell surface receptors. cAMP is subsequently degraded by a set of phosphodiesterases (PDEs) which display cell-type specific expression and may also affect baseline levels of the messenger. To study which specific PDEs contribute most to cAMP regulation, we knocked down individual PDEs and recorded breakdown rates of cAMP levels following transient stimulation in HeLa cells stably expressing the FRET/FLIM sensor, Epac-SH189. Many hundreds of cells were recorded at 5 s intervals for each condition. FLIM time traces were calculated for every cell, and decay kinetics were obtained. cAMP clearance was significantly slower when PDE3A and, to a lesser amount, PDE10A were knocked down, identifying these isoforms as dominant in HeLa cells. However, taking advantage of the quantitative FLIM data, we found that knockdown of individual PDEs has a very limited effect on baseline cAMP levels. By combining photon-efficient FLIM instrumentation with optimized sensors, systematic gene knockdown and an automated open-source analysis pipeline, our study demonstrates that dynamic screening of transient cell signals has become feasible. The quantitative platform described here provides detailed kinetic analysis of cellular signals in individual cells with unprecedented throughput.

Rho GTPases are regulatory proteins, which orchestrate cell features such as morphology, polarity and movement. Therefore, probing Rho GTPase activity is key to understanding processes such as development and cell migration. Localization-based reporters for active Rho GTPases are attractive probes to study Rho GTPase-mediated processes in real time with subcellular resolution in living cells and tissue. Until now, relocation Rho biosensors (sensors that relocalize to the native location of active Rho GTPase) seem to have been only useful in certain organisms and have not been characterized well. In this paper, we systematically examined the contribution of the fluorescent protein and Rho-binding peptides on the performance of localization-based sensors. To test the performance, we compared relocation efficiency and specificity in cell-based assays. We identified several improved localization-based, genetically encoded fluorescent biosensors for detecting endogenous Rho activity. This enables a broader application of Rho relocation biosensors, which was demonstrated by using the improved biosensor to visualize Rho activity during several cellular processes, such as cell division, migration and G protein-coupled receptor signaling. Owing to the improved avidity of the new biosensors for Rho activity, cellular processes regulated by Rho can be better understood. This article has an associated First Person interview with the first author of the paper., Summary: The dT-2xrGBD location-based Rho biosensor relocalizes more efficiently than other sensors of this type, and this sensor enables the observation of endogenous Rho activity in cultured cells.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

WNT/CTNNB1 signaling regulates tissue development and homeostasis in all multicellular animals, but the underlying molecular mechanism remains incompletely understood. Specifically, quantitative insight into endogenous protein behavior is missing. Here, we combine CRISPR/Cas9-mediated genome editing and quantitative live-cell microscopy to measure the dynamics, diffusion characteristics and absolute concentrations of fluorescently tagged, endogenous CTNNB1 in human cells under both physiological and oncogenic conditions. State-of-the-art imaging reveals that a substantial fraction of CTNNB1 resides in slow-diffusing cytoplasmic complexes, irrespective of the activation status of the pathway. This cytoplasmic CTNNB1 complex undergoes a major reduction in size when WNT/CTNNB1 is (hyper)activated. Based on our biophysical measurements, we build a computational model of WNT/CTNNB1 signaling. Our integrated experimental and computational approach reveals that WNT pathway activation regulates the dynamic distribution of free and complexed CTNNB1 across different subcellular compartments through three regulatory nodes: the destruction complex, nucleocytoplasmic shuttling, and nuclear retention.

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

Summary Fluorescent proteins (FPs) are of vital importance to biomedical research. Many of the currently available fluorescent proteins do not fluoresce when expressed in non‐native environments, such as the bacterial periplasm. This strongly limits the options for applications that employ multiple FPs, such as multiplex imaging and Förster resonance energy transfer (FRET). To address this issue, we have engineered a new cyan fluorescent protein based on mTurquoise2 (mTq2). The new variant is dubbed superfolder turquoise2ox (sfTq2ox) and is able to withstand challenging, oxidizing environments. sfTq2ox has improved folding capabilities and can be expressed in the periplasm at higher concentrations without toxicity. This was tied to the replacement of native cysteines that may otherwise form promiscuous disulfide bonds. The improved sfTq2ox has the same spectroscopic properties as mTq2, that is, high fluorescence lifetime and quantum yield. The sfTq2ox‐mNeongreen FRET pair allows the detection of periplasmic protein‐protein interactions with energy transfer rates exceeding 40%. Employing the new FRET pair, we show the direct interaction of two essential periplasmic cell division proteins FtsL and FtsB and disrupt it by mutations, paving the way for in vivo antibiotic screening. Significance The periplasmic space of Gram‐negative bacteria contains many regulatory, transport and cell wall‐maintaining proteins. A preferred method to investigate these proteins in vivo is by the detection of fluorescent protein fusions. This is challenging since most fluorescent proteins do not fluoresce in the oxidative environment of the periplasm. We assayed popular fluorescent proteins for periplasmic functionality and describe key factors responsible for periplasmic fluorescence. Using this knowledge, we engineered superfolder mTurquoise2ox (sfTq2ox), a new cyan fluorescent protein, capable of bright fluorescence in the periplasm. We show that our improvements come without a trade‐off from its parent mTurquoise2. Employing sfTq2ox as FRET donor, we show the direct in vivo interaction and disruption of unique periplasmic antibiotic targets FtsB and FtsL., The periplasm of Gram‐negative bacteria is challenging to research as most fluorescent proteins do not function in its environment. We developed a new fluorescent protein, sfTq2ox, that is able to efficiently transport to, fold and mature in the periplasm. The beneficial properties of sfTq2ox come without a trade‐off allowing the development of a highly efficient FRET assay for the periplasm.

We report the identification of high-affinity and selectivity integrin α5β1-binding bicyclic peptides via “designed random libraries”, that is, the screening of libraries comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop in combination with a randomized sequence (XXX) in the second loop. Screening of first-generation libraries for α5β1-binding peptides yielded a triple-digit nanomolar bicyclic α5β1-binder (CT3RGDcT3AYGCT3, IC50 = 406 nM). Next-generation libraries were designed by partially varying the structure of the strongest first-generation lead inhibitor and screened for improved affinities and selectivities for this receptor. In this way, we identified three high-affinity α5β1-binders (CT3RGDcT3AYJCT3, J = D-Leu, IC50 = 90 nM; CT3RGDcT3AYaCT3, IC50 = 156 nM; CT3RGDcT3AWGCT3, IC50 = 173 nM), of which one even showed a higher α5β1-affinity than the 32 amino acid benchmark peptide knottin-RGD (IC50 = 114 nM). Affinity for α5β1-integrin was confirmed by SPFS analysis showing a Kd of 4.1 nM for Cy5-labeled RGD-bicycle CT3RGDcT3AYJCT3 (J = D-Leu) and a somewhat higher Kd (9.0 nM) for Cy5-labeled knottin-RGD. The α5β1-bicycles, for example, CT3RGDcT3AYJCT3 (J = D-Leu), showed excellent selectivities over αvβ5 (IC50 ratio α5β1/αvβ5 between 50 ratios α5β1/αvβ3 between 0.090 and 0.157). In vitro staining of adipose-derived stem cells with Cy5-labeled peptides using confocal microscopy revealed strong binding of the α5β1-selective bicycle CT3RGDcT3AWGCT3 to integrins in their natural environment, illustrating the high potential of these RGD bicycles as markers for α5β1-integrin expression.

Developmental biology addresses one of the most beautiful miracles of life: how just one little cell can turn into an entire organism, built out of billions of cells. To answer the fundamental question of how this happens, it is essential to visualize cells, tissues and organs – allowing us to follow them during the process of tissue morphogenesis. In this day and age, scientists are also increasingly interested in understanding what goes on inside each of these cells, since cell behaviour is ultimately organised at the molecular level through specific gene expression and protein activity patterns. Knowing how an organism is built is not only interesting, but also essential to intervene when these processes go wrong, for example when developmental defects or uncontrolled cell growth occur. Cells are tightly controlled by the actions of several signal transduction pathways that maintain an intricate balance of cell proliferation, differentiation and survival. The WNT signalling pathway is essential in these processes, driving both embryonic development and tissue homeostasis throughout the multicellular animal kingdom. When Wnt signalling goes awry, this can result in a range of diseases, including cancer. Over forty years of WNT signalling research has resulted in a solid blueprint of the pathway and detailed insight into its core working mechanism. However, many details remain incompletely understood, especially in the context of how Wnt signalling drives division and differentiation of stem cells in complex, mammalian tissues. This brings the challenge of studying often subtle signalling events in wholetissue samples. Therefore, in this thesis, we have developed novel tools to study endogenous WNT signalling in mice.

The mammary gland is a complex and dynamic tissue. Its development and homeostasis require extensive and controlled communication between different cells. To regulate cell behavior, signaling molecules have to facilitate communication between cells at exactly the right time and place. WNT ligands are an example of such signaling molecules, and they are part of a conserved intercellular signaling pathway. Despite our knowledge of the WNT/CTNNB1 pathway, surprisingly little is known about how spatiotemporal expression of Wnt genes is regulated. To understand the basis of spatiotemporal specific cell-cell communication, we investigated how cell type specific Wnt expression is regulated in the mammary gland. An extensive transcriptomic analysis of the postnatal mammary gland showed specific expression patterns of Wnt genes in different cell populations. Generally speaking, specific gene expression patterns are thought to be established by regulatory elements that are scattered throughout the genome. We present a three-step plan on the identification of enhancers, which constitute a subcategory of regulatory elements that have the capacity to induce transcription of one or more genes. We used our workflow to find Wnt2 and Wnt4 enhancers that are active in the mammary gland. This resulted in the identification of an active enhancer hub upstream of the Wnt4 gene, and the identification of a Wnt2 enhancer, 15.4 kb upstream of the Wnt2 promoter. This has given us new insights into how cell type specific expression patterns of Wnt genes are established in the mammary gland.

In molecular cell biology, reporter assays are frequently used to investigate gene expression levels. Reporter assays employ a gene that encodes a light-emitting protein, of which the luminescence is quantified as a proxy of gene expression. Commercial parties provide reporter assay kits that include protocols and specialized detection machinery. However, downstream analysis of the output data and their presentation are not standardized. We have developed plotXpress to fill this gap, providing a free, open-source platform for the semi-automated analysis and standardized visualisation of experimental gene reporter data. Users can upload raw luminescence data acquired from a reporter gene assay with an internal control. In plotXpress, the data is corrected for sample variation with the internal control and the average for each condition is calculated. When a reference condition is selected the fold change is calculated for all other conditions, based on the selected reference. The results are shown as dot plots with a statistical summary, which can be adjusted to create publication-grade plots without requiring coding skills. Altogether, plotXpress is an open-source, low-threshold, web-based tool, that promotes a standardized and reproducible analysis while providing an appealing visualization of reporter data. The webtool can be accessed at: https://huygens.science.uva.nl/PlotXpress/

Comparative genome- and proteome-wide screens yield large amounts of data. To efficiently present such datasets and to simplify the identification of hits, the results are often presented in a type of scatterplot known as a volcano plot, which shows a measure of effect size versus a measure of significance. The data points with the largest effect size and a statistical significance beyond a user-defined threshold are considered as hits. Such hits are usually annotated in the plot by a label with their name. Volcano plots can represent ten thousands of data points, of which typically only a handful is annotated. The information of data that is not annotated is hardly or not accessible. To simplify access to the data and enable its re-use, we have developed an open source and online web tool with R/Shiny. The web app is named VolcaNoseR and it can be used to create, explore, label and share volcano plots (https://huygens.science.uva.nl/VolcaNoseR). When the data is stored in an online data repository, the web app can retrieve that data together with user-defined settings to generate a customized, interactive volcano plot. Users can interact with the data, adjust the plot and share their modified plot together with the underlying data. Therefore, VolcaNoseR increases the transparency and re-use of large comparative genome- and proteome-wide datasets.

CTNNB1 (catenin β-1, also known as β-catenin) plays a dual role in the cell. It is the key effector of WNT/CTNNB1 signalling, acting as a transcriptional co-activator of TCF/LEF target genes. It is also crucial for cell adhesion and a critical component of cadherin-based adherens junctions. Two functional pools of CTNNB1, a transcriptionally active and an adhesive pool, can therefore be distinguished. Whether cells merely balance the distribution of available CTNNB1 between these functional pools or whether interplay occurs between them has long been studied and debated. While interplay has been indicated upon artificial modulation of cadherin expression levels and during epithelial–mesenchymal transition, it is unclear to what extent CTNNB1 exchange occurs under physiological conditions and in response to WNT stimulation. Here, we review the available evidence for both of these models, discuss how CTNNB1 binding to its many interaction partners is controlled and propose avenues for future studies.

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/β‐catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here, we report the generation and characterization of a new knock‐in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi‐cistronic targeting cassette at the 3′ end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock‐in allele expresses a bright fluorescent reporter (3xNLS‐SGFP2) and a doxycycline‐inducible driver for lineage tracing (rtTA3). We show that the Axin2P2A‐rtTA3‐T2A‐3xNLS‐SGFP2 strain labels WNT/CTNNB1 responsive cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.

This year's Gairdner Foundation Award for Biomedical Research goes to Roel Nusse for his pioneering work on the Wnt signaling pathway and its many roles in development, cancer, and stem cells.

Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-inducible domains respond to the same wavelength as is required for excitation of popular CFP/YFP-based FRET pairs, rendering the techniques incompatible with each other. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor. Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. The new sensor was named ROPY and it visualizes the interaction between the microtubule regulator stathmin/OP18 and free tubulin heterodimers. We show that through phosphorylation of the ROPY sensor, its tubulin sequestering ability can be locally regulated by photo-activatable Rac1 (PARac1), independent of the FRET readout. Together, ROPY and PARac1 provide spatiotemporal control over free tubulin levels. ROPY/PARac1-based optogenetic regulation of free tubulin levels allowed us to demonstrate that depletion of free tubulin prevents the formation of pioneer microtubules, while local upregulation of tubulin concentration allows localized microtubule extensions to support the lamellipodia.

Experimental data can broadly be divided in discrete or continuous data. Continuous data are obtained from measurements that are performed as a function of another quantitative variable, e.g., time, length, concentration, or wavelength. The results from these types of experiments are often used to generate plots that visualize the measured variable on a continuous, quantitative scale. To simplify state-of-the-art data visualization and annotation of data from such experiments, an open-source tool was created with R/shiny that does not require coding skills to operate it. The freely available web app accepts wide (spreadsheet) and tidy data and offers a range of options to normalize the data. The data from individual objects can be shown in 3 different ways: (1) lines with unique colors, (2) small multiples, and (3) heatmap-style display. Next to this, the mean can be displayed with a 95% confidence interval for the visual comparison of different conditions. Several color-blind-friendly palettes are available to label the data and/or statistics. The plots can be annotated with graphical features and/or text to indicate any perturbations that are relevant. All user-defined settings can be stored for reproducibility of the data visualization. The app is dubbed PlotTwist and runs locally or online: https://huygens.science.uva.nl/PlotTwist, This Community Page article describes PlotTwist, an open source app created with R/shiny to simplify state-of-the-art visualization and annotation of results from experiments that generate continuous data; the app runs locally or online, and no coding skills are needed to use it.

Light-induced dimerizing systems, e.g. iLID, are an increasingly utilized optogenetics tool to perturb cellular signaling. The major benefit of this technique is that it allows external spatiotemporal control over protein localization with sub-cellular specificity. However, when it comes to local recruitment of signaling components to the plasmamembrane, this precision in localization is easily lost due to rapid diffusion of the membrane anchor. In this study, we explore different approaches of countering the diffusion of peripheral membrane anchors, to the point where we detect immobilized fractions with iFRAP on a timescale of several minutes. One method involves simultaneous binding of the membrane anchor to a secondary structure, the microtubules. The other strategy utilizes clustering of the anchor into large immobile structures, which can also be interlinked by employing tandem recruitable domains. For both approaches, the anchors are peripheral membrane constructs, which also makes them suitable for in vitro use. Upon combining these slower diffusing anchors with recruitable guanine exchange factors (GEFs), we show that we can elicit much more localized morphological responses from Rac1 and Cdc42 as compared to a regular CAAX-box based membrane anchor in living cells. Thanks to these new slow diffusing anchors, more precisely defined membrane recruitment experiments are now possible.

The aim of the research presented in this thesis was to gain a better understanding of how intracellular signaling by GPCRs is regulated. We used fluorescence microscopy to study GPCR signaling in live cells. In order to study protein-protein interactions involved in GPCR signaling, a high spatiotemporal resolution is required. This because these interactions are often short-lived and occur at

This thesis is part of a project with the goal of establishing a simple artificial cell containing a minimum of required components to control microtubule growth and adjust the morphology of the encapsulating membrane in vitro. First, we focused on creating a working system in vivo. Regulation of protein activity in the system was achieved through the use of optogenetics. All signaling is subjected to mechanisms such as diffusion, even the optogenetic systems, which is why we altered the diffusion rate of components in chapter 2. We developed a strategy with different types of membrane anchors which decrease lateral diffusion and were able to demonstrate their effect through light-induced recruitment of signaling proteins to the plasma membrane. Chapter 3 addresses the problem of combining FRET sensors with blue light sensitive optogenetic domains through redshifted fluorescent proteins. With this method we explored the effect of photoregulatable Rac1 on stathmin and microtubules. In chapter 4 we created a photoregulatable kinase that directly phosphorylates stathmin, in order to reduce the amount of intermediates between stimuli and microtubule response. Finally, in chapter 5 we offer a way to directly regulate the stathmin molecule itself via blue light. This lead to the first light-induced, reversible mechanism to capture and release endogenous tubulin at will. These tools are a first step towards the creation of a minimalistic membrane regulating in vitro system.

Diacylglycerol (DAG) is an important intermediate in lipid biosynthesis and plays key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difficult because biochemical fractionation affects the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in living plant cells. The sensor is composed of a fusion between yellow fluorescent protein and the C1a domain of protein kinase C (YFP–C1aPKC) that specifically binds DAG, and was stably expressed in suspension-cultured tobacco BY-2 cells and whole Arabidopsis thaliana plants. Confocal imaging revealed that the majority of the YFP–C1aPKC fluorescence did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaflet of membranes (i.e. accessible to the biosensor) is in general too low, and confirms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans-Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo.

The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein–coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer–based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR1-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαqactivity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR1-Gαi-Cdc42 pathway.

Intracellular pH (pH i ) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pH i homeostasis. Unfortunately, accurate pH i quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pH i at single-cell levels in Bacillus subtilis . Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pH i in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pH i and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pH i regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. IMPORTANCE This study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives.

Wnt/ß-catenin signalling is crucial for maintaining the balance between cell proliferation and differentiation, both during tissue morphogenesis and in tissue maintenance throughout postnatal life. Whereas the signalling activities of the core Wnt/ß-catenin pathway components are understood in great detail, far less is known about the precise role and regulation of the many different modulators of Wnt/ß-catenin signalling that have been identified to date. Here we describe TMEM98, a putative transmembrane protein of unknown function, as an interaction partner and regulator of the GSK3-binding protein FRAT2. We show that TMEM98 reduces FRAT2 protein levels and, accordingly, inhibits the FRAT2-mediated induction of ß-catenin/TCF signalling. We also characterize the intracellular trafficking of TMEM98 in more detail and show that it is recycled between the plasma membrane and the Golgi. Together, our findings not only reveal a new layer of regulation for Wnt/ß-catenin signalling, but also a new biological activity for TMEM98.

Endothelial cells (ECs) line all blood vessels and dynamically regulate the endothelial barrier function, which is critical for proper functioning of the vasculature and human health. ECs continuously sense and respond to various stimuli that can either strengthen or disrupt the endothelium, depending on the output of large and complex signaling networks that become activated. Activation of these respective networks often include regulation via GPCRs, one of the major groups of cell surface receptors that respond to a variety of stimuli and of which ECs express many different subtypes. In general, GPCRs initiate Rho GTPase signaling cascades, thereby acting on the actin cytoskeleton and subsequently regulating EC-cell contacts. Since the latter is a direct measure of permeability and endothelial barrier function, these signaling mechanisms are indispensable for a healthy endothelium and vasculature. Despite the interest regarding GPCR-Rho GTPase-mediated signaling networks in the endothelium, critical, molecualr details are lacking. Therefore, this thesis aims to reveal novel molecular players in GPCR-Rho GTPase signaling networks that control the endothelial barrier function. In short, we revealed novel regulators and dynamic signaling networks that control the endothelial barrier function via GPCR-Rho GTPase signaling. We specified major regulators at the level of receptors (e.g. PAR-1 and S1PRs), GEFs (e.g. pRex1) and Rho GTPases (e.g. Rac1, Cdc42 and Rho) and furthermore highlighted the dynamics of the endothelium. Thereby, our research has provided new, molecular insights into the physiology of the endothelium and endothelial barrier control, and furthermore identifies novel therapeutic targets in vascular, endothelialbased pathologies.

Fluorescent proteins (FPs) are used as genetic labels to study processes in live cells by using fluorescence microscopy. Multiple processes can be observed simultaneously, when FPs in multiple colors are used. Currently, red fluorescent proteins (RFPs) are not optimal for quantitative cellular imaging. The aim of the research presented in this thesis is to create an improved monomeric red fluorescent protein (mRFP). Initially, we applied established methods to the development of mRFPs. However, we found that the development of mRFPs was more complex and screening on merely one parameter is not sufficient. Therefore, we developed a multi-parameter screening method (Chapter 1). The development of this multi-parameter screening method coincided with the development of improved mRFPs and led to the mScarlet family (Chapter 2). mScarlet is the brightest mRFP with a record quantum yield in its spectral class. Two other important variants are developed: mScarlet-I which shows enhanced maturation and mScarlet-H which displays extreme photostability. We quantitively compared the new mScarlets to existing RFPs in vitro and in vivo. Moreover, photophysical properties of the mScarlet family and several well-known RFPs are studied (Chapter 3). Many photophysical properties critically depend on both pH and illumination and are different for each RFP variant. This makes it a major challenge to optimize and screen for RFPs with a particular combination of favorable photophysical properties. The developed methods and new insights will assist future development of FPs of any color. Ultimately, mScarlet itself can serve as a new template for mRFP optimization.

Tunneling membrane nanotubes (TnTs) are membrane protrusions connecting nearby or distant cells in vitro and in vivo. Functions of TnTs in cellular processes are various and rely on TnT structure, which also depends on cytoskeletal composition. In the present study, we focused on the organization of microtubules (MTs) and intermediate filaments (IFs) in TnTs of urothelial cells. We analysed TnTs of normal porcine urothelial cells, which morphologically and physiologically closely resemble normal human urothelial cells, and of cancer cells derived from invasive human urothelial neoplasm. Wide-field fluorescence, confocal and super-resolution microscopy techniques, together with image analyses and 3D reconstructions enlightened specific MT-IF organization in TnTs, and for the first time revealed that MTs and IFs co-occur in the majority of normal and cancer urothelial cell TnTs. Our findings show that in the initiation segment of TnTs, MTs are cross-linked with each other into filamentous network, however in the middle and the attaching segment of TnT, MTs can helically enwrap IFs, the phenomenon that has not been shown before within the TnTs. In this study, we assess MT-IF co-occurrence in TnTs and present evidence that such helical organization of MTs enwrapping IFs is only occurring in a minority of the TnTs. We also discuss the possible cell-biological and physiological reasons for helical organization of MTs in TnTs.

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) can visualize responses of individual cells in real time. Here, we evaluated whether FRET-based biosensors provide sufficient contrast and specificity to measure activity of G-protein–coupled receptors. The four histamine receptor subtypes (H1R, H2R, H3R, and H4R) respond to the ligand histamine by activating three canonical heterotrimeric G-protein–mediated signaling pathways with a reported high degree of specificity. Using FRET-based biosensors, we demonstrate that H1R activates Gαq. We also observed that H1R activates Gαi, albeit at a 10-fold lower potency. In addition to increasing cAMP levels, most likely via Gαs, we found that the H2R induces Gαq-mediated calcium release. The H3R and H4R activated Gαi with high specificity and a high potency. We demonstrate that a number of FRET sensors provide sufficient contrast to: 1) analyze the specificity of the histamine receptor subtypes for different heterotrimeric G-protein families with single-cell resolution, 2) probe for antagonist specificity, and 3) allow the measurement of single-cell concentration-response curves.

We developed single-image fluorescence lifetime imaging microscopy (siFLIM), a method for acquiring quantitative lifetime images from a single exposure. siFLIM takes advantage of a new generation of dedicated cameras that simultaneously record two 180 degrees -phase-shifted images, and it allows for video-rate lifetime imaging with minimal phototoxicity and bleaching. siFLIM is also inherently immune to artifacts stemming from rapid cellular movements and signal transients.

There were multiple prerequisites to the evolution of multicellular animal life, including the generation of multiple cell fates ("cellular diversity") and their patterned spatial arrangement ("spatial form"). Wnt proteins operate as primordial symmetry-breaking signals. By virtue of their short-range nature and their capacity to activate both lineage-specifying and cell-polarizing intracellular signaling cascades, Wnts can polarize cells at their site of contact, orienting the axis of cell division while simultaneously programming daughter cells to adopt diverging fates in a spatially stereotyped way. By coupling cell fate to position, symmetry-breaking Wnt signals were pivotal in constructing the metazoan body by generating cellular diversity and spatial form.

In the process of leukocyte migration from the circulation across the vascular wall, the crosstalk with endothelial cells that line the blood vessels is essential. It is now firmly established that in endothelial cells important signaling events are initiated upon leukocyte adhesion that impinge on the regulation of cell-cell contact and control the efficiency of transendothelial migration. In addition, several external factors such as shear force and vascular stiffness were recently identified as important regulators of endothelial signaling and, consequently, leukocyte transmigration. Here, I review recent insights into endothelial signaling events that are linked to leukocyte migration across the vessel wall. In this field, protein phosphorylation and Rho-mediated cytoskeletal dynamics are still widely studied using increasingly sophisticated mouse models. In addition, activation of tyrosine phosphatases, changes in endothelial cell stiffness as well as different vascular beds have all been established as important factors in endothelial signaling and leukocyte transmigration. Finally, I address less-well-studied but interesting components in the endothelium that also control transendothelial migration, such as the ephrins and their Eph receptors, that provide novel insights in the complexity associated with this process.

Bacillus subtilis spores are a problem for the food industry as they are able to survive preservation processes. The spores often reside in food products, where their inherent protection against various stress treatments causes food spoilage. Sorbic acid is widely used as a weak acid preservative in the food industry. Its effect on spore germination and outgrowth in a combined, 'hurdle', preservation setting has gained limited attention. Therefore, the effects of mild sorbic acid (3 mM), heat-treatment (85 degrees C for 10 min) and a combination of both mild stresses on germination and outgrowth of B. subtilis 1A700 spores were analysed at single spore level. The heat-treatment of the spore population resulted in a germination efficiency of 46.8% and an outgrowth efficiency of 32.9%. In the presence of sorbic acid (3 mM), the germination and outgrowth efficiency was 93.3% and 80.4% respectively whereas the combined heat and sorbic acid stress led to germination and outgrowth efficiencies of 52.7% and 27.0% respectively. The heat treatment clearly primarily affected the germination process, while sorbic acid affected the outgrowth and generation time. In addition a new 'burst' time-point was defined as the time-point at which the spore coat visibly breaks and/or is shed. The combined stresses had a synergistic effect on the time of the end of germination to the burst time-point, increasing both the mean and its variation more than either of the single stresses did.

Fluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.

BACKGROUND: Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity.METHODS: Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest.RESULTS: The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGATΔ15, lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGATΔ15 variant. Synthetic recruitment of TGATΔ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane.CONCLUSION: Together, these results show that membrane association of TGAT is critical for its activity.