Fluorescence Recovery After Photobleaching Research Articles

Smad2/3/4 complex could undergo liquid liquid phase separation and induce apoptosis through TAT in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-β signaling pathway may be associated with tumor progression. However, the relationship between TGF-β signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-β signaling pathway on HCC progression as well as the potential regulatory mechanism involved. MethodsWe conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni–NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo.ResultsWe found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is −1032 to −1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis.ConclusionThis study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-β signaling pathway and HCC, which contributes to discover the mechanism of HCC development.

Supported Biomembrane Systems Incorporating Multiarm Polymers and Bioorthogonal Tethering.

To functionalize interfaces with supported biomembranes and membrane proteins, the challenge is to build stabilized and supported systems that mimic the native lipid microenvironment. Our objective is to control substrate-to-biomembrane spacing and the tethering chemistry so proteoliposomes can be fused and conjugated without perturbation of membrane protein function. Furthermore, the substrates need to exhibit low protein and antibody nonspecific binding to use these systems in assays. We have employed protein orthogonal coupling schemes in concert with multiarm poly(ethylene glycol) (PEG) technology to build supported biomembranes on microspheres. The lipid bilayer structures and tailored substrates of the microsphere-supported biomembranes were analyzed via flow cytometry, confocal fluorescence, and super-resolution imaging microscopy, and the lateral fluidity was quantified using fluorescence recovery after photobleaching (FRAP) techniques. Under these conditions, the 4-arm-PEG20,000-NH2 based configuration gave the most desirable tethering system based on lateral diffusivity and coverage.

Bottlebrush Block Copolymers at the Interface of Immiscible Liquids: Adsorption and Lateral Packing.

Amphiphilic bottlebrush block copolymers (BBCPs), having a hydrophilic bottlebrush polymer (BP) linked covalently to a hydrophobic BP, were found to segregate to liquid-liquid interfaces to minimize the free energy of the system. The key parameter influencing the outcome of the experiments is the ratio between the degree of polymerization of the backbone (NBB) and that of the side-chain brushes (NSC). Specifically, a spherical, star-like configuration results when NBB < NSC, while a cylindrical, bottlebrush-like shape is preferred when NBB > NSC. Dynamic interfacial tension (γ) and fluorescence recovery after photobleaching (FRAP) measurements show that the BBCP configuration influences the areal density and in-plane diffusion at the fluid interface. The characteristic relaxation times associated with BBCP adsorption (τA) and reorganization (τR) were determined by fitting time-dependent interfacial tension measurements to a sum of two exponential relaxation functions. Both τA and τR initially increased with NBB up to 92 repeat units, due to the larger hydrodynamic radius in solution and slower in-plane diffusivity, attributed to a shorter cross-sectional diameter of the side-chains near the block junction. This trend reversed at NBB = 190, with shorter τA and τR attributed to increased segregation strength and exposure of the bare water/toluene interface due to tilting and/or wiggling of the backbone chains, respectively. The adsorption energy barrier decreased with higher NBB, due to a reduced BBCP packing density at the fluid interface. This study provides fundamental insights into macromolecular assembly at fluid interfaces, as it pertains to unique bottlebrush block architectures.

Ionic Effect on the Microenvironment of Biomolecular Condensates.

Biomolecules such as proteins and RNA could organize to form condensates with distinct microenvironments through liquid-liquid phase separation (LLPS). Recent works have demonstrated that the microenvironment of biomolecular condensates plays a crucial role in mediating biological activities, such as the partition of biomolecules, and the subphase organization of the multiphasic condensates. Ions could influence the phase transition point of LLPS, following the Hofmeister series. However, the ion-specific effect on the microenvironment of biomolecular condensates remains unknown. In this study, we utilized fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and microrheology techniques to investigate the ion effect on the microenvironment of condensates. We found that ions significantly affect the microenvironment of biomolecular condensates: salting-in ions increase micropolarity and reduce the microviscosity of the condensate, while salting-out ions induce opposing effects. Furthermore, we manipulate the miscibility and multilayering behavior of condensates through ion-specific effects. In summary, our work provides the first quantitative survey of the microenvironment of protein condensates in the presence of ions from the Hofmeister series, demonstrating how ions impact micropolarity, microviscosity, and viscoelasticity of condensates. Our results bear implications on how membrane-less organelles would exhibit varying microenvironments in the presence of continuously changing cellular conditions.

Network pharmacology of Dracaena sp. in Guangxi and its related species leaf secondary metabolites possess antioxidant properties

In this study, metabolite maps were conducted on leaf samples accessions of Dracaena sp. in Guangxi (C6, C13, CZ4, JX2, RM1, and BM3) and six related species including D. angustifolia (CH), D. elliptica (XZ), D. cochinchinensis (CB), D. cambodiana (HN), D. marginata (HB), and Yucca schidigera (SL). We identified 2,971 compounds including carboxylic acids and their derivatives, organooxygen compounds, prenol lipids, benzenes and their substituted derivatives, and flavonoids. Of those, 73 characteristic secondary metabolites were screened out by weighted correlation network analysis (WGCNA). Pterostilbene, 9-cis-retinal, and dracorubin were unique to the Dracaena sp. in Guangxi, and 3-hydroxytridecanoic acid and chrysin were highly abundant in them. An in vitro antioxidant activity assay disclosed that all 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging rates and all fluorescence recovery after photobleaching (FRAP) rates were in the ranges of 75–87 % and 3.10–3.25 μmol/mL, respectively, for the Dracaena sp. in Guangxi. Hence, the latter have relatively high and stable foliar antioxidant activity. A network pharmacological analysis of the top 20 metabolites in Dracaena leaf and their 446 antioxidant-related disease targets revealed ten core action targets, including GAPDH, AKT1, MAPK3, VEGFA, CASP3, TNF, MAPK1, SRC, EGFR, and MAPK8. An antioxidant-related compound-target-pathway network was constructed and verified by molecular docking. The results of this study provide a theoretical basis for mining and exploiting the foliar secondary metabolites of Dracaena.

Spaced training activates Miro/Milton-dependent mitochondrial dynamics in neuronal axons to sustain long-term memory

Neurons have differential and fluctuating energy needs across distinct cellular compartments, shaped by brain electrochemical activity associated with cognition. Invitro studies show that mitochondria transport from soma to axons is key to maintaining neuronal energy homeostasis. Nevertheless, whether the spatial distribution of neuronal mitochondria is dynamically adjusted invivo in an experience-dependent manner remains unknown. In Drosophila, associative long-term memory (LTM) formation is initiated by an early and persistent upregulation of mitochondrial pyruvate flux in the axonal compartment of neurons in the mushroom body (MB). Through behavior experiments, super-resolution analysis of mitochondria morphology in the neuronal soma and invivo mitochondrial fluorescence recovery after photobleaching (FRAP) measurements in the axons, we show that LTM induction, contrary to shorter-lived memories, is sustained by the departure of some mitochondria from MB neuronal soma and increased mitochondrial dynamics in the axonal compartment. Accordingly, impairing mitochondrial dynamics abolished the increased pyruvate consumption, specifically after spaced training and in the MB axonal compartment, thereby preventing LTM formation. Our results thus promote reorganization of the mitochondrial network in neurons as an integral step in elaborating high-order cognitive processes.

Abstract 193: Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9

Abstract Cell shape change and motility are essential in metastasis and involve remodeling of the actin cytoskeleton, cell adhesions, and plasma membrane. How these cytoskeletal and membrane remodeling are regulated and coordinated remains largely unknown. We previously identified TRIM9 as a regulator of actin dynamics and exocytosis in developing neurons. Deletion of murine Trim9 impairs neuronal migration, netrin-induced axon turning, and axonal and dendritic branching, and increased exocytosis and filopodial stability. In addition, we have shown TRIM9 alters dynamics of the actin polymerase VASP at filopodia tips via non-degrative mono-ubiquitination. TRIM9 is expressed in other motile cells, but the non-neuronal role of TRIM9 remains unknown. TRIM9 was identified as a possible prognostic biomarker in melanoma and high expression correlates with low patient survival. Melanomas undergo phenotype switching, where three distinct phenotypes exist that are associated with differences in gene expression. The three phenotypes consist of a melanocytic proliferative state, a neural crest-like invasive state, an intermediate state. Single cell RNAseq (scRNASeq) data from patient-derived melanoma indicate TRIM9 is highly expressed in the proliferative melanocytic state, which correlates with poor prognosis. We hypothesize that TRIM9 coordinates actin dynamics, adhesion, and exocytosis in melanoma. We show that TRIM9 protein is enriched in several human melanoma lines. Here we examine the role of TRIM9 in regulation of focal adhesions, exocytosis, migration, and invasion in two of these lines. Genetic loss of TRIM9 increased random migration velocity but reduced directional persistence. Interestingly, we find that TRIM9 plays a role in promoting bleb-like morphology and inhibits the ability to durotax on shallow gradients on soft substrate. Fluorescence recovery after photobleaching (FRAP), Total internal reflection fluorescence microscopy (TIRF), and widefield microscopy revealed that loss of TRIM9 results in increased focal adhesions, cell size, and altered dynamics of focal adhesion proteins VASP, zyxin, and paxillin dynamics. In addition, TRIM9 knockout cells exhibit reduced filopodial length and density and altered filopodial localization of VASP. In-gel zymography indicates that TRIM9 knockout cells also display an increased degradative capacity. Current studies are investigating how loss of TRIM9 alters parameters of cell contractility, lamellipodia, invadopodia, and exocytosis to define the role of TRIM9 in melanoma motility. Together these findings suggest TRIM9 reduces adhesion and migration, increases proliferation and blebbing and may be an important regulator of phenotype switching in melanoma. Citation Format: Kimberly Lukasik, Jordan Brooks, Sam Redinbo, James Bear, Stephanie Gupton. Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 193.

Diffusion of Multiple Species Resolved by Fluorescence Lifetime Recovery after Photobleaching (FLRAP).

Fluorescence recovery after photobleaching (FRAP) is now an indispensable tool to analyze the diffusion of molecules in vivo and in vitro. However, a conventional fluorescence intensity-based approach has difficulty in analyzing the diffusion of multiple species simultaneously. Here, we report fluorescence lifetime recovery after photobleaching (FLRAP) that incorporates fluorescence lifetime information into FRAP. By using FLRAP, the fluorescence intensity-recovery curves of each species can be successfully extracted from the ensemble photon data by utilizing their species-specific fluorescence decay curves, which are verified by applying FLRAP to two heterogeneous systems. Thus, FLRAP can be a powerful tool to quantitatively elucidate the molecular diffusion of multiple species in complex systems such as in living cells.

Effect of Pore Confinement of Ionic Liquids on Solute Diffusion within Mesoporous Silica Microparticles.

The transport properties of the ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) confined within silica microparticles with well-ordered, accessible mesopores (5.4 or 9 nm diameter) were investigated. [BMIM][PF6] confinement was confirmed by using differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy. The transport properties of the confined IL were studied using the neutral and cationic fluorescent probes 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) and rhodamine 6G, respectively, through fluorescence recovery after photobleaching (FRAP) in confocal microscopy. The diffusivity of DCM in 9 nm pores is 0.026 ± 0.0091 μm2/s, which is 2 orders of magnitude less than in the bulk ionic liquid. The pore size did not affect the diffusivity of DCM in unmodified silica nanopores. The diffusivity of the cationic probe is reduced by 63% relative to that of the neutral probe. Diffusivity is increased with water content, where equilibrium hydration of the system leads to a 37% increase in DCM diffusivity. The most dramatic impact on diffusivity was caused by tethering an IL-like methylimidazolium chloride group to the pores, which increased the pore hydrophobicity and resulted in 3-fold higher diffusivity of DCM compared to bare silica pores. Subsequent exchange of the chloride anion from the tethering group with PF6- decreased the diffusivity to half that of bare silica. The diffusion of probe molecules is affected most strongly by the pore wall effects on probe interactions rather than by the pore size itself, which suggests that understanding pore wall diffusion is critical to the design of nanoconfined ILs for separations, catalysis, and energy storage.

Efficient Estimates of Surface Diffusion Parameters for Spatio-Temporally Resolved Virus Replication Dynamics.

Advanced methods of treatment are needed to fight the threats of virus-transmitted diseases and pandemics. Often, they are based on an improved biophysical understanding of virus replication strategies and processes in their host cells. For instance, an essential component of the replication of the hepatitis C virus (HCV) proceeds under the influence of nonstructural HCV proteins (NSPs) that are anchored to the endoplasmatic reticulum (ER), such as the NS5A protein. The diffusion of NSPs has been studied by in vitro fluorescence recovery after photobleaching (FRAP) experiments. The diffusive evolution of the concentration field of NSPs on the ER can be described by means of surface partial differential equations (sufPDEs). Previous work estimated the diffusion coefficient of the NS5A protein by minimizing the discrepancy between an extended set of sufPDE simulations and experimental FRAP time-series data. Here, we provide a scaling analysis of the sufPDEs that describe the diffusive evolution of the concentration field of NSPs on the ER. This analysis provides an estimate of the diffusion coefficient that is based only on the ratio of the membrane surface area in the FRAP region to its contour length. The quality of this estimate is explored by a comparison to numerical solutions of the sufPDE for a flat geometry and for ten different 3D embedded 2D ER grids that are derived from fluorescence z-stack data of the ER. Finally, we apply the new data analysis to the experimental FRAP time-series data analyzed in our previous paper, and we discuss the opportunities of the new approach.

Parameter Identifiability in PDE Models of Fluorescence Recovery After Photobleaching.

Identifying unique parameters for mathematical models describing biological data can be challenging and often impossible. Parameter identifiability for partial differential equations models in cell biology is especially difficult given that many established in vivo measurements of protein dynamics average out the spatial dimensions. Here, we are motivated by recent experiments on the binding dynamics of the RNA-binding protein PTBP3 in RNP granules of frog oocytes based on fluorescence recovery after photobleaching (FRAP) measurements. FRAP is a widely-used experimental technique for probing protein dynamics in living cells, and is often modeled using simple reaction-diffusion models of the protein dynamics. We show that current methods of structural and practical parameter identifiability provide limited insights into identifiability of kinetic parameters for these PDE models and spatially-averaged FRAP data. We thus propose a pipeline for assessing parameter identifiability and for learning parameter combinations based on re-parametrization and profile likelihoods analysis. We show that this method is able to recover parameter combinations for synthetic FRAP datasets and investigate its application to real experimental data.

Fraping: A computational tool for detecting slight differences in fluorescence recovery after photobleaching (FRAP) data for actin polymerization analysis.

Fluorescence recovery after photobleaching (FRAP) is a laser method of light microscopy to evaluate the rapid movement of fluorescent molecules. To have a more reliable approach to analyze data from FRAP, we designed Fraping, a free access R library to data analysis obtained from FRAP. Unlike other programs, Fraping has a new form of analyzing curves of FRAP using statistical analysis based on the average curve difference. To evaluate our library, we analyzed the differences of actin polymerization in real time between dendrites and secondary neurites of cultured neuron transfected with LifeAct to track F-actin changes of neurites. We found that Fraping provided greater sensitivity than the conventional model using mobile fraction analysis. Likewise, this approach allowed us to normalize the fluorescence to the size area of interest and adjust data curves choosing the best parametric model. In addition, this library was supplemented with data simulation to have a more significant enrichment for the analysis behavior. We concluded that Fraping is a method that reduces bias when analyzing two data groups as compared with the conventional methods. This method also allows the users to choose a more suitable analysis approach according to their requirements. RESEARCH HIGHLIGHTS: Fraping is a new programming tool to analyze FRAP data to normalize fluorescence recovery curves. The conventional method uses one-point analysis, and the new one compares all the points to define the similarity of the fluorescence recovery.

Understanding and manipulating extracellular behaviors of Wnt ligands.

Wnt, a family of secreted signaling proteins, serves diverse functions in embryogenesis, organogenesis, cancer, and stem cell functions. In the context of development, Wnt has been considered a representative morphogen, forming concentration gradients to give positional information to cells or tissues. However, although gradients are often illustrated in schemata, the reality of concentration gradients, or in other words, actual spatial distribution of Wnt ligands, and their behaviors in the extracellular space still remain poorly known. To understand extracellular behavior of Wnt ligands, quantitative analyses such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) are highly informative because Wnt dispersal involves physical and biochemical processes, such as diffusion and binding to or dissociation from cell surface molecules, including heparan sulfate proteoglycans (HSPGs). Here, I briefly discuss representative methods to quantify morphogen dynamics. In addition, I discuss molecular manipulations of morphogens, mainly focusing on use of protein binders, and synthetic biology of morphogens as indicators of current and future directions in this field.

Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy.

PET hydrolases are an emerging class of enzymes that are being heavily researched for their use in bioprocessing polyethylene terephthalate (PET). While work has been done in studying the binding of PET oligomers to the active site of these enzymes, the dynamics of PET hydrolases binding to a bulk PET surface is an unexplored area. Here, methods were developed for total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) microscopy to study the adsorption and desorption dynamics of these proteins onto a PET surface. TIRF microscopy was employed to measure both on and off rates of two of the most commonly studied PET hydrolases, PHL7 and LCC, on a PET surface. It was found that these proteins have a much slower off rates on the order of 10-3 s-1 , comparable to non-productive binding in enzymes such as cellulose. In combination with FRAP microscopy, a dynamic model is proposed in which adsorption and desorption dominates over lateral diffusion over the surface. The results of this study could have implications for the future engineering of PET hydrolases, either to target them to a PET surface or to modulate interaction with their substrate.

GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact.

Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells. Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP). Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact. Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.

De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.

The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A.

Mutations in activin-like kinase 2 (ALK2), e.g., ALK2-R206H, induce aberrant signaling to SMAD1/5/8, leading to Fibrodysplasia Ossificans Progressiva (FOP). In spite of extensive studies, the underlying mechanism is still unclear. Here, we quantified the homomeric and heteromeric interactions of ACVR2A, ACVR2B, ALK2-WT, and ALK2-R206H by combining IgG-mediated immobilization of one receptor with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of a co-expressed receptor. ACVR2B formed stable homomeric complexes that were enhanced by Activin A (ActA), while ACVR2A required ActA for homodimerization. ALK2-WT, but not ALK2-R206H, exhibited homomeric complexes unaffected by ActA. ACVR2B formed ActA-enhanced heterocomplexes with ALK2-R206H or ALK2-WT, while ACVR2A interacted mainly with ALK2-WT. The extent of the homomeric complex formation of ACVR2A or ACVR2B was reflected in their ability to induce the oligomerization of ALK2-R206H and ALK2-WT. Thus, ACVR2B, which forms dimers without ligand, induced ActA-independent ALK2-R206H clustering but required ActA for enhancing the oligomerization of the largely dimeric ALK2-WT. In contrast, ACVR2A, which undergoes homodimerization in response to ActA, required ActA to induce ALK2-R206H oligomerization. To investigate whether these interactions are translated into signaling, we studied signaling by the FOP-inducing hyperactive ALK2-R206H mutant, with ALK2-WT signaling as control. The activation of SMAD1/5/8 signaling in cells expressing ALK2-R206H alone or together with ACVR2A or ACVR2B was measured by blotting for pSMAD1/5/8 and by transcriptional activation assays using BRE-Luc reporter. In line with the biophysical studies, ACVR2B activated ALK2-R206H without ligand, while activation by ACVR2A was weaker and required ActA. We propose that the homodimerization of ACVR2B or ACVR2A dictates their ability to recruit ALK2-R206H into higher complexes, enabling the homomeric interactions of ALK2-R206H receptors and, subsequently, their activation.

NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting

Endothelial cells express neuropilin 1 (NRP1), endoglin (ENG) and vascular endothelial growth factor receptor 2 (VEGFR2), which regulate VEGF-A-mediated vascular development and angiogenesis. However, the link between complex formation among these receptors with VEGF-A-induced signaling and biology is yet unclear. Here, we quantify surface receptor interactions by IgG-mediated immobilization of one receptor, and fluorescence recovery after photobleaching (FRAP) measurements of the mobility of another coexpressed receptor. We observe stable ENG/NRP1, ENG/VEGFR2, and NRP1/VEGFR2 complexes, which are enhanced by VEGF-A. ENG augments NRP1/VEGFR2 interactions, suggesting formation of tripartite complexes bridged by ENG. Effects on signaling are measured in murine embryonic endothelial cells expressing (MEEC+/+) or lacking (MEEC-/-) ENG, along with NRP1 and/or ENG overexpression or knockdown. We find that optimal VEGF-A-mediated phosphorylation of VEGFR2 and Erk1/2 requires ENG and NRP1. ENG or NRP1 increase VEGF-A-induced sprouting, becoming optimal in cells expressing all three receptors, and both processes are inhibited by a MEK1/2 inhibitor. We propose a model where the maximal potency of VEGF-A involves a tripartite complex where ENG bridges VEGFR2 and NRP1, providing an attractive therapeutic target for modulation of VEGF-A signaling and biological responses.

Live-cell imaging of endogenous CSB-mScarletI as a sensitive marker for DNA-damage-induced transcription stress

Transcription by RNA polymerase II (RNA Pol II) is crucial for cellular function, but DNA damage severely impedes this process. Thus far, transcription-blocking DNA lesions (TBLs) and their repair have been difficult to quantify in living cells. To overcome this, we generated, using CRISPR-Cas9-mediated gene editing, mScarletI-tagged Cockayne syndrome group B protein (CSB) and UV-stimulated scaffold protein A (UVSSA) knockin cells. These cells allowed us to study the binding dynamics of CSB and UVSSA to lesion-stalled RNA Pol II using fluorescence recovery after photobleaching (FRAP). We show that especially CSB mobility is a sensitive transcription stress marker at physiologically relevant DNA damage levels. Transcription-coupled nucleotide excision repair (TC-NER)-mediated repair can be assessed by studying CSB immobilization over time. Additionally, flow cytometry reveals the regulation of CSB protein levels by CRL4CSA-mediated ubiquitylation and deubiquitylation by USP7. This approach allows the sensitive detection of TBLs and their repair and the study of TC-NER complex assembly and stability in living cells.

Dancing with the Stars: Using Image Analysis to Study the Choreography of the Endoplasmic Reticulum and Its Partners and of Movement Within Its Tubules.

In this chapter, approaches to the image analysis of the choreography of the plant endoplasmic reticulum (ER) labeled with fluorescent fusion proteins ("stars," if you wish) are presented. The approaches include the analyses of those parts of the ER that are attached through membrane contact sites to moving or non-moving partners (other "stars"). Image analysis is also used to understand the nature of the tubular polygonal network, the hallmark of this organelle, and how the polygons change over time due to tubule sliding or motion. Furthermore, the remodeling polygons of the ER interact with regions of fundamentally different topologies, the ER cisternae, and image analysis can be used to separate the tubules from the cisternae. ER cisternae, like polygons and tubules, can be motile or stationary. To study which parts are attached to non-moving partners, such as domains of the ER that form membrane contact sites with the plasma membrane/cell wall, an image analysis approach called persistency mapping has been used. To study the domains of the ER that move rapidly and stream through the cell, image analysis of optic flow has been used. However, optic flow approaches confuse the movement of the ER itself with the movement of proteins within the ER. As an overall measure of ER dynamics, optic flow approaches are of value, but their limitation as to what exactly is "flowing" needs to be specified. Finally, there are important imaging approaches that directly address the movement of fluorescent proteins within the ER lumen or in the membrane of the ER. Of these, fluorescence recovery after photobleaching (FRAP), inverse FRAP (iFRAP), and single particle tracking approaches are described.