ABSTRACT
Morphogens are intercellular signaling molecules providing spatial information to cells in developing tissues to coordinate cell fate decisions. The spatial information is encoded within long-ranged concentration gradients of the morphogen. Direct measurement of morphogen dynamics in a range of systems suggests that local and global diffusion coefficients can differ by orders of magnitude. Further, local diffusivity can be large, which would potentially abolish any concentration gradient rapidly. Such observations have led to alternative transport models being proposed, including transcytosis and cytonemes. Here, we show that accounting for tissue architecture combined with receptor binding is sufficient to hinder the diffusive dynamics of morphogens, leading to an order of magnitude decrease in the effective diffusion coefficient from local to global scales. In particular, we built a realistic in silico architecture of the extracellular spaces of the zebrafish brain using light and electron microscopy data. Simulations on realistic architectures demonstrate that tortuosity and receptor binding within these spaces are sufficient to reproduce experimentally measured morphogen dynamics. Importantly, this work demonstrates that hindered diffusion is a viable mechanism for gradient formation, without requiring additional regulatory control.
ABSTRACT
Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.
Subject(s)
Drosophila Proteins , Homeodomain Proteins , Animals , Body Patterning/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Embryo, Nonmammalian/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
Imaging fluorescence correlation spectroscopy (FCS) is a powerful tool to extract information on molecular mobilities, actions, and interactions in live cells, tissues, and organisms. Nevertheless, several limitations restrict its applicability. First, FCS is data hungry, requiring 50,000 frames at 1-ms time resolution to obtain accurate parameter estimates. Second, the data size makes evaluation slow. Third, as FCS evaluation is model dependent, data evaluation is significantly slowed unless analytic models are available. Here, we introduce two convolutional neural networks-FCSNet and ImFCSNet-for correlation and intensity trace analysis, respectively. FCSNet robustly predicts parameters in 2D and 3D live samples. ImFCSNet reduces the amount of data required for accurate parameter retrieval by at least one order of magnitude and makes correct estimates even in moderately defocused samples. Both convolutional neural networks are trained on simulated data, are model agnostic, and allow autonomous, real-time evaluation of imaging FCS measurements.
Subject(s)
Deep Learning , Spectrometry, Fluorescence/methodsABSTRACT
Fluorescence correlation spectroscopy (FCS) is a single molecule sensitive tool for the quantitative measurement of biomolecular dynamics and interactions. Improvements in biology, computation, and detection technology enable real-time FCS experiments with multiplexed detection even in vivo. These new imaging modalities of FCS generate data at the rate of hundreds of MB/s requiring efficient data processing tools to extract information. Here, we briefly review FCS's capabilities and limitations before discussing recent directions that address these limitations with a focus on imaging modalities of FCS, their combinations with super-resolution microscopy, new evaluation strategies, especially machine learning, and applications in vivo.
Subject(s)
Microscopy, Fluorescence , Microscopy, Fluorescence/methods , Spectrometry, Fluorescence/methodsABSTRACT
Number and brightness (N&B) analysis is a fluorescence spectroscopy technique to quantify oligomerization of the mobile fraction of proteins. Accurate results, however, rely on a good knowledge of nonfluorescent states of the fluorescent labels, especially of fluorescent proteins, which are widely used in biology. Fluorescent proteins have been characterized for confocal, but not camera-based, N&B, which allows, in principle, faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment are still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptor's oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization is not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, lead to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization dependent, involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation are the outcomes of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains.
Subject(s)
ErbB ReceptorsABSTRACT
Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid-based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry. Additionally, we explored the effect of this interaction on infectivity and movement of the virus during infection was explored using plaque assay and fluorescence-based imaging and single particle tracking. GM1a was deemed to interact with DENV at domain I (DI) and domain II (DII) of the E protein of the protein coat at quaternary contacts of a fully assembled virus, leading to a 10-fold and 7-fold increase in infectivity for DENV1 and DENV2 in mammalian cell systems, respectively. We determined that the interaction of the virus with GM1a triggers a speeding up of virus movement on live cell surfaces, possibly resulting from a reduction in rigidity of cellular rafts during infection. Collectively, our results suggest that GM1a functions as a coreceptor/attachment factor for DENV during infection in mammalian systems.
Subject(s)
Dengue Virus , Dengue , Flavivirus , Animals , Humans , Dengue Virus/metabolism , Viral Envelope Proteins/metabolism , Gangliosides/metabolism , Flavivirus/metabolism , Mammals/metabolismABSTRACT
Modern electron-multiplying charge-coupled device (EMCCD) and scientific complementary metal-oxide semiconductor (sCMOS) cameras read out fluorescence data with single-molecule sensitivity at thousands of frames per second. Exploiting these capabilities in full requires data evaluation in real time. The direct camera-read-out tool presented here allows access to the data while the camera is recording. This provides simplified and accurate alignment procedures for total internal reflection fluorescence microscopy (TIRFM) and single-plane illumination microscopy (SPIM), and simplifies and accelerates fluorescence experiments. The tool handles a range of widely used EMCCD and sCMOS cameras and uses imaging fluorescence correlation spectroscopy for its evaluation. It is easily extendable to other camera models and other techniques and is a base for automated TIRFM and SPIM data acquisition.
Subject(s)
Electrons , Semiconductors , Equipment Design , Microscopy, Fluorescence/methods , Spectrometry, FluorescenceABSTRACT
The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer-induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10-15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.
Subject(s)
Cholesterol , Membrane Microdomains , Cell Membrane/chemistry , Cholesterol/metabolism , Heptanol/analysis , Heptanol/metabolism , Lipid Bilayers/metabolism , Membrane Microdomains/metabolism , Membrane Proteins/metabolismABSTRACT
Membrane contact between intracellular organelles is important in mediating organelle communication. However, the assembly of molecular machinery at membrane contact site and its internal organization correlating with its functional activity remain unclear. Here, we demonstrate that a gel-like condensation of Cidec, a crucial protein for obesity development by facilitating lipid droplet (LD) fusion, occurs at the LD-LD contact site (LDCS) through phase separation. The homomeric interaction between the multivalent N terminus of Cidec is sufficient to promote its phase separation both in vivo and in vitro. Interestingly, Cidec condensation at LDCSs generates highly plastic and lipid-permeable fusion plates that are geometrically constrained by donor LDs. In addition, Cidec condensates are distributed unevenly in the fusion plate generating stochastic sub-compartments that may represent unique lipid passageways during LD fusion. We have thus uncovered the organization and functional significance of geometry-constrained Cidec phase separation in mediating LD fusion and lipid homeostasis.
Subject(s)
Lipid Droplets/metabolism , Lipid Metabolism/physiology , Lipids , Obesity/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Cell Differentiation/physiology , Homeostasis/physiology , Humans , MiceABSTRACT
Spinal Muscular Atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons that is caused by a deficiency in ubiquitously expressed Survival Motor Neuron (SMN) protein. Two mutually exclusive hypotheses have been discussed to explain increased motor neuron vulnerability in SMA. Reduced SMN levels have been proposed to lead to defective snRNP assembly and aberrant splicing of transcripts that are essential for motor neuron maintenance. An alternative hypothesis proposes a motor neuron-specific function for SMN in axonal transport of mRNAs and/or RNPs. To address these possibilities, we used a novel in vivo approach with fluorescence correlation spectroscopy (FCS) in transgenic zebrafish embryos to assess the subcellular dynamics of Smn in motor neuron cell bodies and axons. Using fluorescently tagged Smn we show that it exists as two freely diffusing components, a monomeric, and a complex-bound, likely oligomeric, component. This oligomer hypothesis was supported by the disappearance of the complex-bound form for a truncated Smn variant that is deficient in oligomerization and a change in its dynamics under endogenous Smn deficient conditions. Surprisingly, our FCS measurements did not provide any evidence for an active transport of Smn in axons. Instead, our in vivo observations are consistent with previous findings that SMN acts as a chaperone for the assembly of snRNP and mRNP complexes.
ABSTRACT
Wnt proteins are a family of hydrophobic cysteine-rich secreted glycoproteins that regulate a gamut of physiological processes involved in embryonic development and tissue homeostasis. Wnt ligands are post-translationally lipidated in the endoplasmic reticulum (ER), a step essential for its membrane targeting, association with lipid domains, secretion and interaction with receptors. However, at which residue(s) Wnts are lipidated remains an open question. Initially it was proposed that Wnts are lipid-modified at their conserved cysteine and serine residues (C77 and S209 in mWnt3a), and mutations in either residue impedes its secretion and activity. Conversely, some studies suggested that serine is the only lipidated residue in Wnts, and substitution of serine with alanine leads to retention of Wnts in the ER. In this work, we investigate whether in zebrafish neural tissues Wnt3 is lipidated at one or both conserved residues. To this end, we substitute the homologous cysteine and serine residues of zebrafish Wnt3 with alanine (C80A and S212A) and investigate their influence on Wnt3 membrane organization, secretion, interaction and signaling activity. Collectively, our results indicate that Wnt3 is lipid modified at its C80 and S212 residues. Further, we find that lipid addition at either C80 or S212 is sufficient for its secretion and membrane organization, while the lipid modification at S212 is indispensable for receptor interaction and signaling.
ABSTRACT
By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5'-3' panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5'-3' panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.
Subject(s)
Capsid Proteins/metabolism , Dengue Virus/metabolism , Molecular Chaperones/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombination, Genetic/genetics , Virus Replication/genetics , Capsid/metabolism , Capsid Proteins/genetics , Codon, Initiator , Cyclization/genetics , Dengue Virus/genetics , Kinetics , Molecular Chaperones/genetics , Nucleic Acid Conformation , Spectrometry, Fluorescence , Spectroscopy, Fourier Transform InfraredABSTRACT
Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.
Subject(s)
Microscopy, Fluorescence/methods , Single Molecule Imaging/methods , Actins/metabolism , Animals , CHO Cells , Cell Membrane , Cricetulus , Diffusion , ErbB Receptors/metabolism , Fluorescence , Humans , Spectrometry, Fluorescence/methodsABSTRACT
Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrPSc), a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. Attachment of PrPC to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrPC into PrPSc. However, the mechanisms governing PrPC conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrPC into PrPSc and offer new therapeutic angles.
Subject(s)
Cytoskeleton/physiology , Membrane Proteins/metabolism , Prions/metabolism , Actins/metabolism , Cell Line , Cell Membrane/metabolism , Cluster Analysis , Cytoskeleton/metabolism , Glycosylphosphatidylinositols/chemistry , Glycosylphosphatidylinositols/metabolism , Humans , Neurons/metabolism , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prion Proteins/metabolism , Protein Isoforms/metabolism , Scrapie/metabolismABSTRACT
The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.
Subject(s)
5' Untranslated Regions/genetics , Capsid Proteins/physiology , Dengue Virus/metabolism , Base Pairing/genetics , Base Sequence , Capsid Proteins/metabolism , Conserved Sequence , Dengue Virus/genetics , Dengue Virus/physiology , Genome, Viral/physiology , Nucleic Acid Conformation , Protein Biosynthesis/genetics , RNA, Circular/chemistry , RNA, Circular/genetics , RNA, Viral/chemistry , RNA, Viral/genetics , Virus Replication/geneticsABSTRACT
Neurexins are presynaptic transmembrane proteins that control synapse activity and are risk factors for autism spectrum disorder. Zebrafish, a popular model for behavioral studies, has six neurexin genes, but their functions in embryogenesis and behavior remain largely unknown. We have previously reported that nrxn2a is aberrantly spliced and specifically dysregulated in motor neurons (MNs) in models of spinal muscular atrophy. In this study, we generated nrxn2aa-/- mutants by CRISPR/Cas9 to understand nrxn2aa function at the zebrafish neuromuscular junction (NMJ) and to determine the effects of its deficiency on adult behavior. Homozygous mutant embryos derived from heterozygous parents did not show obvious defects in axon outgrowth or synaptogenesis of MNs. In contrast, maternal-zygotic (MZ) nrxn2aa-/- mutants displayed extensively branched axons and defective MNs, suggesting a cell-autonomous role for maternally provided nrxn2aa in MN development. Analysis of the NMJs revealed enlarged choice points in MNs of mutant larvae and reduced co-localization of pre- and post-synaptic terminals, indicating impaired synapse formation. Severe early NMJ defects partially recovered in late embryos when mutant transcripts became strongly upregulated. Ultimately, however, the induced defects resulted in muscular atrophy symptoms in adult MZ mutants. Zygotic homozygous mutants developed normally but displayed increased anxiety at adult stages. Together, our data demonstrate an essential role for maternal nrxn2aa in NMJ synapse establishment, while zygotic nrxn2aa expression appears dispensable for synapse maintenance. The viable nrxn2aa-/- mutant furthermore serves as a novel model to study how an increase in anxiety-like behaviors impacts other deficits.
Subject(s)
Anxiety/pathology , Axon Guidance , Gene Expression Regulation, Developmental , Motor Neurons/pathology , Nerve Tissue Proteins/deficiency , Neurogenesis , Zebrafish Proteins/deficiency , Animals , Anxiety/etiology , Anxiety/metabolism , CRISPR-Cas Systems , Motor Neurons/metabolism , Nerve Tissue Proteins/genetics , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
Wnt3 proteins are lipidated and glycosylated signaling molecules that play an important role in zebrafish neural patterning and brain development. However, the transport mechanism of lipid-modified Wnts through the hydrophilic extracellular environment for long-range action remains unresolved. Here we determine how Wnt3 accomplishes long-range distribution in the zebrafish brain. First, we characterize the Wnt3-producing source and Wnt3-receiving target regions. Subsequently, we analyze Wnt3 mobility at different length scales by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. We demonstrate that Wnt3 spreads extracellularly and interacts with heparan sulfate proteoglycans (HSPG). We then determine the binding affinity of Wnt3 to its receptor, Frizzled1 (Fzd1), using fluorescence cross-correlation spectroscopy and show that the co-receptor, low-density lipoprotein receptor-related protein 5 (Lrp5), is required for Wnt3-Fzd1 interaction. Our results are consistent with the extracellular distribution of Wnt3 by a diffusive mechanism that is modified by tissue morphology, interactions with HSPG, and Lrp5-mediated receptor binding, to regulate zebrafish brain development.
Subject(s)
Brain/metabolism , Gene Expression Regulation, Developmental/physiology , Wnt3 Protein/metabolism , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Brain/embryology , Embryo, Nonmammalian , Fluorescence Recovery After Photobleaching , Frizzled Receptors/genetics , Frizzled Receptors/metabolism , Heparan Sulfate Proteoglycans/metabolism , Low Density Lipoprotein Receptor-Related Protein-5/genetics , Low Density Lipoprotein Receptor-Related Protein-5/metabolism , Microscopy, Confocal , Protein Binding , Wnt3 Protein/genetics , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
G-Protein-coupled receptors (GPCRs) are ubiquitous within eukaryotes, responsible for a wide array of physiological and pathological processes. Indeed, the fact that they are the most drugged target in the human genome is indicative of their importance. Despite the clear interest in GPCRs, most information regarding their activity has been so far obtained by analyzing the response from a "bulk medium". As such, this Perspective summarizes some of the common methods for this indirect observation. Nonetheless, by inspecting approaches applying super-resolution imaging, we argue that imaging is perfectly situated to obtain more detailed structural and spatial information, assisting in the development of new GPCR-targeted drugs and clinical strategies. The benefits of direct optical visualization of GPCRs are analyzed in the context of potential future directions in the field.