Biology subjects

Ma, W.

Publications and source records attributed to Ma, W..

At least 217 records · Page 12Linked to original sources

bioRxiv2020

Myomegalin regulates Hedgehog pathway by controlling PDE4D at the centrosome

Mutations in the Hedgehog (Hh) signaling are implicated in birth defects and cancers, including medulloblastoma, one of the most malignant pediatric brain tumors. Current Hh inhibitors face the challenge of drug resistance and tumor relapse, urging new insights in the Hh pathway regulation. Our previous study revealed how PDE4D controls global levels of cAMP in the cytoplasm to positively regulate Hh signaling; in the present study we found that a specific isoform PDE4D3 is tethered to the centrosome by myomegalin, a centrosome/Golgi associated protein. Myomegalin loss dislocates PDE4D3 from the centrosome, leading to local PKA over-activation and inhibition of the Hh signaling, leaving other PKA-related pathways unaffected. Myomegalin loss suppresses the proliferation of granule neuron precursors, and blocks the growth of medulloblastoma in mouse model. Our findings specify a new regulatory mechanism of the Hh pathway, and highlight an exciting therapeutic avenue for Hh-related cancers with reduced side effects.

cell biology↗

bioRxiv2020

Genomic Architecture of Cells in Tissues (GeACT): Study of Human Mid-gestation Fetus

By circumventing cellular heterogeneity, single cell omics have now been widely utilized for cell typing in human tissues, culminating with the undertaking of human cell atlas aimed at characterizing all human cell types. However, more important are the probing of gene regulatory networks, underlying chromatin architecture and critical transcription factors for each cell type. Here we report the Genomic Architecture of Cells in Tissues (GeACT), a comprehensive genomic data base that collectively address the above needs with the goal of understanding the functional genome in action. GeACT was made possible by our novel single-cell RNA-seq (MALBAC-DT) and ATAC-seq (METATAC) methods of high detectability and precision. We exemplified GeACT by first studying representative organs in human mid-gestation fetus. In particular, correlated gene modules (CGMs) are observed and found to be cell-type-dependent. We linked gene expression profiles to the underlying chromatin states, and found the key transcription factors for representative CGMs. HighlightsO_LIGenomic Architecture of Cells in Tissues (GeACT) data for human mid-gestation fetus C_LIO_LIDetermining correlated gene modules (CGMs) in different cell types by MALBAC-DT C_LIO_LIMeasuring chromatin open regions in single cells with high detectability by METATAC C_LIO_LIIntegrating transcriptomics and chromatin accessibility to reveal key TFs for a CGM C_LI

genomics↗

bioRxiv2020

Critical role for cold shock protein YB-1 in cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and cancer progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cell lines resulted in cytokinesis failure, multinucleation and an increase in G1 transit time. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging of cells undergoing cytokinesis both in vitro and in zebrafish embryos, we found that YB-1 was critical for microtubule organization during cytokinesis. Using mass spectrometry we identified multiple novel phosphorylation sites on YB-1. We show that phosphorylation of YB-1 at multiple serine residues was essential for its function during cytokinesis. Using atomistic modelling we show how multiple phosphorylations alter YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining for the first time precisely how YB-1 regulates cell division. SUMMARYY-box-binding protein-1, YB-1, is essential for cell division, but it is not clear how it functions. Using live imaging and confocal microscopy we show that YB-1 functions only in the last step of division, specifically being required to initiate cytokinesis.

cell biology↗

bioRxiv2020

Precision engineering of the transcription factor Cre1 in Hypocrea jecorina (Trichoderma reesei) for efficient cellulase production in the presence of glucose

In Trichoderma reesei, carbon catabolite repression (CCR) significantly downregulates the transcription of cellulolytic enzymes, which is usually mediated by the zinc finger protein Cre1. It was found that there is a conserved region at the C-terminus of Cre1/CreA in several cellulase-producing fungi that contains up to three continuous S/T phosphorylation sites. Here, S387, S388, T389, and T390 at the C-terminus of Cre1 in T. reesei were mutated to valine for mimicking an unphosphorylated state, thereby generating the transformants Tr_Cre1S387V, Tr_Cre1S388V, Tr_Cre1T389V, and Tr_Cre1T390V, respectively. Transcription of cel7a in Tr_ Cre1S388V was markedly higher than that of the parent strain when grown in glucose-containing media. Under these conditions, both filter paperase (FPase) and p-nitrophenyl-{beta}-D-cellobioside (pNPCase) activities, as well as soluble proteins from Tr_Cre1S388V were significantly increased by up to 2- to 3-fold compared with that of other transformants and the parent strain. To our knowledge, this is the first report demonstrating an improvement of cellulase production in fungal species under CCR by mimicking dephosphorylation at the C-terminus of Cre1. Taken together, we developed a precision engineering strategy based on the modification of phosphorylation sites of Cre1 transcription factor to enhance the production of cellulase in fungal species under CCR.

bioengineering↗

bioRxiv2020

In vivo x-ray diffraction and simultaneous EMG reveal the time course of myofilament lattice dilation and filament stretch

Muscles function within an organism depends on the feedback between molecular to meter-scale processes. While the motions of muscles contractile machinery are well described in isolated preparations, only a handful of experiments have documented the kinematics of the lattice occurring when multi-scale interactions are fully intact. We used time-resolved x-ray diffraction to record the kinematics of the myofilament lattice within a normal operating context: the tethered flight of Manduca sexta. Since the primary flight muscles of Manduca sexta are synchronous, we used these results to reveal the timing of in vivo cross-bridge recruitment, which occurred 24 (s.d. 26) ms following activation. In addition, the thick filaments stretched an average of 0.75 (s.d. 0.32)% and thin filaments stretched 1.11 (s.d. 0.65)%. In contrast to other in vivo preparations, lattice spacing changed an average of 2.72 (s.d. 1.47)%. Lattice dilation of this magnitude significantly impacts shortening velocity and force generation, and filament stretching tunes force generation. While kinematics were consistent within individual trials, there was extensive variation between trials. Using a mechanism-free machine learning model we searched for patterns within and across trials. While lattice kinematics were predictable within trials, the model could not create predictions across trials. This indicates that the variability we see across trials may be explained by latent variables occurring in this naturally functioning system. The diverse kinematic combinations we documented mirror muscles adaptability and may facilitate its robust function in unpredictable conditions.

biophysics↗

bioRxiv2020

BARTweb: a web server for transcription factor association analysis

Identifying active transcription factors (TFs) that bind to cis-regulatory regions in the genome to regulate differential gene expression is a key task in gene regulation research. TF binding profiles from numerous existing ChIP-seq data can be utilized for association analysis with query data for TF identification, as alternative to DNA sequence motif analysis. Here, we present BARTweb, an interactive webserver for identifying TFs whose genomic binding patterns associate with input genomic features, by leveraging over 13,000 public ChIP-seq datasets for human and mouse. Using an updated Binding Analysis for Regulation of Transcription (BART) algorithm, BARTweb can identify functional TFs that regulate a gene set, or have a binding profile correlated with a ChIP-seq profile or enriched in a genomic region set, without a priori information of the cell type. Compared with the original BART package, BARTweb substantially reduces the execution time of a typical job by two orders of magnitude. We also show that BARTweb outperforms other existing tools in identifying true TFs from collected experimental data. BARTweb is a useful webserver for performing functional analysis of gene regulation. BARTweb is freely available at http://bartweb.org.

bioinformatics↗

bioRxiv2020

Unstructured mRNAs form multivalent RNA-RNA interactions to generate TIS granule networks

The TIS granule network is a constitutively expressed membraneless organelle that concentrates mRNAs with AU-rich elements and interacts with the major site of protein synthesis, the rough endoplasmic reticulum. Most known biomolecular condensates are sphere-like, but TIS granules have a mesh-like morphology. Through in vivo and in vitro reconstitution experiments we discovered that this shape is generated by extensive intermolecular RNA-RNA interactions. They are mostly accomplished by mRNAs with large unstructured regions in their 3'UTRs that we call intrinsically disordered regions (IDRs). As AU-rich RNA is a potent chaperone that suppresses protein aggregation and is overrepresented in mRNAs with IDRs, our data suggests that TIS granules concentrate mRNAs that assist protein folding. In addition, the proximity of translating mRNAs in TIS granule networks may enable co-translational protein complex formation.

molecular biology↗

bioRxiv2020

A synergistic action of sirtuins directly recognizes DNA breaks and potentiates DNA damage response and repair

The DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to {gamma}H2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1 deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair.

biochemistry↗

bioRxiv2020

Transmission dynamics of 2019 novel coronavirus (2019-nCoV)

RationaleSeveral studies have estimated basic production number of novel coronavirus pneumonia (NCP). However, the time-varying transmission dynamics of NCP during the outbreak remain unclear. ObjectivesWe aimed to estimate the basic and time-varying transmission dynamics of NCP across China, and compared them with SARS. MethodsData on NCP cases by February 7, 2020 were collected from epidemiological investigations or official websites. Data on severe acute respiratory syndrome (SARS) cases in Guangdong Province, Beijing and Hong Kong during 2002-2003 were also obtained. We estimated the doubling time, basic reproduction number (R0) and time-varying reproduction number (Rt) of NCP and SARS. Measurements and main resultsAs of February 7, 2020, 34,598 NCP cases were identified in China, and daily confirmed cases decreased after February 4. The doubling time of NCP nationwide was 2.4 days which was shorter than that of SARS in Guangdong (14.3 days), Hong Kong (5.7 days) and Beijing (12.4 days). The R0 of NCP cases nationwide and in Wuhan were 4.5 and 4.4 respectively, which were higher than R0 of SARS in Guangdong (R0=2.3), Hongkong (R0=2.3), and Beijing (R0=2.6). The Rt for NCP continuously decreased especially after January 16 nationwide and in Wuhan. The R0 for secondary NCP cases in Guangdong was 0.6, and the Rt values were less than 1 during the epidemic. ConclusionsNCP may have a higher transmissibility than SARS, and the efforts of containing the outbreak are effective. However, the efforts are needed to persist in for reducing time-varying reproduction number below one. At a Glance CommentaryO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSSince December 29, 2019, pneumonia infection with 2019-nCoV, now named as Novel Coronavirus Pneumonia (NCP), occurred in Wuhan, Hubei Province, China. The disease has rapidly spread from Wuhan to other areas. As a novel virus, the time-varying transmission dynamics of NCP remain unclear, and it is also important to compare it with SARS. What This Study Adds to the FieldWe compared the transmission dynamics of NCP with SARS, and found that NCP has a higher transmissibility than SARS. Time-varying production number indicates that rigorous control measures taken by governments are effective across China, and persistent efforts are needed to be taken for reducing instantaneous reproduction number below one.

systems biology↗

bioRxiv2020

Correlated Gene Modules Uncovered by Single-Cell Transcriptomics with High Detectability and Accuracy

Single cell transcriptome sequencing has become extremely useful for cell typing. However, such differential expression data has shed little light on regulatory relationships among genes. Here, by examining pairwise correlations between mRNA levels of any two genes under steady-state conditions, we uncovered correlated gene modules (CGMs), clusters of intercorrelated genes that carry out certain biological functions together. We report a novel single-cell RNA-seq method called MALBAC-DT with higher detectability and accuracy, allowing determination of the covariance matrix of the expressed mRNAs for a homogenous cell population. We observed a prevalence of positive correlations between pairs of genes, with higher correlations corresponding to higher likelihoods of protein-protein interactions. Some CGMs, such as the p53 module in a cancer cell line, are cell type specific, while others, such as the protein synthesis CGM, are shared by different cell types. CGMs distinguished direct targets of p53 and exposed different modes of regulation of these genes in different cell types. Our covariance analyses of steady-state fluctuations provides a powerful way to advance our functional understanding of gene-to-gene interactions.

genomics↗

bioRxiv2019

Cytological abnormalities and pollen abortion in interspecific hybrids of Nicotiana

Interspecific cross breeding introduces superior agronomic traits into cultivated species; however, problematic pollen sterility occurs in the hybrids. Our previous study obtained interspecific hybrids from the cross between a cytoplasmic male sterility line of Nicotiana tabacum and Nicotiana alata, and some of the hybrids were pollen sterile. Here, we conducted an in-depth cellular study to understand the cytological mechanism of pollen abortion in these hybrids (F1-D sterile) compared with pollen development in the fertile hybrids (F1-S sterile) from the same cross. The ultrastructure observation showed that the membrane of microspore in F1-D sterile hybrid was deficient at all represented developmental stages. Chromosome behavior during meiosis was studied by carbol fuchsin staining, which indicated that cytomixis, chromosome leakage and asymmetric callose wall deposition occurred with high frequency in the microsporocyte of F1-D sterile. The results of the ultrastructure and 6-diamidino-2-phenylindole (DAPI) analyses also showed that the cytoplasm and nucleus were unstable and extruded from F1-D sterile microspore during the developmental process, leading to mature pollen grains that were vacuous and collapsed in the aperture region. In addition, delayed tapetum degradation was also detected in the anther of F1-D sterile, and might be associated with irregular sporopollenin deposition in the aperture region of F1-D sterile pollen. Genetic unbalance and cytomembrane deficiency might both be responsible for the instability of the chromosome, nuclear and cytoplasm, and resulted in pollen abortion in F1-D sterile hybrids, and irregular tapetum degradation might also be related with pollen sterility.

developmental biology↗

bioRxiv2019

Na2CO3-Responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

Alkali-salinity exerts severe osmotic, ionic and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na2CO3-responsive proteins. A total of 104 and 102 Na2CO3-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na2CO3-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na2CO3 tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na2CO3 stress, and they have improved our understanding of the Na2CO3-responsive mechanisms in halophytes.

plant biology↗

bioRxiv2019

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

\"Bairihua\", a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development. Here, a SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in Bairihua and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in \"Bairihua\". Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time. Taken together, our results implied a novel pathway that ChuSPL9 regulated flowering development, but not flowering transition, with the participation of ChuHMGA. Further investments need to be done to verify the details of this pathway.

plant biology↗

bioRxiv2019

Lattice arrangement of myosin filaments correlates with fiber type in rat skeletal muscle

The thick (myosin-containing) filaments of vertebrate skeletal muscle are arranged in a hexagonal lattice, interleaved with an array of thin (actin-containing) filaments with which they interact to produce contraction. X-ray diffraction and electron microscopy have shown that there are two types of thick filament lattice. In the simple lattice, all filaments have the same orientation about their long axis, while in the super lattice, nearest neighbors have rotations differing by 60{degrees}. Tetrapods (amphibians, reptiles, birds, mammals) typically have only a super lattice, while the simple lattice is confined to fish. We have carried out X-ray diffraction and electron microscopy of the soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat and found that while the EDL has a super-lattice, as expected, the SOL has a simple lattice. The EDL and SOL of the rat are unusual in being essentially pure fast and slow muscles respectively. The mixed fiber content of most tetrapod muscles and/or lattice disorder may explain why the simple lattice has not been apparent in these vertebrates before. This is supported by only weak simple lattice diffraction in the X-ray pattern of mouse SOL, which has a greater mix of fiber types than rat. We conclude that the simple lattice might be common in tetrapods. The correlation between fiber type and filament lattice arrangement suggests that the lattice arrangement may contribute to the functional properties of a muscle.\n\nSummaryThe three-dimensional arrangement of thick filaments in skeletal muscle is studied by X-ray diffraction and electron microscopy. A correlation is found between thick filament lattice type (simple or super lattice) and fiber type (fast/slow). This suggests that lattice organization contributes to muscle functional properties

biophysics↗

bioRxiv2019

The Toxin Antitoxin MazEF Drives Staphylococcus aureus Chronic Infection

Staphylococcus aureus is the major organism responsible for surgical implant infections. Antimicrobial treatment of these infections often fails leading to expensive surgical intervention and increased risk of mortality to the patient. The challenge in treating these infections is associated with the high tolerance of S. aureus biofilm to antibiotics. MazEF, a toxin-antitoxin system, is thought to be an important regulator of this phenotype, but its physiological function in S. aureus is controversial. Here, we examined the role of MazEF in developing chronic infections by comparing growth and antibiotic tolerance phenotypes in three S. aureus strains to their corresponding strains with disruption of mazF expression. Strains lacking mazF production showed increased biofilm growth, and decreased biofilm antibiotic tolerance. Deletion of icaADBC in the mazF::tn background suppressed the growth phenotype observed with mazF-disrupted strains, suggesting the phenotype was ica-dependent. We confirmed these phenotypes in our murine animal model. Loss of mazF resulted in increased bacterial burden and decreased survival rate compared to its wild-type strain demonstrating that loss of the mazF gene caused an increase in S. aureus virulence. Although lack of mazF gene expression increased S. aureus virulence, it was more susceptible to antibiotics in vivo. Combined, the ability of mazF to inhibit biofilm formation and promote biofilm antibiotic tolerance plays a critical role in transitioning from an acute to chronic infection that is difficult to eradicate with antibiotics alone.\n\nImportanceSurgical infections are one of the most common types of infections obtained in a hospital. Staphylococcus aureus is the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate as the bacteria form a biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria floating in liquid, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate that mazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allows S. aureus to transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host.

microbiology↗

bioRxiv2019

Connecting gene regulatory relationships to neurobiological mechanisms of brain disorders

Despite being clinically distinguishable, many neuropsychiatric disorders display a remarked level of genetic correlation and overlapping symptoms. Deciphering neurobiological mechanisms underlying potential shared genetic etiology is challenging because (1) most common risk variants reside in the non-coding region of the genome, and (2) a genome-wide framework is required to compare genome-wide association studies (GWAS) having different power. To address these challenges, we developed a platform, Hi-C coupled MAGMA (H-MAGMA), that converts SNP-level summary statistics into gene-level association statistics by assigning non-coding SNPs to their cognate genes based on chromatin interactions. We applied H-MAGMA to five psychiatric disorders and four neurodegenerative disorders to interrogate biological pathways, developmental windows, and cell types implicated for each disorder. We found that neuropsychiatric disorder-associated genes coalesce at the level of developmental windows (mid-gestation) and cell-type specificity (excitatory neurons). On the contrary, neurodegenerative disorder-associated genes show more diverse cell type specific, and increasing expression over time, consistent with the age-associated elevated risk of developing neurodegenerative disorders. Genes associated with Alzheimers disease were not only highly expressed in microglia, but also subject to microglia and oligodendrocyte-specific dysregulation, highlighting the importance of understanding the cellular context in which risk variants exert their effects. We also obtained a set of pleiotropic genes that are shared across multiple psychiatric disorders and may form the basis for common neurobiological susceptibility. Pleiotropic genes are associated with neural activity and gene regulation, with selective expression in corticothalamic projection neurons. These results show how H-MAGMA adds to existing frameworks to help identify the neurobiological basis of shared and distinct genetic architecture of brain disorders.

neuroscience↗

bioRxiv2019

A nanometer difference in myofilament lattice spacing of two cockroach leg muscles explains their different functions

Muscle is highly organized across scales. Consequently, small changes in arrangement of myofilaments can influence macroscopic function. Two leg muscles of a cockroach, have identical innervation, mass, twitch responses, length-tension curves, and force-velocity relationships. However, during running, one muscle is dissipative, while the other produces significant positive mechanical work. Using time resolved x-ray diffraction in intact, contracting muscle, we simultaneously measured the myofilament lattice spacing, packing structure, and macroscopic force production of these muscle to test if nanoscale differences could account for this conundrum. While the packing patterns are the same, one muscle has 1 nm smaller lattice spacing at rest. Under isometric activation, the difference in lattice spacing disappeared explaining the two muscles identical steady state behavior. During periodic contractions, one muscle undergoes a 1 nm greater change in lattice spacing, which correlates with force. This is the first identified feature that can account for the muscles different functions.

biophysics↗

bioRxiv2019

Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation

Dvl (Dishevelled) is one of several essential non-enzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits other components of the destruction complex leading to inactivation of {beta}-catenin degradation. Although this model is widespread, direct evidence for this process is lacking. In this study, we tagged mEGFP to C-terminus of dishevlled2 gene using CRISPR/Cas9 induced homologous recombination and observed its dynamics directly at the single molecule level with Total Internal Reflection Fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and the dynamic features of membrane-associated Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane bound Dvl2 is predominantly monomer in the absent of Wnt (mean size 1.10). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.08/m2 to 0.34/m2. Wnt3a also leads to increased oligomerization which raises the weighted averaged mean size of Dvl2 complexes to 1.4; with 65% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of Dvl2 at the membrane caused by increased affinity of Dvl2 for Fzd, the Dvl2 and Fzd binding is independent of LRP5/6. The oligomerized Dvl2 complexes have greatly increased dwell time, 2~3 minutes compared to less than 1 second for monomeric Dvl2. These properties make Dvl a unique scaffold dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.\n\nSignificance StatementCanonical Wnt signaling is one of the most widely distributed pathways in metazoan development. Despite intense genetic and biochemical study for over 35 years, the major features of signaling across the plasma membrane are still poorly understood. Dishevelled serves as an essential bridge between the membrane receptors and downstream signaling components. Attempts to reconstruct the pathway and analyze its biochemical features in vitro have been hampered by Dishevelleds tendency to aggregate in vitro and to form large aggregates of dubious significance in vivo. To obtain a molecular understanding of the role of Dvl in Wnt signaling, while circumventing these aggregation problems we have expressed a fluorescent tagged Dishevelled in cells at their physiological concentration and quantified the size distribution of Dishevelled before and after Wnt treatment. We found that limited oligomerization in response to the Wnt ligand is very dynamic and provides a key step of signal transduction.

systems biology↗