Role of a Pdlim5:PalmD complex in directing dendrite morphology.

Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further, in multiple neurodevelopmental and aging disorders, disruptions of dendrite formation or shaping is associated with atypical neuronal connectivity. We showed previously that Pdlim5 binds delta-catenin and promotes dendrite branching. We report here that Pdlim5 interacts with PalmD, a protein previously suggested by others to interact with the cytoskeleton (e.g., via adducin/spectrin) and to regulate membrane shaping. Functionally, the knockdown of PalmD or Pdlim5 in rat primary hippocampal neurons dramatically reduces branching and conversely, PalmD exogenous expression promotes dendrite branching as does Pdlim5. Further, we show that each proteins' effects are dependent on the presence of the other. In summary, using primary rat hippocampal neurons we reveal the contributions of a novel Pdlim5:PalmD protein complex, composed of functionally inter-dependent components responsible for shaping neuronal dendrites.

Sprr1 and miR-130b contribute to the senescence-like phenotype in aging.

Aging is an inevitable process with senescence being one of its hallmarks. Recent advances have indicated that the elimination of senescent cells can reduce the signs of aging and increase healthy life span. Here, we identify a negative modulator of aging, Sprr1a, and in turn a negative modulator of Sprr1a, miR-130b. We show that reductions in Sprr1a levels, including via miR-130b expression, promotes cell senescence-like phenotype. We find that mediators of senescence, such as inflammatory cytokines and cell cycle regulators, are modulated by the miR-130b and Sprr1a-related pathway. For example, the levels of p16, p53 and p21 become decreased or increased upon the respective expression of Sprr1a versus miR-130b. Further, as shown in relation to p16 levels and ß-galactosidase levels, cells expressing Sprr1a exhibit significant protection from senescence-inducing factors such as radiation or Doxorubicin, suggesting that Sprr1a might contribute to protection against age-related pathologies. Taken together, we introduce two modulators of properties associated with senescence-like phenotype.

Role of a Pdlim5:PalmD complex in directing dendrite morphology.

Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further, in multiple neurodevelopmental and aging disorders, disruptions of dendrite formation or shaping is associated with atypical neuronal connectivity. We showed previously that Pdlim5 binds delta-catenin and promotes dendrite branching (Baumert et al., J Cell Biol 2020). We report here that Pdlim5 interacts with PalmD, a protein previously suggested by others to interact with the cytoskeleton (e.g., via adducin/ spectrin) and to regulate membrane shaping. Functionally, the knockdown of PalmD or Pdlim5 in rat primary hippocampal neurons dramatically reduces branching and conversely, PalmD exogenous expression promotes dendrite branching as does Pdlim5. Further, we show that effects of each protein are dependent on the presence of the other. In summary, using primary rat hippocampal neurons we reveal the contributions of a novel Pdlim5:PalmD protein complex, composed of functionally inter-dependent components responsible for shaping neuronal dendrites.

p120-catenin subfamily members have distinct as well as shared effects on dendrite morphology during neuron development in vitro.

Dendritic arborization is essential for proper neuronal connectivity and function. Conversely, abnormal dendrite morphology is associated with several neurological pathologies like Alzheimer's disease and schizophrenia. Among major intrinsic mechanisms that determine the extent of the dendritic arbor is cytoskeletal remodeling. Here, we characterize and compare the impact of the four proteins involved in cytoskeletal remodeling-vertebrate members of the p120-catenin subfamily-on neuronal dendrite morphology. In relation to each of their own distributions, we find that p120-catenin and delta-catenin are expressed at relatively higher proportions in growth cones compared to ARVCF-catenin and p0071-catenin; ARVCF-catenin is expressed at relatively high proportions in the nucleus; and all catenins are expressed in dendritic processes and the soma. Through altering the expression of each p120-subfamily catenin in neurons, we find that exogenous expression of either p120-catenin or delta-catenin correlates with increased dendritic length and branching, whereas their respective depletion decreases dendritic length and branching. While increasing ARVCF-catenin expression also increases dendritic length and branching, decreasing expression has no grossly observable morphological effect. Finally, increasing p0071-catenin expression increases dendritic branching, but not length, while decreasing expression decreases dendritic length and branching. These distinct localization patterns and morphological effects during neuron development suggest that these catenins have both shared and distinct roles in the context of dendrite morphogenesis.

In-silico probing of AML related RUNX1 cancer-associated missense mutations: Predicted relationships to DNA binding and drug interactions.

The molecular consequences of cancer associated mutations in Acute myeloid leukemia (AML) linked factors are not very well understood. Here, we interrogated the COSMIC database for missense mutations associated with the RUNX1 protein, that is frequently mis-regulated in AML, where we sought to identify recurrently mutated positions at the DNA-interacting interface. Indeed, six of the mutated residues, out of a total 417 residues examined within the DNA binding domain, evidenced reduced DNA association in in silico predictions. Further, given the prominence of RUNX1's compromised function in AML, we asked the question if the mutations themselves might alter RUNX1's interaction (off-target) with known FDA-approved drug molecules, including three currently used in treating AML. We identified several AML-associated mutations in RUNX1 that were calculated to enhance RUNX1's interaction with specific drugs. Specifically, we retrieved data from the COSMIC database for cancer-associated mutations of RUNX1 by using R package "data.table" and "ggplot2" modules. In the presence of DNA and/or drug, we used docking scores and energetics of the complexes as tools to evaluate predicted interaction strengths with RUNX1. For example, we performed predictions of drug binding pockets involving Enasidenib, Giltertinib, and Midostaurin (AML associated), as well as ten different published cancer associated drug compounds. Docking of wild type RUNX1 with these 13 different cancer-associated drugs indicates that wild-type RUNX1 has a lower efficiency of binding while RUNX1 mutants R142K, D171N, R174Q, P176H, and R177Q suggested higher affinity of drug association. Literature evidence support our prediction and suggests the mutation R174Q affects RUNX1 DNA binding and could lead to compromised function. We conclude that specific RUNX1 mutations that lessen DNA binding facilitate the binding of a number of tested drug molecules. Further, we propose that molecular modeling and docking studies for RUNX1 in the presence of DNA and/or drugs enables evaluation of the potential impact of RUNX1 cancer associated mutations in AML.

Delta-Catenin as a Modulator of Rho GTPases in Neurons.

Small Rho GTPases are molecular switches that are involved in multiple processes including regulation of the actin cytoskeleton. These GTPases are activated (turned on) and inactivated (turned off) through various upstream effector molecules to carry out many cellular functions. One such upstream modulator of small Rho GTPase activity is delta-catenin, which is a protein in the p120-catenin subfamily that is enriched in the central nervous system. Delta-catenin affects small GTPase activity to assist in the developmental formation of dendrites and dendritic spines and to maintain them once they mature. As the dendritic arbor and spine density are crucial for synapse formation and plasticity, delta-catenin's ability to modulate small Rho GTPases is necessary for proper learning and memory. Accordingly, the misregulation of delta-catenin and small Rho GTPases has been implicated in several neurological and non-neurological pathologies. While links between delta-catenin and small Rho GTPases have yet to be studied in many contexts, known associations include some cancers, Alzheimer's disease (AD), Cri-du-chat syndrome, and autism spectrum disorder (ASD). Drawing from established studies and recent discoveries, this review explores how delta-catenin modulates small Rho GTPase activity. Future studies will likely elucidate how PDZ proteins that bind delta-catenin further influence small Rho GTPases, how delta-catenin may affect small GTPase activity at adherens junctions when bound to N-cadherin, mechanisms behind delta-catenin's ability to modulate Rac1 and Cdc42, and delta-catenin's ability to modulate small Rho GTPases in the context of diseases, such as cancer and AD.

A catenin of the plakophilin-subfamily, Pkp3, responds to canonical-Wnt pathway components and signals.

Vertebrate beta-catenin plays a key role as a transducer of canonical-Wnt signals. We earlier reported that, similar to beta-catenin, the cytoplasmic signaling pool of p120-catenin-isoform1 is stabilized in response to canonical-Wnt signals. To obtain a yet broader view of the Wnt-pathway's impact upon catenin proteins, we focused upon plakophilin3 (plakophilin-3; Pkp3) as a representative of the plakophilin-catenin subfamily. Promoting tissue integrity, the plakophilins assist in linking desmosomal cadherins to intermediate filaments at desmosome junctions, and in common with other catenins they perform additional functions including in the nucleus. In this report, we test whether canonical-Wnt pathway components modulate Pkp3 protein levels. We find that in common with beta-catenin and p120-catenin-isoform1, Pkp3 is stabilized in the presence of a Wnt-ligand or a dominant-active form of the LRP6 receptor. Pkp3's levels are conversely lowered upon expressing destruction-complex components such as GSK3ß and Axin, and in further likeness to beta-catenin and p120-isoform1, Pkp3 associates with GSK3beta and Axin. Finally, we note that Pkp3-catenin trans-localizes into the nucleus in response to Wnt-ligand and its exogenous expression stimulates an accepted Wnt reporter. These findings fit an expanded model where context-dependent Wnt-signals or pathway components modulate Pkp3-catenin levels. Future studies will be needed to assess potential gene regulatory, cell adhesive, or cytoskeletal effects.

Novel phospho-switch function of delta-catenin in dendrite development.

In neurons, dendrites form the major sites of information receipt and integration. It is thus vital that, during development, the dendritic arbor is adequately formed to enable proper neural circuit formation and function. While several known processes shape the arbor, little is known of those that govern dendrite branching versus extension. Here, we report a new mechanism instructing dendrites to branch versus extend. In it, glutamate signaling activates mGluR5 receptors to promote Ckd5-mediated phosphorylation of the C-terminal PDZ-binding motif of delta-catenin. The phosphorylation state of this motif determines delta-catenin's ability to bind either Pdlim5 or Magi1. Whereas the delta:Pdlim5 complex enhances dendrite branching at the expense of elongation, the delta:Magi1 complex instead promotes lengthening. Our data suggest that these complexes affect dendrite development by differentially regulating the small-GTPase RhoA and actin-associated protein Cortactin. We thus reveal a "phospho-switch" within delta-catenin, subject to a glutamate-mediated signaling pathway, that assists in balancing the branching versus extension of dendrites during neural development.

Divergent roles of the Wnt/PCP Formin Daam1 in renal ciliogenesis.

Kidneys are composed of numerous ciliated epithelial tubules called nephrons. Each nephron functions to reabsorb nutrients and concentrate waste products into urine. Defects in primary cilia are associated with abnormal formation of nephrons and cyst formation in a wide range of kidney disorders. Previous work in Xenopus laevis and zebrafish embryos established that loss of components that make up the Wnt/PCP pathway, Daam1 and ArhGEF19 (wGEF) perturb kidney tubulogenesis. Dishevelled, which activates both the canonical and non-canonical Wnt/PCP pathway, affect cilia formation in multiciliated cells. In this study, we investigated the role of the noncanoncial Wnt/PCP components Daam1 and ArhGEF19 (wGEF) in renal ciliogenesis utilizing polarized mammalian kidney epithelia cells (MDCKII and IMCD3) and Xenopus laevis embryonic kidney. We demonstrate that knockdown of Daam1 and ArhGEF19 in MDCKII and IMCD3 cells leads to loss of cilia, and Daam1's effect on ciliogenesis is mediated by the formin-activity of Daam1. Moreover, Daam1 co-localizes with the ciliary transport protein Ift88 and is present in cilia. Interestingly, knocking down Daam1 in Xenopus kidney does not lead to loss of cilia. These data suggests a new role for Daam1 in the formation of primary cilia.

TMEM9 promotes intestinal tumorigenesis through vacuolar-ATPase-activated Wnt/ß-catenin signalling.

Vesicular acidification and trafficking are associated with various cellular processes. However, their pathologic relevance to cancer remains elusive. We identified transmembrane protein 9 (TMEM9) as a vesicular acidification regulator. TMEM9 is highly upregulated in colorectal cancer. Proteomic and biochemical analyses show that TMEM9 binds to and facilitates assembly of vacuolar-ATPase (v-ATPase), a vacuolar proton pump, resulting in enhanced vesicular acidification and trafficking. TMEM9-v-ATPase hyperactivates Wnt/ß-catenin signalling via lysosomal degradation of adenomatous polyposis coli (APC). Moreover, TMEM9 transactivated by ß-catenin functions as a positive feedback regulator of Wnt signalling in colorectal cancer. Genetic ablation of TMEM9 inhibits colorectal cancer cell proliferation in vitro, ex vivo and in vivo mouse models. Moreover, administration of v-ATPase inhibitors suppresses intestinal tumorigenesis of APC mouse models and human patient-derived xenografts. Our results reveal the unexpected roles of TMEM9-controlled vesicular acidification in hyperactivating Wnt/ß-catenin signalling through APC degradation, and propose the blockade of TMEM9-v-ATPase as a viable option for colorectal cancer treatment.

Deregulation of CRAD-controlled cytoskeleton initiates mucinous colorectal cancer via ß-catenin.

Epithelial integrity is maintained by the cytoskeleton and through cell adhesion. However, it is not yet known how a deregulated cytoskeleton is associated with cancer. We identified cancer-related regulator of actin dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer. We found that CRAD stabilizes the cadherin-catenin-actin complex via capping protein inhibition. The loss of CRAD inhibits F-actin polymerization and subsequently disrupts the cadherin-catenin-actin complex, which leads to ß-catenin release and Wnt signalling hyperactivation. In mice, CRAD knockout induces epithelial cell integrity loss and Wnt signalling activation, resulting in the development of intestinal mucinous adenoma. With APC mutation, CRAD knockout initiates and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a tumour suppressor, the inactivation of which deregulates the cytoskeleton and hyperactivates Wnt signalling thus initiating mucinous colorectal cancer. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity and suppressing tumorigenesis.

LIG4 mediates Wnt signalling-induced radioresistance.

Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of ß-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by ß-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/ß-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with ß-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration.

Phosphorylation and isoform use in p120-catenin during development and tumorigenesis.

P120-catenin is essential to vertebrate development, modulating cadherin and small-GTPase functions, and growing evidence points also to roles in the nucleus. A complexity in addressing p120-catenin's functions is its many isoforms, including optional splicing events, alternative points of translational initiation, and secondary modifications. In this review, we focus upon how choices in the initiation of protein translation, or the earlier splicing of the RNA transcript, relates to primary sequences that harbor established or putative regulatory phosphorylation sites. While certain p120 phosphorylation events arise via known kinases/phosphatases and have defined outcomes, in most cases the functional consequences are still to be established. In this review, we provide examples of p120-isoforms as they relate to phosphorylation events, and thereby to isoform dependent protein-protein associations and downstream functions. We also provide a view of upstream pathways that determine p120's phosphorylation state, and that have an impact upon development and disease. Because other members of the p120 subfamily undergo similar processing and phosphorylation, as well as related catenins of the plakophilin subfamily, what is learned regarding p120 will by extension have wide relevance in vertebrates.

Beyond ß-catenin: prospects for a larger catenin network in the nucleus.

ß-catenin is widely regarded as the primary transducer of canonical WNT signals to the nucleus. In most vertebrates, there are eight additional catenins that are structurally related to ß-catenin, and three α-catenin genes encoding actin-binding proteins that are structurally related to vinculin. Although these catenins were initially identified in association with cadherins at cell-cell junctions, more recent evidence suggests that the majority of catenins also localize to the nucleus and regulate gene expression. Moreover, the number of catenins reported to be responsive to canonical WNT signals is increasing. Here, we posit that multiple catenins form a functional network in the nucleus, possibly engaging in conserved protein-protein interactions that are currently better characterized in the context of actin-based cell junctions.

FOXKs promote Wnt/ß-catenin signaling by translocating DVL into the nucleus.

Dishevelled (DVL) proteins serve as crucial regulators that transduce canonical Wnt signals to the GSK3ß-destruction complex, resulting in the stabilization of ß-catenin. Emerging evidence underscores the nuclear functions of DVLs, which are critical for Wnt/ß-catenin signaling. However, the mechanism underlying DVL nuclear localization remains poorly understood. Here we discovered two Forkhead box (FOX) transcription factors, FOXK1 and FOXK2, as bona fide DVL-interacting proteins. FOXK1 and FOXK2 positively regulate Wnt/ß-catenin signaling by translocating DVL into the nucleus. Moreover, FOXK1 and FOXK2 protein levels are elevated in human colorectal cancers and correlate with DVL nuclear localization. Conditional expression of Foxk2 in mice induced intestinal hyper-proliferation that featured enhanced DVL nuclear localization and upregulated Wnt/ß-catenin signaling. Together, our results not only reveal a mechanism by which DVL is translocated into the nucleus but also suggest unexpected roles of FOXK1 and FOXK2 in regulating Wnt/ß-catenin signaling.

Nuclear signaling from cadherin adhesion complexes.

The arrival of multicellularity in evolution facilitated cell-cell signaling in conjunction with adhesion. As the ectodomains of cadherins interact with each other directly in trans (as well as in cis), spanning the plasma membrane and associating with multiple other entities, cadherins enable the transduction of "outside-in" or "inside-out" signals. We focus this review on signals that originate from the larger family of cadherins that are inwardly directed to the nucleus, and thus have roles in gene control or nuclear structure-function. The nature of cadherin complexes varies considerably depending on the type of cadherin and its context, and we will address some of these variables for classical cadherins versus other family members. Substantial but still fragmentary progress has been made in understanding the signaling mediators used by varied cadherin complexes to coordinate the state of cell-cell adhesion with gene expression. Evidence that cadherin intracellular binding partners also localize to the nucleus is a major point of interest. In some models, catenins show reduced binding to cadherin cytoplasmic tails favoring their engagement in gene control. When bound, cadherins may serve as stoichiometric competitors of nuclear signals. Cadherins also directly or indirectly affect numerous signaling pathways (e.g., Wnt, receptor tyrosine kinase, Hippo, NFκB, and JAK/STAT), enabling cell-cell contacts to touch upon multiple biological outcomes in embryonic development and tissue homeostasis.

p120-catenin regulates REST and CoREST, and modulates mouse embryonic stem cell differentiation.

Although the canonical Wnt pathway and ß-catenin have been extensively studied, less is known about the role of p120-catenin (also known as δ1-catenin) in the nuclear compartment. Here, we report that p120-catenin binds and negatively regulates REST and CoREST (also known as Rcor1), a repressive transcriptional complex that has diverse developmental and pathological roles. Using mouse embryonic stem cells (mESCs), mammalian cell lines, Xenopus embryos and in vitro systems, we find that p120-catenin directly binds the REST-CoREST complex, displacing it from established gene targets to permit their transcriptional activation. Importantly, p120-catenin levels further modulate the mRNA and protein levels of Oct4 (also known as POU5F1), Nanog and Sox2, and have an impact upon the differentiation of mESCs towards neural fates. In assessing potential upstream inputs to this new p120-catenin-REST-CoREST pathway, REST gene targets were found to respond to the level of E-cadherin, with evidence suggesting that p120-catenin transduces signals between E-cadherin and the nucleus. In summary, we provide the first evidence for a direct upstream modulator and/or pathway regulating REST-CoREST, and reveal a substantial role for p120-catenin in the modulation of stem cell differentiation.

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development.

Plakophilin-3 catenin associates with the ETV1/ER81 transcription factor to positively modulate gene activity.

Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm ("animal cap") cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.