Proximity proteomics of synaptopodin provides insight into the molecular composition of the spine apparatus of dendritic spines.

The spine apparatus is a specialized compartment of the neuronal smooth endoplasmic reticulum (ER) located in a subset of dendritic spines. It consists of stacks of ER cisterns that are interconnected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft. While this organelle was first observed over 60 y ago, its molecular organization remains a mystery. Here, we performed in vivo proximity proteomics to gain some insight into its molecular components. To do so, we used the only known spine apparatus-specific protein, synaptopodin, to target a biotinylating enzyme to this organelle. We validated the specific localization in dendritic spines of a small subset of proteins identified by this approach, and we further showed their colocalization with synaptopodin when expressed in nonneuronal cells. One such protein is Pdlim7, an actin binding protein not previously identified in spines. Pdlim7, which we found to interact with synaptopodin through multiple domains, also colocalizes with synaptopodin on the cisternal organelle, a peculiar stack of ER cisterns resembling the spine apparatus and found at axon initial segments of a subset of neurons. Moreover, Pdlim7 has an expression pattern similar to that of synaptopodin in the brain, highlighting a functional partnership between the two proteins. The components of the spine apparatus identified in this work will help elucidate mechanisms in the biogenesis and maintenance of this enigmatic structure with implications for the function of dendritic spines in physiology and disease.

Afadin cooperates with Claudin-2 to promote breast cancer metastasis.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that the PDZ-binding motif of Claudin-2 is necessary for anchorage-independent growth of cancer cells and is required for liver metastasis. Several PDZ domain-containing proteins were identified that interact with the PDZ-binding motif of Claudin-2 in liver metastatic breast cancer cells, including Afadin, Arhgap21, Pdlim2, Pdlim7, Rims2, Scrib, and ZO-1. We specifically examined the role of Afadin as a potential Claudin-2-interacting partner that promotes breast cancer liver metastasis. Afadin associates with Claudin-2, an interaction that requires the PDZ-binding motif of Claudin-2. Loss of Afadin also impairs the ability of breast cancer cells to form colonies in soft agar and metastasize to the lungs or liver. Immunohistochemical analysis of Claudin-2 and/or Afadin expression in 206 metastatic breast cancer tumors revealed that high levels of both Claudin-2 and Afadin in primary tumors were associated with poor disease-specific survival, relapse-free survival, lung-specific relapse, and liver-specific relapse. Our findings indicate that signaling downstream from a Claudin-2/Afadin complex enables the efficient formation of breast cancer metastases. Moreover, combining Claudin-2 and Afadin as prognostic markers better predicts the potential of breast cancer to metastasize to soft tissues.

Rebooting the collagen gel: Artificial hydrogels for the study of epithelial mesenchymal transformation.

The collagen gel has been used to study epithelial-mesenchymal transformation (EMT) for over 30 years. With advances in the field of materials sciences, new options are available to design optically clear, three-dimensional nature-inspired matrix mimetics to study EMT. Here, we review the history of the collagen gel assay, discuss its current use and how newer artificial matrices can be built to simulate in vivo extracellular environments and investigate important current questions in the EMT field. We suggest that further collaborations between materials scientists and biologists will be critical to move the field of EMT forward. Developmental Dynamics 247:332-339, 2018. © 2017 Wiley Periodicals, Inc.

Proteomic characterization of epicardial-myocardial signaling reveals novel regulatory networks including a role for NF-κB in epicardial EMT.

Signaling between the epicardium and underlying myocardium is crucial for proper heart development. The complex molecular interactions and regulatory networks involved in this communication are not well understood. In this study, we integrated mass spectrometry with bioinformatics to systematically characterize the secretome of embryonic chicken EPDC-heart explant (EHE) co-cultures. The 150-protein secretome dataset established greatly expands the knowledge base of the molecular players involved in epicardial-myocardial signaling. We identified proteins and pathways that are implicated in epicardial-myocardial signaling for the first time, as well as new components of pathways that are known to regulate the crosstalk between epicardium and myocardium. The large size of the dataset enabled bioinformatics analysis to deduce networks for the regulation of specific biological processes and predicted signal transduction nodes within the networks. We performed functional analysis on one of the predicted nodes, NF-κB, and demonstrate that NF-κB activation is an essential step in TGFß2/PDGFBB-induced cardiac epithelial-to-mesenchymal transition. In summary, we have generated a global perspective of epicardial-myocardial signaling for the first time, and our findings open exciting new avenues for investigating the molecular basis of heart development and regeneration.

Pdlim7 Regulates Arf6-Dependent Actin Dynamics and Is Required for Platelet-Mediated Thrombosis in Mice.

Upon vessel injury, platelets become activated and rapidly reorganize their actin cytoskeleton to adhere to the site of endothelial damage, triggering the formation of a fibrin-rich plug to prevent further blood loss. Inactivation of Pdlim7 provides the new perspective that regulation of actin cytoskeletal changes in platelets is dependent on the encoded PDZ-LIM protein. Loss-of-function of Pdlim7 triggers hypercoagulopathy and causes significant perinatal lethality in mice. Our in vivo and in vitro studies reveal that Pdlim7 is dynamically distributed along actin fibers, and lack of Pdlim7 leads to a marked inability to rearrange the actin cytoskeleton. Specifically, the absence of Pdlim7 prevents platelets from bundling actin fibers into a concentric ring that defines the round spread shape of activated platelets. Similarly, in mouse embryonic fibroblasts, loss of Pdlim7 abolishes the formation of stress fibers needed to adopt the typical elongated fibroblast shape. In addition to revealing a fundamental cell biological role in actin cytoskeletal organization, we also demonstrate a function of Pdlim7 in regulating the cycling between the GTP/GDP-bound states of Arf6. The small GTPase Arf6 is an essential factor required for actin dynamics, cytoskeletal rearrangements, and platelet activation. Consistent with our findings of significantly elevated initial F-actin ratios and subsequent morphological aberrations, loss of Pdlim7 causes a shift in balance towards an increased Arf6-GTP level in resting platelets. These findings identify a new Pdlim7-Arf6 axis controlling actin dynamics and implicate Pdlim7 as a primary endogenous regulator of platelet-dependent hemostasis.

Maintaining Eastern newts (Notophthalmus viridescens) for regeneration research.

The adult Eastern newt, Notophthalmus viridescens, has long served as a model for appendage as well as heart muscle regeneration studies. Newt tissues include all major cell types known in other vertebrates and mammals, including bone, cartilage, tendon, muscle, nerves, dermis, and epidermis. Therefore, these aquatic salamanders make an excellent model for studying the regeneration of complex tissues. Regeneration of adult tissues requires the integration of new tissues with preexisting tissues to form a functioning unit through a process that is not yet well understood. Scale is also an issue, because the regenerating tissues or structures are magnitudes larger than their embryonic counterparts during development, and therefore, it is likely that different physics and mechanics apply. Regardless, regeneration recapitulates to some degree developmental processes. In this chapter, we will describe basic methods for maintaining adult Eastern newts in the laboratory for the study of regeneration. To determine similarities and differences between development and regeneration at the cellular and molecular level, there is also a need for embryonic newt tissue. We therefore also outline a relatively simple way to produce and raise newt embryos in the laboratory.

Assessing cardiomyocyte proliferative capacity in the newt heart and primary culture.

Unlike humans, adult newts possess extraordinary abilities to functionally regenerate lost and injured organs, including cardiac muscle. The most remarkable feature of mature newt cardiomyocytes is their ability to reenter the cell cycle, undergo cell division, and serve as a reservoir for progenitor cells. There are, however, a number of unsolved questions concerning the cellular and molecular mechanisms that underlie this plasticity; for example, we still lack a deeper understanding of the cell-inherent properties of newt cardiomyocytes and to what degree they differ from their mammalian counterparts. Along with considerable morphological changes at the wound site, a striking feature shared by different regenerating tissues in the newt is an extensive and dynamic remodeling of the extracellular environment. The dynamic signaling between cardiomyocytes and extracellular environment is of eminent importance in the control of the differentiated state of the cell, but the molecular details remain elusive. In this chapter, we describe methods to assess cardiomyocyte proliferation in vivo and enrich primary cardiomyocytes from newt hearts to study their behavior, taking extracellular matrix components into consideration.

Loss of the cytoskeletal protein Pdlim7 predisposes mice to heart defects and hemostatic dysfunction.

The actin-associated protein Pdlim7 is essential for heart and fin development in zebrafish; however, the expression and function of this PDZ-LIM family member in the mammal has remained unclear. Here, we show that Pdlim7 predominantly localizes to actin-rich structures in mice including the heart, vascular smooth muscle, and platelets. To test the requirement for Pdlim7 in mammalian development and function, we analyzed a mouse strain with global genetic inactivation of Pdlim7. We demonstrate that Pdlim7 loss-of-function leads to significant postnatal mortality. Inactivation of Pdlim7 does not disrupt cardiac development, but causes mild cardiac dysfunction in adult mice. Adult Pdlim7(-/-) mice displayed increased mitral and tricuspid valve annulus to body weight ratios. These structural aberrations in Pdlim7(-/-) mice were supported by three-dimensional reconstructions of adult cardiac valves, which revealed increased surface area to volume ratios for the mitral and tricuspid valve leaflets. Unexpectedly, we found that loss of Pdlim7 triggers systemic venous and arterial thrombosis, leading to significant mortality shortly after birth in Pdlim7(+/-) (11/60) and Pdlim7(-/-) (19/35) mice. In line with a prothrombotic phenotype, adult Pdlim7(-/-) mice exhibit dramatically decreased tail bleed times compared to controls. These findings reveal a novel and unexpected function for Pdlim7 in maintaining proper hemostasis in neonatal and adult mice.

A dynamic spatiotemporal extracellular matrix facilitates epicardial-mediated vertebrate heart regeneration.

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.

Biochemical and mechanical environment cooperatively regulate skeletal muscle regeneration.

During forelimb regeneration in the newt Notophthalmus viridescens, the dynamic expression of a transitional matrix rich in hyaluronic acid, tenascin-C, and fibronectin controls muscle cell behavior in vivo and in vitro. However, the influence of extracellular matrix (ECM) remodeling on tissue stiffness and the cellular response to mechanical variations during regeneration was unknown. By measuring the transverse stiffness of tissues in situ, we found undifferentiated regenerative blastemas were less stiff than differentiated stump muscle (13.3±1.6 vs. 16.6±1.2 kPa). To directly determine how ECM and stiffness combine to affect skeletal muscle fragmentation, migration, and fusion, we coated silicone-based substrates ranging from 2 to 100 kPa with matrices representative of transitional (tenascin-C and fibronectin) and differentiated environments (laminin and Matrigel). Using live-cell imaging, we found softer tenascin-C-coated substrates significantly enhanced migration and fragmentation of primary newt muscle cells. In contrast, stiffer substrates coated with laminin, Matrigel, or fibronectin increased differentiation while suppressing migration and fragmentation. These data support our in vivo observations that a transitional matrix of reduced stiffness regulates muscle plasticity and progenitor cell recruitment into the regenerative blastema. These new findings will enable the determination of how biochemical and mechanical cues from the ECM control genetic pathways that drive regeneration.

Salamanders and fish can regenerate lost structures--why can't we?

The recent introduction of in vivo lineage-tracing techniques using fluorescently labeled cells challenged the long-standing view that complete dedifferentiation is a major force driving vertebrate tissue regeneration. The report in BMC Developmental Biology by Juan Carlos Izpisúa Belmonte and colleagues adds a new twist to a rapidly evolving view of the origin of blastemal cells. As classic and recent experimental findings are considered together, a new perspective on vertebrate muscle regeneration is emerging.See research article http://www.biomedcentral.com/1471-213X/12/9.

Multi-tissue microarray analysis identifies a molecular signature of regeneration.

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.

High resolution three-dimensional imaging: Evidence for cell cycle reentry in regenerating skeletal muscle.

Newts and other urodele amphibians can replace lost structures including limbs, providing a vertebrate model for the study of regeneration of complex tissues. The composite of different cell and tissue types in the limb, however, presents a challenge for their imaging in three-dimensions (3D) at cellular level resolution. To observe myofibers in vivo without distortion, we developed a streamlined protocol whereby 80 µm thick cryosections are mounted on slides, processed for immunohistochemistry, imaged using confocal microscopy and z-stacks rendered in 3D. This methodology enabled precise in situ rendering of regenerating muscle, demonstrating cell cycle reentry of nuclei within the myofiber syncytium. The high resolution imaging of muscle or comparable tissue types as intact 3D entities in the context of extracellular and intracellular molecules allows for the determination of signaling and cell response pathways, making this method useful for studies that attempt to characterize rare physiological events in vivo.

Pdlim7 is required for maintenance of the mesenchymal/epidermal Fgf signaling feedback loop during zebrafish pectoral fin development.

BACKGROUND: Vertebrate limb development involves a reciprocal feedback loop between limb mesenchyme and the overlying apical ectodermal ridge (AER). Several gene pathways participate in this feedback loop, including Fgf signaling. In the forelimb lateral plate mesenchyme, Tbx5 activates Fgf10 expression, which in turn initiates and maintains the mesenchyme/AER Fgf signaling loop. Recent findings have revealed that Tbx5 transcriptional activity is regulated by dynamic nucleocytoplasmic shuttling and interaction with Pdlim7, a PDZ-LIM protein family member, along actin filaments. This Tbx5 regulation is critical in heart formation, but the coexpression of both proteins in other developing tissues suggests a broader functional role. RESULTS: Knock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype. While early gene induction and patterning in the presumptive fin field appear normal, the pectoral fin precursor cells display compaction and migration defects between 18 and 24 hours post-fertilization (hpf). During fin growth fgf24 is sequentially expressed in the mesenchyme and then in the apical ectodermal ridge (AER). However, in pdlim7 antisense morpholino-treated embryos this switch of expression is prevented and fgf24 remains ectopically active in the mesenchymal cells. Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme. As a consequence of perturbed AER function in the absence of Pdlim7, pathway components in the fin mesenchyme are misregulated or absent, indicating a breakdown of the Fgf signaling feedback loop, which is ultimately responsible for the loss of fin outgrowth. CONCLUSION: This work provides the first evidence for the involvement of Pdlim7 in pectoral fin development. Proper fin outgrowth requires fgf24 downregulation in the fin mesenchyme with subsequent activation in the AER, and Pdlim7 appears to regulate this transition, potentially through Tbx5 regulation. By controlling Tbx5 subcellular localization and transcriptional activity and possibly additional yet unknown means, Pdlim7 is required for proper development of the heart and the fins. These new regulatory mechanisms may have important implications how we interpret Tbx5 function in congenital hand/heart syndromes in humans.

A transitional extracellular matrix instructs cell behavior during muscle regeneration.

Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved.

Nucleocytoplasmic functions of the PDZ-LIM protein family: new insights into organ development.

Recent work on the PDZ-LIM protein family has revealed that it has important activities at the cellular level, mediating signals between the nucleus and the cytoskeleton, with significant impact on organ development. We review and integrate current knowledge about the PDZ-LIM protein family and propose a new functional role, sequestering nuclear factors in the cytoplasm. Characterized by their PDZ and LIM domains, the PDZ-LIM family is comprised of evolutionarily conserved proteins found throughout the animal kingdom, from worms to humans. Combining two functional domains in one protein, PDZ-LIM proteins have wide-ranging and multi-compartmental cell functions during development and homeostasis. In contrast, misregulation can lead to cancer formation and progression. New emerging roles include interactions with integrins, T-box transcription factors, and receptor tyrosine kinases. Facilitating the assembly of protein complexes, PDZ-LIM proteins can act as signal modulators, influence actin dynamics, regulate cell architecture, and control gene transcription.

Pdlim7 (LMP4) regulation of Tbx5 specifies zebrafish heart atrio-ventricular boundary and valve formation.

Tbx5 is involved in congenital heart disease, however, the mechanisms leading to organ malformation are greatly unknown. We hypothesized a model by which the Tbx5 binding protein Pdlim7 controls nuclear/cytoplasmic shuttling and function of the transcription factor. Using the zebrafish, we present in vivo significance for an essential role of Tbx5/Pdlim7 protein interaction in the regulation of cardiac formation. Knock-down of Pdlim7 results in a non-looped heart, strikingly reminiscent of the tbx5 heartstrings mutant phenotype. However, while misregulation of Pdlim7 and Tbx5 produce similar aberrant cardiac morphology, molecular and histological analysis uncovered that the Pdlim7 and Tbx5 cardiac phenotypes are due to opposite effects on valve development. Loss of Pdlim7 function causes no valve tissue to develop while lack of Tbx5 results in increased valve tissue. These opposing defects are evident before valve formation and are the result of distinct gene misregulation during specification of the atrio-ventricular (AV) boundary. We show that Pdlim7/Tbx5 interactions affect the expression of Tbx5 target genes nppa and tbx2b at the AV boundary, and their domains of misexpression directly correlate with the identified valve defects. These studies demonstrate that controlling the correct balance of Tbx5 activity is crucial for the specification of the AV boundary and valve formation.

An evolutionarily conserved nuclear export signal facilitates cytoplasmic localization of the Tbx5 transcription factor.

During cardiac development, the T-box transcription factor Tbx5 displays dynamic changes in localization from strictly nuclear to both nuclear and cytoplasmic to exclusively cytoplasmic along the actin cytoskeleton in cells coexpressing its binding protein LMP4. Although nuclear localization signals (NLSs) have been described, the mechanism by which Tbx5 exits the nucleus remained elusive. Here, we describe for Tbx5 a nuclear export signal (NES) that is recognized by the CRM1 export protein. Site-directed mutagenesis of a critical amino acid(s) within this sequence determined the functionality of this NES. Confocal localization studies and luciferase transcriptional reporter assays with NES mutant Tbx5 forms demonstrated retention in the nucleus, regardless of the presence of LMP4. Coimmunoprecipitation and pharmacological interference studies demonstrated a direct interaction between Tbx5 and CRM1, revealing that Tbx5 is using the CRM1 pathway for nuclear export. In addition to Tbx5, we identified NESs in all T-box proteins and demonstrated interaction of the family members Tbx3 and Brachyury with the CRM1 exporter, suggesting general significance. This first demonstration of evolutionarily conserved NESs in all T-box proteins in conjunction with NLSs indicates a primordial function of T-box proteins to dynamically shuttle between nuclear and cytoplasmic compartments of the cell.

Differential regulation of Tbx5 protein expression and sub-cellular localization during heart development.

The T-box transcription factor Tbx5 can interact with Nkx2.5 and Gata4 transcription factors to synergistically regulate heart-specific genes in the nucleus. While a nuclear role for Tbx5 is clearly defined, we have previously shown that Tbx5 shuttles from nuclear to cytoplasmic sites, forming a complex with the PDZ-LIM protein LMP4 on the actin cytoskeleton. In this study, using a developmental series of chicken hearts, we provide the first evidence for differential Tbx5 protein expression and sub-cellular localization during cardiogenesis. At the tissue level, we show temporally and spatially restricted Tbx5 co-expression with LMP4. In cells co-expressing LMP4 and Tbx5 we demonstrate dynamic Tbx5 re-localization from exclusively nuclear to nuclear and cytoplasmic expression in the atrio-ventricular cushion. Furthermore, in coronary vessel development we show exclusive cytoplasmic localization of Tbx5, indicating a function for Tbx5 in the cytoplasm. In addition, we discover unknown regulation of Tbx5 and LMP4 expression in epicardial tissue, suggesting a specific role for Tbx5 in epicardial formation. These studies provide in vivo significance of the LMP4/Tbx5 protein interaction, suggesting both nuclear and cytoplasmic roles for Tbx5. The shuttling between nuclear and cytoplasmic sites reveals a novel mechanism for Tbx transcription factor regulation in chicken heart development allowing new insights for a better understanding of the molecular basis of hand/heart birth defects associated with TBX5 mutations.