Cell-matrix Adhesion Molecules Research Articles

#718 N-Ethylmaleimide Sensitive Factor (NSF) is essential for maintaining podocyte focal adhesion

Abstract Background and Aims Cell-matrix adhesion molecules in podocytes play a critical role in attaching these cells to the glomerular basement membrane, ensuring the proper functioning of the glomerular filtration barrier. Previous studies have shown that patients with proteinuric glomerular diseases often have higher levels of podocyturia. This increase is notably linked to a reduction in focal adhesion proteins, especially in cases of focal segmental glomerulosclerosis. Our study aims to explore the regulation of these focal adhesion molecules in podocytes. Method Doxycycline (Dox) inducible podocyte-specific Tln1 knockout mice (Tln1fl/fl Nphs2-rtTA TetO-Cre (Tln1 KO)) were used as the model for podocyte injury. These mice are genetically modified to lack Talin 1, a key protein in focal adhesion. The absence of Talin 1 in these mice leads to the development of pathological changes akin to focal segmental glomerulosclerosis, characterized by extensive proteinuria, thereby providing a valuable model for understanding this kidney disease (J Clin Invest. 2019 1;129(3):1295-13). As Tln1 KO mice 10 days after Dox induction (Tln1 KO 10 d) showed proteinuria without elevated serum creatinine, these mice were considered as the early phase of podocyte injury. Single-cell RNA sequencing (scRNAseq) was performed using these mice kidney samples, followed by differentially expressed genes (DEG) analyses. Podocyte-specific Nsf KO (Nphs2-Cre Rosa-DTRflox Nsf fl/fl (Nsf cKO)) mice were generated based on the result of the DEG analyses and the effect of NSF on focal adhesion was examined using primary podocyte culture. Results Nsf was identified as one of the most significantly down-regulated DEGs in the podocytes ofTln1 KO 10d mice. Further analysis of public scRNAseq data revealed a decrease in Nsf mRNA expression levels also in lipopolysaccharide-treated mouse podocytes. Notably, proteinuria was observed in Nsf conditional knockout (cKO) mice as early as 2 weeks of age, with all Nsf cKO mice succumbing to end-stage kidney disease by 6 weeks post-birth. Examination of isolated mutant podocytes showed a marked reduction in cell spreading. Additionally, a significant decrease in Integrin β1 expression was observed in primary podocytes of Nsf cKO mice, as evidenced by immunoblot analysis. In vivo studies using transmission electron microscopy revealed endoplasmic reticulum (ER) expansion in the podocytes of Nsf cKO mice. Immunofluorescence staining indicated a substantial increase in the ER and ER-Golgi intermediate compartment (ERGIC) in isolated Nsf cKO podocytes. These findings suggest that the deletion of Nsf in podocytes leads to a disruption in ER-to-Golgi protein transport, subsequently impairing the trafficking of focal adhesion proteins. Conclusion This study provides new insights into the molecular mechanisms underlying podocyte injury and the progression of kidney diseases. NSF plays a pivotal role in maintaining podocyte focal adhesion.

N-cadherin and β1 integrin coordinately regulate growth plate cartilage architecture.

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives growth of many bones. One essential event to generate the ordered cell array characterizing growth plate cartilage is the formation of chondrocyte columns in the proliferative zone via 90-degree rotation of daughter cells to align with the long axis of the bone. Previous studies have suggested crucial roles for cadherins and integrin β1 in column formation. The purpose of this study was to determine the relative contributions of cadherin- and integrin-mediated cell adhesion in column formation. Here we present new mechanistic insights generated by application of live time-lapse confocal microscopy of cranial base explant cultures, robust genetic mouse models, and new quantitative methods to analyze cell behavior. We show that conditional deletion of either the cell-cell adhesion molecule Cdh2 or the cell-matrix adhesion molecule Itgb1 disrupts column formation. Compound mutants were used to determine a potential reciprocal regulatory interaction between the two adhesion surfaces and identified that defective chondrocyte rotation in a N-cadherin mutant was restored by a heterozygous loss of integrin β1. Our results support a model for which integrin β1, and not N-cadherin, drives chondrocyte rotation and for which N-cadherin is a potential negative regulator of integrin β1 function.

IL-36γ and IL-36Ra Reciprocally Regulate Colon Inflammation and Tumorigenesis by Modulating the Cell-Matrix Adhesion Network and Wnt Signaling.

Inflammatory bowel disease and colorectal cancer are associated with dysregulation of cytokine networks. However, it is challenging to target cytokines for effective intervention because of the overlapping functions and unpredictable interactions of cytokines in such diverse networks. Here, it is shown that IL‐36γ and IL‐36Ra, an agonist and an antagonist for IL‐36R signaling respectively, reciprocally regulate the experimental colitis and the colon cancer development in mice. Knockout or neutralization of IL‐36γ alleviates dextran sulfate sodium (DSS)‐induced colitis and inhibits colon cancer development, whereas knockout of IL‐36Ra exacerbates DSS‐induced colitis and promotes colonic tumorigenesis in multiple colon cancer models in mice. Mechanistically, IL‐36γ upregulates extracellular matrix and cell–matrix adhesion molecules and facilitates Wnt signaling, which is mitigated by IL‐36Ra or IL‐36γ neutralizing antibody. Consistently, IL‐36γ levels are positively correlated with extracellular matrix levels and β‐catenin levels in human colorectal tumor biopsies. These findings suggest the critical role of IL‐36γ and IL‐36Ra in gut inflammation and tumorigenesis and indicate that targeting the IL‐36γ/IL‐36Ra signal balance provides potential therapeutic strategy for inflammatory bowel disease and gastrointestinal cancers.

The dysfunction of BP180/collagen XVII in keratinocytes promotes melanoma progression.

BP180, also termed collagen XVII, is a hemidesmosomal transmembrane glycoprotein expressed in basal keratinocytes, and functions as a cell-matrix adhesion molecule in the dermal-epidermal junction of the skin. Its function other than cell-matrix adhesion remains unclear. We generated a mouse strain with BP180 dysfunction (termed ∆NC16A), which develops spontaneous skin inflammation accompanied by an influx of myeloid derived suppressor cells (MDSCs). We utilized the B16 mouse melanoma model to demonstrate that BP180 dysfunction in either skin or basal keratinocytes promotes MDSC influx into skin and tumor progression. MDSC depletion reduced tumor progression in ∆NC16A mice, demonstrating a critical role for BP180 dysfunction-driven MDSCs in melanoma progression. This study provides the first direct evidence that BP180, a cell-cell matrix adhesion molecule, possesses anti-tumor function through modulating infiltration of MDSCs. Basal keratinocytes actively participate in skin microenvironment changes caused by BP180 dysfunction. ∆NC16A mice could be a new animal model to study the melanoma microenvironment.

Integrin as a Molecular Target for Anti-cancer Approaches in Lung Cancer.

Integrins are cell-matrix adhesion molecules providing both mechanical engagement of cell to extracellular matrix, and generation of cellular signals that are implicated in cancer malignancies. The concept that integrins play important roles in cell survival, proliferation, motility, differentiation, and ensuring appropriate cell localization, leads to the hypothesis that inhibition of certain integrins would benefit cancer therapy. In lung cancer, integrins αv, α5, β1, β3, and β5 have been shown to augment survival and metastatic potential of cancer cells. This review presents data suggesting integrins as molecular targets for anti-cancer approaches, and the mechanisms through which integrins confer resistance of lung cancer to chemotherapeutics and metastasis. The better understanding of these key molecules may benefit the discovery of anti-cancer drugs and strategies.

Gon4l regulates notochord boundary formation and cell polarity underlying axis extension by repressing adhesion genes

Anteroposterior (AP) axis extension during gastrulation requires embryonic patterning and morphogenesis to be spatiotemporally coordinated, but the underlying genetic mechanisms remain poorly understood. Here we define a role for the conserved chromatin factor Gon4l, encoded by ugly duckling (udu), in coordinating tissue patterning and axis extension during zebrafish gastrulation through direct positive and negative regulation of gene expression. Although identified as a recessive enhancer of impaired axis extension in planar cell polarity (PCP) mutants, udu functions in a genetically independent, partially overlapping fashion with PCP signaling to regulate mediolateral cell polarity underlying axis extension in part by promoting notochord boundary formation. Gon4l limits expression of the cell–cell and cell–matrix adhesion molecules EpCAM and Integrinα3b, excesses of which perturb the notochord boundary via tension-dependent and -independent mechanisms, respectively. By promoting formation of this AP-aligned boundary and associated cell polarity, Gon4l cooperates with PCP signaling to coordinate morphogenesis along the AP embryonic axis.

Tetraspanin-enriched microdomains regulate digitation junctions.

Tetraspanins co-emerged with multi-cellular organisms during evolution and are typically localized at the cell–cell interface, [corrected] and form tetraspanin-enriched microdomains (TEMs) by associating with each other and other membrane molecules. Tetraspanins affect various biological functions, but how tetraspanins engage in multi-faceted functions at the cellular level is largely unknown. When cells interact, the membrane microextrusions at the cell-cell interfaces form dynamic, digit-like structures between cells, which we term digitation junctions (DJs). We found that (1) tetraspanins CD9, CD81, and CD82 and (2) TEM-associated molecules integrin α3β1, CD44, EWI2/PGRL, and PI-4P are present in DJs of epithelial, endothelial, and cancer cells. Tetraspanins and their associated molecules also regulate the formation and development of DJs. Moreover, (1) actin cytoskeleton, RhoA, and actomyosin activities and (2) growth factorreceptor-Src-MAP kinase signaling, but not PI-3 kinase, regulate DJs. Finally, we showed that DJs consist of various forms in different cells. Thus, DJs are common, interactive structures between cells, and likely affect cell adhesion, migration, and communication. TEMs probably modulate various cell functions through DJs. Our findings highlight that DJ morphogenesis reflects the transition between cell-matrix adhesion and cell-cell adhesion and involves both cell-cell and cell-matrix adhesion molecules.

The Differential Expression of Adhesion Molecule and Extracellular Matrix Genes in Mesenchymal Stromal Cells after Interaction with Cord Blood Hematopoietic Progenitors.

The dynamics of the expression of genes encoding adhesion molecules, molecules of the connective tissue matrix, and its remodeling enzymes was studied in multipotent mesenchymal stromal cells (MSCs) from human adipose tissue after interaction with cord blood hematopoietic progenitors (HSPCs). An upregulation of ICAM1 and VCAM1, directly proportional to the coculture time (24-72 h), was found. After 72 h of culturing, a downregulation of the genes encoding the majority of matrix molecules (SPP1; COL6A2,7A1; MMP1,3; TIMP1,3; and HAS1) and cell-matrix adhesion molecules (ITGs) was revealed. The detected changes may ensure the realization of the stromal MSC function due to improvement of adhesion and transmigration of HSPCs into the subcellular space.

Expression of ICAM-1, E-cadherin, periostin and midkine in metastases of pancreatic ductal adenocarcinomas

Development and progression of malignant tumors is in part characterized by the ability of a tumor cell to overcome cell-cell and cell-matrix adhesion and to disseminate in organs distinct from that in which they originated. This study was undertaken to analyze the clinical significance of the expression of the following cell-cell and cell-matrix adhesion molecules in pancreatic ductal adenocarcinomas (PDACs) and synchronous liver metastases: intercellular adhesion molecule 1 (ICAM-1), E-cadherin, periostin, and midkine (MK). ICAM-1, E-cadherin, periostin and MK expression was analyzed by immunohistochemistry on a tissue microarray containing 34 PDACs and 12 liver metastasis specimens. ICAM-1 expression was predominantly localized in the membranes of the cells and was found in weak to moderate intensities in PDACs and liver metastases. E-cadherin expression was absent in the majority of PDACs and corresponding liver metastases. The secreted proteins periostin and MK were expressed in various intensities in primary cancers and liver metastases. Statistical analysis demonstrated that the expression levels of the analyzed markers were neither significantly associated with metastasis in PDACs nor with clinical outcome of patients. Our study shows that the expression of the cell-cell and cell-matrix adhesion molecules ICAM-1, E-cadherin, periostin and MK was not significantly linked to metastatic disease in PDACs. Moreover, our study excludes the analyzed markers as prognostic markers in PDACs.

Actin cytoskeleton regulates functional anchorage-migration switch during T-cadherin-induced phenotype modulation of vascular smooth muscle cells

ABSTRACTVascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics per se play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.

Cellular mechano-environment regulates the mammary circadian clock

Circadian clocks drive ∼24 h rhythms in tissue physiology. They rely on transcriptional/translational feedback loops driven by interacting networks of clock complexes. However, little is known about how cell-intrinsic circadian clocks sense and respond to their microenvironment. Here, we reveal that the breast epithelial clock is regulated by the mechano-chemical stiffness of the cellular microenvironment in primary cell culture. Moreover, the mammary clock is controlled by the periductal extracellular matrix in vivo, which contributes to a dampened circadian rhythm during ageing. Mechanistically, the tension sensing cell-matrix adhesion molecule, vinculin, and the Rho/ROCK pathway, which transduces signals provided by extracellular stiffness into cells, regulate the activity of the core circadian clock complex. We also show that genetic perturbation, or age-associated disruption of self-sustained clocks, compromises the self-renewal capacity of mammary epithelia. Thus, circadian clocks are mechano-sensitive, providing a potential mechanism to explain how ageing influences their amplitude and function.

Proteome and Secretome Characterization of Glioblastoma-Derived Neural Stem Cells.

Glioblastoma multiforme (GBM) (grade IV astrocytoma) is the most common and aggressive primary brain tumor. GBM consists of heterogeneous cell types including a subset of stem cell-like cells thought to sustain tumor growth. These tumor-initiating glioblastoma multiforme-derived neural stem (GNS) cells as well as their genetically normal neural stem (NS) counterparts can be propagated in culture as relatively pure populations. Here, we perform quantitative proteomics to globally characterize and compare total proteome plus the secreted proteome (secretome) between GNS cells and NS cells. Proteins and pathways that distinguish malignant cancer (GNS) stem cells from their genetically normal counterparts (NS cells) might have value as new biomarkers or therapeutic targets. Our analysis identified and quantified ∼7,500 proteins in the proteome and ∼2,000 in the secretome, 447 and 138 of which were differentially expressed, respectively. Notable tumor-associated processes identified using gene set enrichment analysis included: extracellular matrix interactions, focal adhesion, cell motility, and cell signaling. We focused on differentially expressed surface proteins, and identified 26 that participate in ligand-receptor pairs that play a prominent role in tumorigenesis. Immunocytochemistry and immunoblotting confirmed that CD9, a recently identified marker of adult subventricular zone NS cells, was consistently enriched across a larger set of primary GNS cell lines. CD9 may, therefore, have value as a GNS-specific surface marker and a candidate therapeutic target. Altogether, these findings support the notion that increased cell-matrix and cell-cell adhesion molecules play a crucial role in promoting the tumor initiating and infiltrative properties of GNS cells. Stem Cells 2017;35:967-980.

Dependence of corneal keratocyte adhesion, spreading, and integrin β1 expression on deacetylated chitosan coating

This study reports, for the first time, the regulation of corneal keratocyte adhesion, spreading, morphology, and integrin gene expression on chitosan coating due to the effects of deacetylation. The degree of deacetylation (DD) in chitosan materials was confirmed by elemental analysis, gel permeation chromatography, and Fourier transform infrared spectroscopy. In this study, chitosan samples with the same molecular weight level but varying DD (74.1±0.5%, 84.4±0.7%, and 94.2±0.5%) were obtained by heat-alkaline treatment under a nitrogen atmosphere. For higher DD groups, the biopolymer carried abundant amino groups since the deacetylation process removed larger amount of acetyl groups from the chitosan molecules. Results showed that the mechanical stability and crystallinity of the chitosan coatings significantly increased with increasing DD value. Fibronectin adsorption, keratocyte adhesion, and cell spreading exhibited a positive correlation with DD due to the chemical functionality of polysaccharides (bearing acetyl and amino groups) and increase of substrate stiffness and crystallinity. In particular, when adhered to chitosan coatings with a DD value of 74.1%, the keratocytes appeared to be fibroblastic, elongated, and spindle shape, indicating a loss of their characteristic dendritic morphology. Furthermore, the gene expression of integrin β1 (i.e., a cell-matrix adhesion molecule) was significantly up-regulated on the chitosan coatings with higher DD, which supports favorable attachment of corneal keratocytes. Our findings suggest that DD-mediated physicochemical properties of chitosan coatings greatly affect cell-substrate crosstalk during corneal keratocyte cultivation.

Initiation of Chondrocyte Self-Assembly Requires an Intact Cytoskeletal Network.

Self-assembly and self-organization have recently emerged as robust scaffold-free tissue engineering methodologies that can be used to generate various tissues, including cartilage, vessel, and liver. Self-assembly, in particular, is a scaffold-free platform for tissue engineering that does not require the input of exogenous energy to the system. Although self-assembly can generate functional tissues, most notably neocartilage, the mechanisms of self-assembly remain unclear. To study the self-assembling process, we used articular chondrocytes as a model to identify parameters that can affect this process. Specifically, the roles of cell-cell and cell-matrix adhesion molecules, surface-bound collagen, and the actin cytoskeletal network were investigated. Using time-lapse imaging, we analyzed the early stages of chondrocyte self-assembly. Within hours, chondrocytes rapidly coalesced into cell clusters before compacting to form tight cellular structures. Chondrocyte self-assembly was found to depend primarily on integrin function and secondarily on cadherin function. In addition, actin or myosin II inhibitors prevented chondrocyte self-assembly, suggesting that cell adhesion alone is not sufficient, but rather the active contractile actin cytoskeleton is essential for proper chondrocyte self-assembly and the formation of neocartilage. Better understanding of the self-assembly mechanisms allows for the rational modulation of this process toward generating neocartilages with improved properties. These findings are germane to understanding self-assembly, an emerging platform for tissue engineering of a plethora of tissues, especially as these neotissues are poised for translation.

Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity

PurposeTo determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. MethodsPrimary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen® Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. ResultsThe FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and β4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and β1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen® Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. ConclusionThe FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea.

Cyclic stretch induced gene expression of extracellular matrix and adhesion molecules in human periodontal ligament cells

ObjectivePeriodontal ligament (PDL) cells play an important role in maintaining periodontal homeostasis upon force loading caused by mastication or orthodontic force. Previous studies revealed stretch-induced realignment of human PDL cells, but the mechanism for this phenomenon still remains unclear. As extracellular matrix (ECM) and adhesion molecules play critical roles in cell migration and alignment, this study aimed to identify mechanoresponsive genes related to ECM and adhesion in human PDL cells. DesignHuman PDL cells were exposed to 10% stretch strain for 6 or 24h, and the expression of 84 genes related to ECM and adhesion were analyzed with real-time PCR array. The protein expression of integrin α5 was examined by Western blot and flow cytometric analysis. ResultsAmong the genes screened, 6 were up-regulated and 3 were down-regulated after 6h stretch. There were 12 up-regulated and 2 down-regulated genes after 24h stretch. These differentially expressed genes included genes encoding cell-cell adhesion molecules (CD44, ICAM1), cell-matrix adhesion molecules (ITGA5, ITGA6, ITGAL, ITGB2, SPP1), basement membrane constituents (SPARC, TNC), collagens and ECM constituents (COL5A1, COL11A1, FN1), ECM proteases (ADAMTS1, ADAMTS8, MMP8) and inhibitors (TIMP1), as well as other adhesion-related molecules (CTGF, CTNND2, TGFBI, CLEC3B). Both the cytosolic and membrane integrin α5 protein levels were up-regulated in response to stretch. ConclusionThis study identified several force-sensitive genes related to ECM and adhesion in stretched human PDL cells and should facilitate future studies on the stretch-induced cell realignment and mechanic force related periodontal remodelling by providing potential target genes.

Reversal of vasohibin-driven negative feedback loop of vascular endothelial growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver diseases

Reversal of vasohibin-driven negative feedback loop of vascular endothelial growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver diseases

CCN1: a novel inflammation-regulated biphasic immune cell migration modulator.

In this study, we performed a comprehensive analysis of the effect of CCN1 on the migration of human immune cells. The molecule CCN1, produced by fibroblasts and endothelial cells, is considered as an important matrix protein promoting tissue repair and immune cell adhesion by binding various integrins. We recently reported that CCN1 therapy is able to suppress acute inflammation in vivo. Here, we show that CCN1 binds to various immune cells including T cells, B cells, NK cells, and monocytes. The addition of CCN1 in vitro enhances both actin polymerization and transwell migration. Prolonged incubation with CCN1, however, results in the inhibition of migration of immune cells by a mechanism that involves downregulation of PI3Kγ, p38, and Akt activation. Furthermore, we observed that immune cells themselves produce constitutively CCN1 and secretion is induced by pro-inflammatory stimuli. In line with this finding, patients suffering from acute inflammation had enhanced serum levels of CCN1. These findings extend the classical concept of CCN1 as a locally produced cell matrix adhesion molecule and suggest that CCN1 plays an important role in regulating immune cell trafficking by attracting and locally immobilizing immune cells.

Crystal Structure of a Rigid Four-Spectrin-Repeat Fragment of the Human Desmoplakin Plakin Domain

The plakin protein family serves to connect cell–cell and cell–matrix adhesion molecules to the intermediate filament cytoskeleton. Desmoplakin (DP) is an integral part of desmosomes, where it links desmosomal cadherins to the intermediate filaments. The 1056-amino-acid N-terminal region of DP contains a plakin domain common to members of the plakin family. Plakin domains contain multiple copies of spectrin repeats (SRs). We determined the crystal structure of a fragment of DP, residues 175–630, consisting of four SRs and an inserted SH3 domain. The four repeats form an elongated, rigid structure. The SH3 domain is present in a loop between two helices of an SR and interacts extensively with the preceding SR in a manner that appears to limit inter-repeat flexibility. The intimate intramolecular association of the SH3 domain with the preceding SR is also observed in plectin, another plakin protein, but not in α-spectrin, suggesting that the SH3 domain of plakins contributes to the stability and rigidity of this subfamily of SR-containing proteins.

Adhesion and growth of dental pulp stem cells on enamel‐like fluorapatite surfaces

To study how apatite crystal alignment of an enamel-like substrate affects DPSC cellular adhesion and growth as a precursor to produce an in vitro enamel/dentin superstructure for future studies. The cells were subcultured in 10% FBS DMEM up to seven weeks on the two surfaces. Specimens were observed under SEM, counted, and analyzed using the human pathway-focused matrix and adhesion PCR array. After three days, the cell number on ordered FA surface was significantly higher than on the disordered surface. Of the 84 focused pathway genes, a total of 20 genes were either up or down regulated in the cells on ordered FA surface compared to the disordered surface. More interestingly, of the cell-matrix adhesion molecules, integrin alpha 7 and 8 (ITGA 7 and 8), integrin beta 3 and 4 (ITGB3 and 4), and the vitronectin receptor-integrin alpha V (ITGAV) and the key adhesion protein-fibronectin1 (FN1) were up-regulated. In SEM, both surfaces showed good biocompatibility and supported long term growth of DPSC cells but with functional cell-matrix interaction on the ordered FA surfaces. The enhanced cellular response of DPSC cell to the ordered FA crystal surface involves a set of delicately regulated matrix and adhesion molecules which could be manipulated by treating the cells with a dentin extract, to produce a dentin/enamel superstructure.