The Role of Palladin in Podocytes.

Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein.Methods We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin.Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and α-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.

Suppression of TGFß-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition.

BACKGROUND: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy. METHODS: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo. RESULTS: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFß-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo. CONCLUSIONS: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.

Actin dynamics regulate subcellular localization of the F-actin-binding protein PALLD in mouse Sertoli cells.

Sertoli cells undergo terminal differentiation at puberty to support all phases of germ cell development, which occurs in the mouse beginning in the second week of life. By ∼18 days postpartum (dpp), nearly all Sertoli cells have ceased proliferation. This terminal differentiation is accompanied by the development of unique and regionally concentrated filamentous actin (F-actin) structures at the basal and apical aspects of the seminiferous epithelium, and this reorganization is likely to involve the action of actin-binding proteins. Palladin (PALLD) is a widely expressed F-actin-binding and bundling protein recently shown to regulate these structures, yet it is predominantly nuclear in Sertoli cells at puberty. We found that PALLD localized within nuclei of primary Sertoli cells grown in serum-free media but relocalized to the cytoplasm upon serum stimulation. We utilized this system with in vivo relevance to Sertoli cell development to investigate mechanisms regulating nuclear localization of this F-actin-binding protein. Our results indicate that PALLD can be shuttled from the nucleus to the cytoplasm, and that this relocalization occurred following depolymerization of the F-actin cytoskeleton in response to cAMP signaling. Nuclear localization was reduced in Hpg-mutant testes, suggesting the involvement of gonadotropin signaling. We found that PALLD nuclear localization was unaffected in testis tissues from LH receptor and androgen receptor-mutant mice. However, PALLD nuclear localization was reduced in the testes of FSH receptor-mutant mice, suggesting that FSH signaling during Sertoli cell maturation regulates this subcellular localization.

Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities.

Palladin (PALLD) belongs to the PALLD/myopalladin (MYPN)/myotilin family of actin-associated immunoglobulin-containing proteins in the sarcomeric Z-line. PALLD is ubiquitously expressed in several isoforms, and its longest 200 kDa isoform, predominantly expressed in striated muscle, shows high structural homology to MYPN. MYPN gene mutations are associated with human cardiomyopathies, whereas the role of PALLD in the heart has remained unknown, partly due to embryonic lethality of PALLD knockout mice. In a yeast two-hybrid screening, CARP/Ankrd1 and FHOD1 were identified as novel interaction partners of PALLD's N-terminal region. To study the role of PALLD in the heart, we generated conditional (cPKO) and inducible (cPKOi) cardiomyocyte-specific PALLD knockout mice. While cPKO mice exhibited no pathological phenotype, ablation of PALLD in adult cPKOi mice caused progressive cardiac dilation and systolic dysfunction, associated with reduced cardiomyocyte contractility, intercalated disc abnormalities, and fibrosis, demonstrating that PALLD is essential for normal cardiac function. Double cPKO and MYPN knockout (MKO) mice exhibited a similar phenotype as MKO mice, suggesting that MYPN does not compensate for the loss of PALLD in cPKO mice. Altered transcript levels of MYPN and PALLD isoforms were found in myocardial tissue from human dilated and ischemic cardiomyopathy patients, whereas their protein expression levels were unaltered.

Comparison of the incidence of pancreatic abnormalities between high risk and control patients: prospective pilot study with 3 Tesla MR imaging.

PURPOSE: To compare the incidence of pancreatic abnormalities detected by MR imaging between high-risk patients and control patients. MATERIALS AND METHODS: Forty-one consecutive patients who had two or more first-degree relatives with pancreatic cancer and who were asymptomatic with no clinical evidence of pancreatic cancer were prospectively included in this study. A control group was obtained by reviewing consecutive patients undergoing 3 Tesla (T) MRI examinations for nonpancreatic indications. On MR imaging, the presence of pancreatic abnormalities were evaluated in consensus by two radiologists who were blinded to clinical history. Pancreatic abnormalities were categorized as developmental abnormalities, mass-type lesions, inflammatory disease, and others. RESULTS: Overall, the incidence of pancreatic abnormalities was greater in the high-risk group than in the control group, but not statistically significant (P = 0.244). In the high-risk group, a total of 16 patients (39%) were diagnosed with pancreatic abnormalities, whereas in the control group, 11 patients (25%) were diagnosed with pancreatic abnormalities. Regarding mass-type lesions, there was a significant difference in incidence between the high-risk group, with a total of seven patients (17%), and the control group, with one patient (2%) (P = 0.028). There were no cases of imaging diagnosis of pancreatic cancer or tissue evaluation by surgical pathology in either group. CONCLUSION: Our prospective pilot study demonstrated a higher incidence of mass-type lesions in patients at increased risk for pancreatic cancer.

Cytoplasmic Ig-domain proteins: cytoskeletal regulators with a role in human disease.

Immunoglobulin domains are found in a wide variety of functionally diverse transmembrane proteins, and also in a smaller number of cytoplasmic proteins. Members of this latter group are usually associated with the actin cytoskeleton, and most of them bind directly to either actin or myosin, or both. Recently, studies of inherited human disorders have identified disease-causing mutations in five cytoplasmic Ig-domain proteins: myosin-binding protein C, titin, myotilin, palladin, and myopalladin. Together with results obtained from cultured cells and mouse models, these clinical studies have yielded novel insights into the unexpected roles of Ig domain proteins in mechanotransduction and signaling to the nucleus. An emerging theme in this field is that cytoskeleton-associated Ig domain proteins are more than structural elements of the cell, and may have evolved to fill different needs in different cellular compartments. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Palladin is a dynamic actin-associated protein in podocytes.

Palladin, a cytoskeletal protein with essential functions for stress fiber formation, is found in developing and mature tissues, including the kidney. To define its role in the kidney, we measured its expression in mouse kidney and found it co-localized with F-actin in smooth muscle cells of renal arterial vessels, mesangial cells, and podocytes but not in tubular epithelium. Using immunoelectron microscopy, we confirmed that palladin was present in podocytes. In cultured mouse podocytes, palladin co-localized with F-actin in dense regions of stress fibers, focal adhesions, cell-cell contacts and motile cell margins. Transfection with the N-terminal half of palladin targeted it to F-actin-containing structures in podocytes while the C-terminal half accumulated in the nucleus, a result also found for endogenous palladin in cultured cells after leptomycin B was used to block nuclear export. Green fluorescent protein (GFP)-tagged palladin was found in dynamic ring-like F-actin structures and ruffles in cultured podocytes after stimulation with epidermal growth factor. Inhibition of palladin expression by transfection of an antisense construct reduced the formation of ring-like structures. Photo-bleaching analysis showed that GFP-palladin turned over with a half-time of 10 s in focal adhesions and dense regions of stress fibers, suggesting that palladin is a dynamic scaffolding protein. Our study shows that palladin is expressed in podocytes and plays an important role in actin dynamics.

Angiotensin II, focal adhesion kinase, and PRX1 enhance smooth muscle expression of lipoma preferred partner and its newly identified binding partner palladin to promote cell migration.

Lipoma preferred partner (LPP) is a proline rich LIM domain family protein highly expressed at plasma membrane dense bodies and focal adhesions in smooth muscle cells.(1) Using the C-terminus of LPP as bait in a yeast two hybrid system, palladin, an actin-associated protein was identified. The palladin interacting region of LPP was mapped to the first and second LIM domains. The N-terminus of palladin interacted with LPP both in vitro and in vivo, but not solely through its FPLPPP and FPPPP motifs. Like LPP, palladin, is highly expressed in differentiated smooth muscle, colocalized at focal adhesions, at isolated lamellipodia and at dense bodies in smooth muscle tissue. Both LPP and palladin enhanced cell migration and spreading. LPP and palladin expression was markedly decreased, in contrast to vinculin or paxillin, in migration defective focal adhesion kinase null cells, but was restored by expression of the paired-related homeobox gene-1 protein. We have previously shown in focal adhesion kinase null cells, that tetracycline induced expression of focal adhesion kinase upregulated expression of LPP(2) and now show upregulation of palladin, and paired-related homeobox gene-1 protein. The expression of both LPP and palladin, like smooth muscle alpha-actin, was increased by angiotensin II, regulated by actin dynamics, upregulated by myocardin and expressed in the neointima of injured aorta. Overall, the data suggest that the function of LPP and palladin is context dependent, that they play a critical role in cytoskeletal remodeling, respond to signals induced by vascular injury as well as signals that induce smooth muscle cell hypertrophy, such as angiotension II.

Tumor-Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma.

CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.

The role of palladin in actin organization and cell motility.

Palladin is a widely expressed protein found in stress fibers, focal adhesions, growth cones, Z-discs, and other actin-based subcellular structures. It belongs to a small gene family that includes the Z-disc proteins myopalladin and myotilin, all of which share similar Ig-like domains. Recent advances have shown that palladin shares with myotilin the ability to bind directly to F-actin, and to crosslink actin filaments into bundles, in vitro. Studies in a variety of cultured cells suggest that the actin-organizing activity of palladin plays a central role in promoting cell motility. Correlative evidence also supports this hypothesis, as palladin levels are typically up-regulated in cells that are actively migrating: in developing vertebrate embryos, in cells along a wound edge, and in metastatic cancer cells. Recently, a mutation in the human palladin gene was implicated in an unusually penetrant form of inherited pancreatic cancer, which has stimulated new ideas about the role of palladin in invasive cancer.

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility.IMPORTANCE Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.

Four and half lim protein 2 (FHL2) stimulates osteoblast differentiation.

UNLABELLED: FHL2, a molecule that interacts with many integrins and transcription factors, was found to play an important role in osteoblast differentiation. Overexpression of FHL2 increases the accumulation of osteoblast differentiation markers and matrix mineralization, whereas FHL2 deficiency results in inhibition of osteoblast differentiation and decreased bone formation. INTRODUCTION: Integrin-matrix interaction plays a critical role in osteoblast function. It has been shown that the cytoplasmic domains of integrin beta subunits mediate signal transduction induced by integrin-matrix interaction. We reasoned that the identification of proteins interacting with beta-cytoplasmic tails followed by analysis of the function of these proteins would enhance our understanding on integrin signaling and the roles of these proteins in osteoblast activities. MATERIALS AND METHODS: Yeast two hybrid assay was used to identify proteins interacting with the cytoplasmic domain of integrin beta5 subunit. The association of these proteins with integrin alphavbeta5 was confirmed by confocal analysis and co-immunoprecipitation. A stable MC3T3-E1 cells line overexpressing Four and Half Lim Protein 2 (FHL2) and mouse osteoblasts deficient in FHL2 were used to study the roles of FHL2 in osteoblast differentiation and bone formation. Matrix protein expression was determined by mRNA analysis and Western blotting. Matrix mineralization was detected by Alizarin red staining. Alkaline phosphatase activity was also measured. muCT was used to determine bone histomorphometry. RESULTS AND CONCLUSIONS: FHL2 and actin-binding proteins, palladin and filamin A, were identified as proteins interacting with beta5 cytoplasmic domain. FHL2 co-localized with alphavbeta5 at the focal adhesion sites in association with palladin and filamin A. FHL2 was also present in nuclei. Osteoblasts overexpressing FHL2 exhibited increased adhesion to and migration on matrix proteins. Conversely, FHL2 stimulation of CREB activity was dependent on integrin function because it was inhibited by Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. The expression of osteoblast differentiation markers and Msx2 was upregulated, and bone matrix mineralization was increased in FHL2 overexpressing cells. In contrast, FHL2-deficient bone marrow cells and osteoblasts displayed decreased osteoblast colony formation and differentiation, respectively, compared with wildtype cells. Moreover, FHL2-deficient female mice exhibited greater bone loss than the wildtype littermates after ovariectomy. Thus, FHL2 plays an important role in osteoblast differentiation and bone formation.

Palladin mutation causes familial pancreatic cancer and suggests a new cancer mechanism.

BACKGROUND: Pancreatic cancer is a deadly disease. Discovery of the mutated genes that cause the inherited form(s) of the disease may shed light on the mechanism(s) of oncogenesis. Previously we isolated a susceptibility locus for familial pancreatic cancer to chromosome location 4q32-34. In this study, our goal was to discover the identity of the familial pancreatic cancer gene on 4q32 and determine the function of that gene. METHODS AND FINDINGS: A customized microarray of the candidate chromosomal region affecting pancreatic cancer susceptibility revealed the greatest expression change in palladin (PALLD), a gene that encodes a component of the cytoskeleton that controls cell shape and motility. A mutation causing a proline (hydrophobic) to serine (hydrophilic) amino acid change (P239S) in a highly conserved region tracked with all affected family members and was absent in the non-affected members. The mutational change is not a known single nucleotide polymorphism. Palladin RNA, measured by quantitative RT-PCR, was overexpressed in the tissues from precancerous dysplasia and pancreatic adenocarcinoma in both familial and sporadic disease. Transfection of wild-type and P239S mutant palladin gene constructs into HeLa cells revealed a clear phenotypic effect: cells expressing P239S palladin exhibited cytoskeletal changes, abnormal actin bundle assembly, and an increased ability to migrate. CONCLUSIONS: These observations suggest that the presence of an abnormal palladin gene in familial pancreatic cancer and the overexpression of palladin protein in sporadic pancreatic cancer cause cytoskeletal changes in pancreatic cancer and may be responsible for or contribute to the tumor's strong invasive and migratory abilities.

The proline-rich protein palladin is a binding partner for profilin.

Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), alpha-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.

The palladin/myotilin/myopalladin family of actin-associated scaffolds.

The dynamic remodeling of the actin cytoskeleton plays a critical role in cellular morphogenesis and cell motility. Actin-associated scaffolds are key to this process, as they recruit cohorts of actin-binding proteins and associated signaling complexes to subcellular sites where remodeling is required. This review is focused on a recently discovered family of three proteins, myotilin, palladin, and myopalladin, all of which function as scaffolds that regulate actin organization. While myotilin and myopalladin are most abundant in skeletal and cardiac muscle, palladin is ubiquitously expressed in the organs of developing vertebrates. Palladin's function has been investigated primarily in the central nervous system and in tissue culture, where it appears to play a key role in cellular morphogenesis. The three family members each interact with specific molecular partners: all three bind to alpha-actinin; in addition, palladin also binds to vasodilator-stimulated phosphoprotein (VASP) and ezrin, myotilin binds to filamin and actin, and myopalladin also binds to nebulin and cardiac ankyrin repeat protein (CARP). Since mutations in myotilin result in two forms of muscle disease, an essential role for this family member in organizing the skeletal muscle sarcomere is implied.

The actin crosslinking protein palladin modulates force generation and mechanosensitivity of tumor associated fibroblasts.

Cells organize actin filaments into higher-order structures by regulating the composition, distribution and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin network and stress fibers, which are critical for mechanosensing of the environment. Palladin also serves as a molecular scaffold for α-actinin, another key actin crosslinker. By virtue of its close interactions with actomyosin structures in the cell, palladin may play an important role in cell mechanics. However, the role of palladin in cellular force generation and mechanosensing has not been studied. Here, we investigate the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. Traction force microscopy revealed that tumor-associated fibroblasts generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells and inhibited their ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in actin organization, adhesion dynamics and altered myosin organization in palladin knock-down cells. Our results suggest that actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

Palladin is upregulated in kidney disease and contributes to epithelial cell migration after injury.

Recovery from acute kidney injury involving tubular epithelial cells requires proliferation and migration of healthy cells to the area of injury. In this study, we show that palladin, a previously characterized cytoskeletal protein, is upregulated in injured tubules and suggest that one of its functions during repair is to facilitate migration of remaining cells to the affected site. In a mouse model of anti-neutrophilic cytoplasmic antibody involving both tubular and glomerular disease, palladin is upregulated in injured tubular cells, crescents and capillary cells with angiitis. In human biopsies of kidneys from patients with other kidney diseases, palladin is also upregulated in crescents and injured tubules. In LLC-PK1 cells, a porcine proximal tubule cell line, stress induced by transforming growth factor-ß1 (TGF-ß1) leads to palladin upregulation. Knockdown of palladin in LLC-PK1 does not disrupt cell morphology but does lead to a defect in cell migration. Furthermore, TGF-ß1 induced increase in the 75 kDa palladin isoform occurs in both the nucleus and the cytoplasm. These data suggest that palladin expression is induced in injured cells and contributes to proper migration of cells in proximal tubules, possibly by regulation of gene expression as part of the healing process after acute injury.

Palladin expression is a conserved characteristic of the desmoplastic tumor microenvironment and contributes to altered gene expression.

The stroma surrounding solid tumors contributes in complex ways to tumor progression. Cancer-associated fibroblasts (CAFs) are the predominant cell type in the tumor stroma. Previous studies have shown that the actin-binding protein palladin is highly expressed in the stroma of pancreas tumors, but the interpretation of these results is complicated by the fact that palladin exists as multiple isoforms. In the current study, the expression and localization of palladin isoform 4 was examined in normal specimens and adenocarcinomas of human pancreas, lung, colon, and stomach samples. Immunohistochemistry with isoform-selective antibodies revealed that expression of palladin isoform 4 was higher in adenocarcinomas versus normal tissues, and highest in CAFs. Immunohistochemistry staining revealed that palladin was present in both the cytoplasm and the nucleus of CAFs, and this was confirmed using immunofluorescence staining and subcellular fractionation of a pancreatic CAF cell line. To investigate the functional significance of nuclear palladin, RNA Seq analysis of palladin knockdown CAFs versus control CAFs was performed, and the results showed that palladin regulates the expression of genes involved in the biosynthesis and assembly of collagen, and organization of the extracellular matrix. These results suggested that palladin isoform 4 may play a conserved role in establishing the phenotype of CAFs in multiple tumor types.

Molecular analysis of the interaction between palladin and alpha-actinin.

Palladin is a novel component of stress fiber dense regions. Antisense and transient overexpression studies have indicated an important role for palladin in the regulation of actin cytoskeleton. Palladin colocalizes and coimmunoprecipitates with alpha-actinin, a dense region component, but the molecular details and functional significance of the interaction have not been studied. We show here a direct association between the two proteins and have mapped the binding site within a short sequence of palladin and in the carboxy-terminal calmodulin domain of alpha-actinin. Using transfection-based targeting assays, we show that palladin is involved in targeting of alpha-actinin to specific subcellular foci indicating a functional interplay between the two actin-associated proteins.

Modulation of corneal fibroblast contractility within fibrillar collagen matrices.

PURPOSE: To investigate the migratory and contractile behavior of isolated human corneal fibroblasts in fibrillar collagen matrices. METHODS: A telomerase-infected, extended-lifespan human corneal fibroblast cell line (HTK) was transfected by using a vector for enhanced green fluorescent protein (GFP)-alpha-actinin. Cells were plated at low density on top of or within 100-microm-thick fibrillar collagen lattices. After 18 hours to 7 days, time-lapse imaging was performed. At each 1- to 3-minute interval, GFP and Nomarski differential interference contrast (DIC) images were acquired in rapid succession. Serum-containing (S+) medium was used initially for perfusion. After 2 hours, perfusion was switched to either serum-free (S-) or S+ medium containing the Rho-kinase inhibitor Y-27632 for 1 to 2 hours. Finally, perfusion was changed back to S+ medium for 1 hour. RESULTS: Two to 4 days after plating, many cells underwent spontaneous contraction and/or relaxation in S+ medium. A decrease in the distance between consecutive alpha-actinin-dense bodies along stress fibers was measured during contraction, and focal adhesion and matrix displacements correlated significantly. Removal of serum or inhibition of Rho-kinase induced cell body elongation and relaxation of matrix stress, as confirmed using finite element modeling. Rapid formation and extension of pseudopodia and filopodia were also observed, and transient tractional forces were generated by these extending processes. CONCLUSIONS: Cultured human corneal fibroblasts can undergo rapid changes in the subcellular pattern of force generation that are mediated, in part, by Rho-kinase. Sarcomeric shortening of stress fibers in contracting corneal fibroblasts is also demonstrated for the first time.