Role Of Palladin Research Articles

Role of palladin in actin dynamics revealed by quantitative TIRFM

Role of palladin in actin dynamics revealed by quantitative TIRFM

Cytoskeletal Protein Palladin in Adult Gliomas Predicts Disease Incidence, Progression, and Prognosis.

Simple SummaryGlioma is a tumor originating from cells supporting the brain and represents a major health challenge. Palladin is a structural protein widely expressed in mammalian tissues and has a pivotal role in cytoskeletal dynamics in health and disease. Palladin is linked to the progression of breast, pancreatic, and renal cancers. However, the role of palladin in gliomas is yet unknown. In this work, we aimed to shed light on palladin’s role in glioma tumors using publicly available data, along with samples obtained from humans and mice. Our findings indicate that palladin expression might be linked to adult glioma progression and is associated with a worse prognosis. Overall, our results introduce the possibility of using palladin as a diagnostic and prognostic marker, as well as a potential future therapeutic target.Brain tumors comprise over 100 types of masses, differing in the following: location; patient age; molecular, histological, and immunohistochemical characteristics; and prognosis and treatment. Glioma tumors originate from neuroglia, cells supporting the brain. Palladin, a structural protein widely expressed in mammalian tissues, has a pivotal role in cytoskeletal dynamics and motility in health and disease. Palladin is linked to the progression of breast, pancreatic, and renal cancers. In the central nervous system, palladin is involved in embryonic development, neuronal maturation, the cell cycle, differentiation, and apoptosis. However, the role of palladin in brain tumors is unknown. In this work, we explored palladin’s role in glioma. We analyzed clinical data, along with bulk and single-cell gene expression. We then validated our results using IHC staining of tumor samples, together with qRT-PCR of glioma cell lines. We determined that wild-type palladin-4 is overexpressed in adult gliomas and is correlated with a decrease in survival. Palladin expression outperformed clinically used prognostic markers and was most prominent in glioblastoma. Finally, we showed that palladin originates from the malignant cell population. Our findings indicate that palladin expression might be linked to adult glioma progression and is associated with prognosis.

Monitoring Palladin's Effect on Actin Dynamics and Organization with TIRF Microscopy

The actin-associated human protein palladin plays a critical role in cytoskeletal organization in both normal and cancerous cells. In breast and pancreatic cancer cell lines, palladin expression levels have been shown to correlate with metastatic potential. We have recently established that palladin contributes to actin dynamics in three distinct ways: nucleation of actin, crosslink formation, and filament stabilization. We hypothesize that palladin directly influences cell motility through simultaneous regulation of actin polymerization and organization. In this work we build upon our kinetic assays of actin polymerization to directly visualize actin assembly and protein dynamics with total internal reflection fluorescence microscopy (TIRFM). Bulk co-sedimentation assays can only monitor an increase in the total amount of actin polymerized, whereas the TIRF assay allows one to follow polymerization dynamics and filament topology simultaneously. To gain insight into the role of palladin in the assembly process and in the dynamics of supramolecular actin structures, we imaged the polymerization of monomeric actin and palladin in real time with TIRFM. Our data show that palladin mediates the formation of junctions between filaments. Analysis of filament morphology reveals that palladin promotes side branching in addition to bundles. Through these methods, we hope to understand how palladin contributes to actin-based cell motility in metastatic cells, which will be important for developing new therapies to specifically target this step in cancer progression.

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.

Abstract 2979: Inhibition of palladin in cancer associated fibroblast impedes pancreatic ductal adenocarcinoma associated desmoplasia

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) claims 97% of patient lives in less than 5 years after diagnosis. Treatment of PDAC is challenged by the collagen-rich, highly dense, fibrous-like extracellular matrix (ECM) known as desmoplasia. Desmoplasia is initiated by activated cancer associated fibroblasts (CAFs) and disseminates throughout the stromal compartment in a vicious cycle involving alterations to the physical/structural and biochemical pathways in neighboring naïve fibroblasts that ultimately enriches the CAF population. This desmoplastic-ECM (D-ECM) encapsulates the tumor mass, thereby limiting therapeutic perfusion. Paradoxically, ablation of D-ECM induces a more aggressive PDAC progression. Although the underlying biology remains unclear, the D-ECM is a consequence of cytoskeletal rearrangements occurring amid the CAFs. The goal of this study is to delineate the cytoskeleton dynamics engaged as a potential mechanism to restore the innate tumor-suppressive properties of a normal ECM. As such, this study focuses on the actin cross-linker palladin which has been identified as an independent prognostic marker in PDAC progression. We postulate palladin has an isoform-specific role in PDAC desmoplasia as well as cancer progression. Using an in vivo-mimetic 3D fibroblast-derived ECM model, we explore the isoform-specific role of palladin during D-ECM onset as well as during D-ECM-induced CAF activation in PDAC patient derived CAFs in which the major palladin isoforms (isoforms 3 and 4) were knocked out via CRISPR/Cas9 gene editing. Using these cells, we examined the activation status of the CAFs while characterizing the ECM produced via quantitative immunoblots, real-time polymerase chain reaction, and detailed microscopy analyses. We then validated the effects of palladin knockout CAFs activity in influencing kras mutated cancer cells’ metabolic activity, invasion and proliferation. Our data indicates palladin isoforms 3 and 4 expression are necessary to drive CAF activation as well as perpetuate the D-ECM phenotype whereby knockdown of either isoform prevents CAF activation while limiting cancer cell behavior. Additionally, the ECMs produced by the palladin knockout CAFs were no longer capable of activating naïve fibroblast in addition to decreasing the inherent aggressive nature of kras-mutant cancer cells. These results are similar to previous findings regarding the restrictive nature of a normal ECM on CAF activation and cancer cell behavior. These findings suggest that palladin isoforms 3 and 4 are required for desmoplastic onset and D-ECM-induced fibroblastic activation and cancer cell progression. Together, these results propose manipulation of specific palladin isoforms could signify a strategy to restore a normal tumor-suppressive stroma in PDAC. Citation Format: Jennifer I. Alexander, Edna Cukierman. Inhibition of palladin in cancer associated fibroblast impedes pancreatic ductal adenocarcinoma associated desmoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2979. doi:10.1158/1538-7445.AM2017-2979

Defining a Conserved Role for Palladin in Tumor Progression

Defining a Conserved Role for Palladin in Tumor Progression

Abstract B46: The actin-binding protein palladin localizes to the nucleus of pancreatic tumor-associated fibroblasts and regulates gene expression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an exceptionally lethal cancer that is characterized by an abundant desmoplastic stroma containing numerous tumor-associated fibroblasts (TAFs). TAFs are believed to contribute to PDAC's rapid progression and its resistance to therapy via multiple mechanisms, including remodeling of the extracellular matrix and secretion of motility-promoting cytokines and growth factors. We recently showed that one isoform of the actin-binding protein palladin is highly expressed in TAFs within the stroma of PDAC patient specimens. To investigate palladin's functional role in this cell type, we obtained a line of immortalized human pancreatic TAFs, and generated a sub-line of palladin knockdown TAFs using lentiviral expression of shRNA. The palladin knockdown TAFs were characterized in a mixed-cell, mouse xenograft model, and the results showed that palladin plays a critical role in the metastasis-promoting activity of TAFs. Recent studies in smooth muscle cells yielded new insights into palladin's potential molecular function in TAFs, as these studies have shown that palladin is detected in the nucleus of smooth muscle cells, where it regulates gene expression through interactions with Myocardin-Related Transcription Factor. As TAFs and smooth muscle cells share similar patterns of gene expression, we set out to test the hypothesis that palladin functions as a transcriptional co-regulator also in TAFs. To address this question, we first investigated palladin's subcellular localization in cultured TAFs using immunofluorescence staining and cellular fractionation combined with immunoblot analysis. The results demonstrated that endogenous palladin is detected in both the stress fibers and the nucleus of TAFs. To ask if palladin plays a role in regulating gene expression in TAFs, total RNA from replicate cultures of knockdown and control cells was isolated and subjected to transcriptomic analysis (RNAseq). The results showed that the levels of expression of over 100 genes were significantly altered when palladin was knocked down in TAFs. Pathway analysis revealed that palladin regulates the expression of multiple genes involved in focal adhesion assembly and cell-matrix interactions, including the matrix metalloproteinases MMP1 and MMP3, and also secreted cytokines that regulate cell motility and epithelial-to-mesenchymal transition, including bone morphogenetic protein 4 (BMP4) and its secreted antagonist Gremlin-1. These novel results define an unexpected role for palladin within the nuclear compartment, and suggest that palladin may regulate multiple TAF behaviors that contribute to tumor progression, including matrix remodeling and cytokine secretion. Future studies will be aimed at investigating the functional roles of palladin-regulated genes in the biology and behavior of TAFs. Citation Format: Meredith Owen, Michael Kerber, Silvia Goicoechea, Rosa F. Hwang, Hong Jin Kim, Carol A. Otey. The actin-binding protein palladin localizes to the nucleus of pancreatic tumor-associated fibroblasts and regulates gene expression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B46.

Actin cross-linking protein palladin and spermatogenesis

In the seminiferous epithelium of the mammalian testis, the most distinctive ultrastructure is the extensive bundles of actin filaments that lie near the Sertoli-spermatid interface and the Sertoli-Sertoli cell interface known as the apical ectoplasmic specialization (apical ES) and the basal ES, respectively. These actin filament bundles not only confer strong adhesion at these sites, they are uniquely found in the testis. Recent studies have shown that ES also confers spermatid and Sertoli cell polarity in the seminiferous epithelium during the epithelial cycle. While these junctions were first described in the 1970s, there are few functional studies in the literature to examine the regulation of these actin filament bundles. It is conceivable that these actin filament bundles at the ES undergo extensive re-organization to accommodate changes in location of developing spermatids during spermiogenesis as spermatids are transported across the seminiferous epithelium. Additionally, these actin filaments are rapidly reorganized during BTB restructuring to accommodate the transit of preleptotene spermatocytes across the barrier at stage VIII of the epithelial cycle. Thus, actin binding and regulatory proteins are likely involved in these events to confer changes in F-actin organization at these sites. Interestingly, there are no reports in the field to study these regulatory proteins until recently. Herein, we summarize some of the latest findings in the field regarding a novel actin cross-linker and actin-bundling protein called palladin. We also discuss in this opinion article the likely role of palladin in regulating actin filament bundles at the ES during spermatogenesis, highlighting the significant of palladin and how this protein is plausibly working in concert with other actin-binding/regulatory proteins and components of polarity proteins to regulate the cyclic events of actin organization and re-organization during the epithelial cycle of spermatogenesis. We also propose a hypothetic model by which palladin regulates ES restructuring during the epithelial cycle of spermatogenesis.

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.

QS296. Differential Expression of Palladin in Breast Cancer and Its Potential Role in Tumor Metastasis

Introduction: Palladin is a cytoskeletal protein involved in actin organization and cell motility. Recent research showed that a mutation in palladin is linked to a form of familial pancreatic cancer, and that palladin may be overexpressed in sporadic pancreatic cancers. The role of palladin in breast cancer has not been well elucidated. Previous work suggests that palladin expression is increased in phenotypically invasive breast cancer cells, while others report a decrease in palladin mRNA levels in metastatic lesions.

The role of palladin in actin organization and cell motility

The role of palladin in actin organization and cell motility