Polo-like kinase 1 (PLK1) is a key cell cycle regulator implicated in the development of various cancers, including prostate cancer. However, the functions of PLK1 beyond cell cycle regulation remain poorly characterized. Here, we report that PLK1 overexpression in prostate epithelial cells triggers oncogenic transformation. It also results in dramatic transcriptional reprogramming of the cells, leading to epithelial-to-mesenchymal transition (EMT) and stimulation of cell migration and invasion. Consistently, PLK1 downregulation in metastatic prostate cancer cells enhances epithelial characteristics and inhibits cell motility. The signaling mechanisms underlying the observed cellular effects of PLK1 involve direct PLK1-dependent phosphorylation of CRAF with subsequent stimulation of the MEK1/2-ERK1/2-Fra1-ZEB1/2 signaling pathway. Our findings highlight novel non-canonical functions of PLK1 as a key regulator of EMT and cell motility in normal prostate epithelium and prostate cancer. This study also uncovers a previously unanticipated role of PLK1 as a potent activator of MAPK signaling.

The morphogenesis of mammalian embryonic external genitalia (eExG) shows dynamic differences between males and females. In genotypic males, eExG are masculinized in response to androgen signaling. Disruption of this process can give rise to multiple male reproductive organ defects. Currently, mechanisms of androgen-driven sexually dimorphic organogenesis are still unclear. We show here that mesenchymal-derived actomyosin contractility, by MYH10, is essential for the masculinization of mouse eExG. MYH10 is expressed prominently in the bilateral mesenchyme of male eExG. Androgen induces MYH10 protein expression and actomyosin contractility in the bilateral mesenchyme. Inhibition of actomyosin contractility through blebbistatin treatment and mesenchymal genetic deletion induced defective urethral masculinization with reduced mesenchymal condensation. We also suggest that actomyosin contractility regulates androgen-dependent mesenchymal directional cell migration to form the condensation in the bilateral mesenchyme leading to changes in urethral plate shape to accomplish urethral masculinization. Thus, mesenchymal-derived actomyosin contractility is indispensable for androgen-driven urethral masculinization.

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis.

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

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMIIA and NMIIB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules.

During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts versus the need to traverse an entire organ. Here we show that in vivo, myosin IIA-deficient T cells had a triad of defects, including overadherence to high-endothelial venules, less interstitial migration and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of three-dimensional motility in microchannels showed that the degree of confinement and myosin IIA function, rather than integrin adhesion (as proposed by the haptokinetic model), optimized motility rate. This motility occurred via a myosin IIA-dependent rapid 'walking' mode with multiple small and simultaneous adhesions to the substrate, which prevented spurious and prolonged adhesions. Adhesion discrimination provided by myosin IIA is thus necessary for the optimization of motility through complex tissues.

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell-matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell-matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell-matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell-matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell-matrix adhesion.

Formation of the epithelial barrier and apico-basal cell polarity represent two characteristics and mutually dependent features of differentiated epithelial monolayers. They are controlled by special adhesive structures, tight junctions (TJs), and polarity protein complexes that define the apical and the basolateral plasma membrane. The functional interplay between TJs and polarity complexes remains poorly understood. We investigated the role of Scribble, a basolateral polarity protein and known tumor suppressor, in regulating TJs in human intestinal epithelium. Scribble was enriched at TJs in T84 and SK-CO15 intestinal epithelial cell monolayers and sections of normal human colonic mucosa. siRNA-mediated knockdown of Scribble in SK-CO15 cells attenuated development of epithelial barrier and inhibited TJ reassembly independently of other basolateral polarity proteins Lgl-1 and Dlg-1. Scribble selectively co-imunoprecipitated with TJ protein ZO-1, and ZO-1 was important for Scribble recruitment to intercellular junctions and TJ reassembly. Lastly, Scribble was mislocalized from TJs and its expression down-regulated in interferon-gamma-treated T84 cell monolayers and inflamed human intestinal mucosa in vivo. We conclude that Scribble is an important regulator of TJ functions and plasticity in the intestinal epithelium. Down-regulation of Scribble may mediate mucosal barrier breakdown during intestinal inflammation.

Epithelial cell motility is critical for self-rejuvenation of normal intestinal mucosa, wound repair, and cancer metastasis. This process is regulated by the reorganization of the F-actin cytoskeleton, which is driven by a myosin II motor. However, the role of myosin II in regulating epithelial cell migration remains poorly understood. This study addressed the role of non-muscle myosin (NM) IIA in two different modes of epithelial cell migration: two-dimensional (2-D) migration that occurs during wound closure and three-dimensional (3-D) migration through a Matrigel matrix that occurs during cancer metastasis. Pharmacological inhibition or siRNA-mediated knockdown of NM IIA in SK-CO15 human colonic epithelial cells resulted in decreased 2-D migration and increased 3-D invasion. The attenuated 2-D migration was associated with increased cell adhesiveness to collagen and laminin and enhanced expression of beta1-integrin and paxillin. On the 2-D surface, NM IIA-deficient SK-CO15 cells failed to assemble focal adhesions and F-actin stress fibers. In contrast, the enhanced invasion of NM IIA-depleted cells was dependent on Raf-ERK1/2 signaling pathway activation, enhanced calpain activity, and increased calpain-2 expression. Our findings suggest that NM IIA promotes 2-D epithelial cell migration but antagonizes 3-D invasion. These observations indicate multiple functions for NM IIA, which, along with the regulation of the F-actin cytoskeleton and cell-matrix adhesions, involve previously unrecognized control of intracellular signaling and protein expression.

Disruption of epithelial cell-cell adhesions represents an early and important stage in tumor metastasis. This process can be modeled in vitro by exposing cells to chemical tumor promoters, phorbol esters and octylindolactam-V (OI-V), known to activate protein kinase C (PKC). However, molecular events mediating PKC-dependent disruption of epithelial cell-cell contact remain poorly understood. In the present study we investigate mechanisms by which PKC activation induces disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium.

The integrity and function of the epithelial barrier is dependent on the apical junctional complex (AJC) composed of tight and adherens junctions and regulated by the underlying actin filaments. A major F-actin motor, myosin II, was previously implicated in regulation of the AJC, however direct evidence of the involvement of myosin II in AJC dynamics are lacking and the molecular identity of the myosin II motor that regulates formation and disassembly of apical junctions in mammalian epithelia is unknown. We investigated the role of nonmuscle myosin II (NMMII) heavy chain isoforms, A, B, and C in regulation of epithelial AJC dynamics and function. Expression of the three NMMII isoforms was observed in model intestinal epithelial cell lines, where all isoforms accumulated within the perijunctional F-actin belt. siRNA-mediated downregulation of NMMIIA, but not NMMIIB or NMMIIC expression in SK-CO15 colonic epithelial cells resulted in profound changes of cell morphology and cell-cell adhesions. These changes included acquisition of a fibroblast-like cell shape, defective paracellular barrier, and substantial attenuation of the assembly and disassembly of both adherens and tight junctions. Impaired assembly of the AJC observed after NMMIIA knock-down involved dramatic disorganization of perijunctional actin filaments. These findings provide the first direct non-pharmacological evidence of myosin II-dependent regulation of AJC dynamics in mammalian epithelia and highlight a unique role of NMMIIA in junctional biogenesis.

All phases of lipopolysaccharide (LPS)-induced fever are mediated by prostaglandin (PG) E2. It is known that the second febrile phase (which starts at approximately 1.5 h post-LPS) and subsequent phases are mediated by PGE2 that originated in endotheliocytes and perivascular cells of the brain. However, the location and phenotypes of the cells that produce PGE2 triggering the first febrile phase (which starts at approximately 0.5 h) remain unknown. By studying PGE2 synthesis at the enzymatic level, we found that it was activated in the lung and liver, but not in the brain, at the onset of the first phase of LPS fever in rats. This activation involved phosphorylation of cytosolic phospholipase A2 (cPLA2) and transcriptional up-regulation of cyclooxygenase (COX)-2. The number of cells displaying COX-2 immunoreactivity surged in the lung and liver (but not in the brain) at the onset of fever, and the majority of these cells were identified as macrophages. When PGE2 synthesis in the periphery was activated, the concentration of PGE2 increased both in the venous blood (which collects PGE2 from tissues) and arterial blood (which delivers PGE2 to the brain). Most importantly, neutralization of circulating PGE2 with an anti-PGE2 antibody both delayed and attenuated LPS fever. It is concluded that fever is initiated by circulating PGE2 synthesized by macrophages of the LPS-processing organs (lung and liver) via phosphorylation of cPLA2 and transcriptional up-regulation of COX-2. Whether PGE2 produced at the level of the blood-brain barrier also contributes to the development of the first phase remains to be clarified.

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

Breast cancer metastasis is driven by a profound remodeling of the cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules, and septin polymers. It is markedly overexpressed in breast cancer, and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer cell migration, growth, and metastasis.

Melanoma differentiation associated gene-7 (mda-7/IL-24) is a member of the IL-10 family of cytokines, with ubiquitous direct and "bystander" tumor-selective killing properties. MDA-7/IL-24 protein binds distinct type II cytokine heterodimeric receptor complexes, IL-20R1/IL-20R2, IL-22R1/IL-20R1 and IL-22R1/IL-20R2. Recombinant MDA-7/IL-24 protein induces endogenous mda-7/IL-24 expression in a receptor-dependent manner; since A549 cells that lack a complete set of cognate receptors are not responsive to exogenous protein. The mechanism of MDA-7/IL-24 ligand-receptor biology is not well understood. We explored the interaction of MDA-7/IL-24 with its' receptors and the consequences of ligand-receptor docking. Using both pharmacological and genetic approaches we demonstrate that MDA-7/IL-24 internalization employs the clathrin-mediated endocytic pathway leading to degradation of receptors via the lysosomal/ubiquitin proteosomal pathway. This clathrin-mediated endocytosis is dynamin-dependent. This study resolves a novel mechanism of MDA-7/IL-24 protein "bystander" function, which involves receptor/protein-mediated internalization and receptor degradation.

Ablation of nonmuscle myosin (NM) II-A or NM II-B results in mouse embryonic lethality. Here, we report the results of ablating NM II-C as well as NM II-C/II-B together in mice. NM II-C ablated mice survive to adulthood and show no obvious defects compared with wild-type littermates. However, ablation of NM II-C in mice expressing only 12% of wild-type amounts of NM II-B results in a marked increase in cardiac myocyte hypertrophy compared with the NM II-B hypomorphic mice alone. In addition, these hearts develop interstitial fibrosis associated with diffuse N-cadherin and β-catenin localization at the intercalated discs, where both NM II-B and II-C are normally concentrated. When both NM II-C and II-B are ablated the B-C-/B-C- cardiac myocytes show major defects in karyokinesis. More than 90% of B-C-/B-C- myocytes demonstrate defects in chromatid segregation and mitotic spindle formation accompanied by increased stability of microtubules and abnormal formation of multiple centrosomes. This requirement for NM II in karyokinesis is further demonstrated in the HL-1 cell line derived from mouse atrial myocytes, by using small interfering RNA knockdown of NM II or treatment with the myosin inhibitor blebbistatin. Our study shows that NM II is involved in regulating cardiac myocyte karyokinesis by affecting microtubule dynamics.

To investigate the contribution of nonmuscle myosin II-A (NM II-A) to early cardiac development we crossed Myh9 floxed mice and Nkx2.5 cre-recombinase mice. Nkx2.5 is expressed in the early heart (E7.5) and later in the tongue epithelium. Mice homozygous for deletion of NM II-A (A(Nkx)/A(Nkx)) are born at the expected ratio with normal hearts, but consistently develop an invasive squamous cell carcinoma (SCC) of the tongue (32/32 A(Nkx)/A(Nkx)) as early as E17.5. To assess reproducibility a second, independent line of Myh9 floxed mice derived from a different embryonic stem cell clone was tested. This second line also develops SCC indistinguishable from the first (15/15). In A(Nkx)/A(Nkx) mouse tongue epithelium, genetic deletion of NM II-A does not affect stabilization of TP53, unlike a previous report for SCC. We attribute the consistent, early formation of SCC with high penetrance to the role of NM II in maintaining mitotic stability during karyokinesis.

Here we identify and characterize a cytoskeletal myosin protein required for IRE1α oligomerization, activation, and signaling. Proteomic screening identified nonmuscle myosin heavy chain IIB (NMHCIIB), a subunit of nonmuscle myosin IIB (NMIIB), as an ER stress-dependent interacting protein specific to IRE1α. Loss of NMIIB compromises XBP1s and UPR target gene expression with no effect on the PERK pathway. Mechanistically, NMIIB is required for IRE1α aggregation and foci formation under ER stress. The NMIIB-mediated effect on IRE1α signaling is in part dependent on the phosphorylation of myosin regulatory light chain and the actomyosin contractility of NMIIB. Biologically, the function of NMIIB in ER stress response is conserved as both mammalian cells and C. elegans lacking NMIIB exhibit hypersensitivity to ER stress. Thus, optimal IRE1α activation and signaling require concerted coordination between the ER and cytoskeleton.