Intercellular Adhesion Molecule-1 Enhances Myonuclear Transcription during Injury-Induced Muscle Regeneration

The local inflammatory environment of injured skeletal muscle contributes to the resolution of the injury by promoting the proliferation of muscle precursor cells during the initial stage of muscle regeneration. However, little is known about the extent to which the inflammatory response influences the later stages of regeneration when newly formed (regenerating myofibers) are accumulating myonuclei and undergoing hypertrophy. Our prior work indicated that the inflammatory molecule ICAM-1 facilitates regenerating myofiber hypertrophy through a process involving myonuclear positioning and/or transcription. The present study tested the hypothesis that ICAM-1 enhances global transcription within regenerating myofibers by augmenting the transcriptional activity of myonuclei positioned in linear arrays (nuclear chains). We found that transcription in regenerating myofibers was ~2-fold higher in wild type compared with ICAM-1-/- mice at 14 and 28 days post-injury. This occurred because the transcriptional activity of individual myonuclei in nuclei chains, nuclear clusters, and a peripheral location were ~2-fold higher in wild type compared with ICAM-1-/- mice during regeneration. ICAM-1’s enhancement of transcription in nuclear chains appears to be an important driver of myofiber hypertrophy as it was statistically associated with an increase in myofiber size during regeneration. Taken together, our findings indicate that ICAM-1 facilitates myofiber hypertrophy after injury by enhancing myonuclear transcription

A microwave-assisted solution combustion synthesis to produce europium-doped calcium phosphate nanowhiskers for bioimaging applications

Biocompatible nanoparticles possessing fluorescent properties offer attractive possibilities for multifunctional bioimaging and/or drug and gene delivery applications. Many of the limitations with current imaging systems center on the properties of the optical probes in relation to equipment technical capabilities. Here we introduce a novel high aspect ratio and highly crystalline europium-doped calcium phosphate nanowhisker produced using a simple microwave-assisted solution combustion synthesis method for use as a multifunctional bioimaging probe. X-ray diffraction confirmed the material phase as europium-doped hydroxyapatite. Fluorescence emission and excitation spectra and their corresponding peaks were identified using spectrofluorimetry and validated with fluorescence, confocal and multiphoton microscopy. The nanowhiskers were found to exhibit red and far red wavelength fluorescence under ultraviolet excitation with an optimal peak emission of 696 nm achieved with a 350 nm excitation. Relatively narrow emission bands were observed, which may permit their use in multicolor imaging applications. Confocal and multiphoton microscopy confirmed that the nanoparticles provide sufficient intensity to be utilized in imaging applications

Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts—An Indicator for Breast Carcinogenesis

Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer

Novel 3D Flipwell system that models gut mucosal microenvironment for studying interactions between gut microbiota, epithelia and immunity

Abstract Gut mucosa consists of stratified layers of microbes, semi-permeable mucus, epithelium and stroma abundant in immune cells. Although tightly regulated, interactions between gut commensals and immune cells play indispensable roles in homeostasis and cancer pathogenesis in the body. Thus, there is a critical need to develop a robust model for the gut mucosal microenvironment. Here, we report our novel co-culture utilizing 3D Flipwell system for establishing the stratified layers of discrete mucosal components. This method allows for analyzing synchronous effects of test stimuli on gut bacteria, mucus, epithelium and immune cells, as well as their crosstalks. In the present report, we tested the immuno-stimulatory effects of sepiapterin (SEP, the precursor of the cofactor of nitric oxide synthase (NOS)—BH4) on the gut mucosal community. We previously reported that SEP effectively reprogrammed tumor-associated macrophages and inhibited breast tumor cell growth. In our co-cultures, SEP largely promoted mucus integrity, bacterial binding, and M1-like polarization of macrophages. Conversely, these phenomena were absent in control-treated cultures. Our results demonstrate that this novel co-culture may serve as a robust in vitro system to recapitulate the effects of pharmacological agents on the gut mucosal microenvironment, and could potentially be expanded to test the effects outside the gut

Dia-Interacting Protein (DIP) Imposes Migratory Plasticity in mDia2-Dependent Tumor Cells in Three-Dimensional Matrices

<div><p>Tumor cells rely upon membrane pliancy to escape primary lesions and invade secondary metastatic sites. This process relies upon localized assembly and disassembly cycles of F-actin that support and underlie the plasma membrane. Dynamic actin generates both spear-like and bleb structures respectively characterizing mesenchymal and amoeboid motility programs utilized by metastatic cells in three-dimensional matrices. The molecular mechanism and physiological trigger(s) driving membrane plasticity are poorly understood. mDia formins are F-actin assembly factors directing membrane pliancy in motile cells. mDia2 is functionally coupled with its binding partner DIP, regulating cortical actin and inducing membrane blebbing in amoeboid cells. Here we show that mDia2 and DIP co-tether to nascent blebs and this linkage is required for bleb formation. DIP controls mesenchymal/amoeboid cell interconvertability, while CXCL12 induces assembly of mDia2:DIP complexes to bleb cortices in 3D matrices. These results demonstrate how DIP-directed mDia2-dependent F-actin dynamics regulate morphological plasticity in motile cancer cells.</p> </div

mDia2:DIP-driven mechanism for bleb and lobopodia formation: bulbous structures driving amoeboid motility in 3D matrices.

<p>1. Cortical Maintenance: activated mDia2 maintains the F-actin cortex underlying the plasma membrane (PM); 2. Cortical disruption and bleb initiation: DIP binding to the mDia2 FH2 domain disrupts F-actin assembly and bundling, initiating cortical weakening and bleb expansion; 3. Re-establishing the bleb F-actin cortex: Upon bleb expansion, mDia2 enters the bleb and through interaction with activated Rho GTPases, re-establishes the F-actin cortex underlying the bleb; and 4a. Bleb retraction ensues, driven, in part, by ROCK and myosin-based contractility; 4b. Compound bleb formation: DIP binding the mDia2 FH2 domain at the rim of the bleb (noted as * in the inset) initiates a secondary break in the bleb cortex, leading to compound bleb, or lobopodia formation.</p

An mDia2/ROCK Signaling Axis Regulates Invasive Egress from Epithelial Ovarian Cancer Spheroids

<div><p>Multi-cellular spheroids are enriched in ascites of epithelial ovarian cancer (OvCa) patients. They represent an invasive and chemoresistant cellular population fundamental to metastatic dissemination. The molecular mechanisms triggering single cell invasive egress from spheroids remain enigmatic. mDia formins are Rho GTPase effectors that are key regulators of F-actin cytoskeletal dynamics. We hypothesized that mDia2-driven F-actin dynamics promote single cell invasive transitions in clinically relevant three-dimensional (3D) OvCa spheroids. The current study is a dissection of the contribution of the F-actin assembly factor mDia2 formin in invasive transitions and using a clinically relevant ovarian cancer spheroid model. We show that RhoA-directed mDia2 activity is required for tight spheroid organization, and enrichment of mDia2 in the invasive cellular protrusions of collagen-embedded OVCA429 spheroids. Depleting mDia2 in ES-2 spheroids enhanced invasive dissemination of single amoeboid-shaped cells. This contrasts with spheroids treated with control siRNA, where a mesenchymal invasion program predominated. Inhibition of another RhoA effector, ROCK, had no impact on ES-2 spheroid formation but dramatically inhibited spheroid invasion through induction of a highly elongated morphology. Concurrent inhibition of ROCK and mDia2 blocked single cell invasion from ES-2 spheroids more effectively than inhibition of either protein alone, indicating that invasive egress of amoeboid cells from mDia2-depleted spheroids is ROCK-dependent. Our findings indicate that multiple GTPase effectors must be suppressed in order to fully block invasive egress from ovarian cancer spheroids. Furthermore, tightly regulated interplay between ROCK and mDia2 signaling pathways dictates the invasive capacities and the type of invasion program utilized by motile spheroid-derived ovarian cancer cells. As loss of the gene encoding mDia2, <i>DRF3,</i> has been linked to cancer progression and metastasis, our results set the stage for understanding molecular mechanisms involved in mDia2-dependent egress of invasive cells from primary epithelial tumors.</p></div

CXCL12 induces an mDia2:DIP complex and membrane blebbing.

<p>(A) MDA-MB-231 cells were plated upon glass coverslips and stimulated for the indicated times with 30 ng/ml CXCL12. Blebbing cells were enumerated in shown triplicate experiments where n>54. Inset: representative blebs in a fixed cell stained with phalloidin. (B) Cells transfected with the designated constructs were stimulated with 25 ng/ml CXCL12. Lysates were co-immunoprecipitated for DIP-associated mDia2 upon CXCL12 stimulation. Tubulin is shown as an i.p input loading control. (C, D) Serum-starved MDA-MB-231 cells were adhered to glass coverslips and stimulated for 30 min with 30 ng/ml CXCL12 before fixation. Endogenous DIP, mDia2 and the F-actin architecture are shown using a 63x oil objective.</p

DIP LRR induces amoeboid motility in invading mesenchymal breast cancer cells.

<p>(A, B) MDA-MB-231 cells were treated with DMSO vehicle, 20 µm MMP-inhibitor GM6001 or were transfected with the indicated CFP-fusion proteins and were embedded in type-I collagen gels and stained as above. Insets validate the CFP-transfection. (C) Elongation indices were calculated as in 3C. (D) MDA-MB-231 cells transfected with either CFP-DIP LRR or CFP-DIP LRR N555A for 48 hrs were embedded in collagen gels overnight, stained with phalloidin and anti-pMLC2 antibodies. (E) MDA-MB-231 cells transfected for 48 hrs with CFP, CFP-DIP LRR or CFP-DIP LRR treated with 90 µM Y27632 reverse-invaded for 24 hrs through transwells coated with collagen I gels. Upon fixation, transwells were imaged by confocal microscopy. CFP pixels were calculated for 10 µm optical slices.</p