Coupling adipose tissue architecture and metabolism via cytoophidia

ABSTRACTTissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here, we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Interestingly, loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, leads to downregulated Collagen-Integrin signaling, weakened adipocyte adhesion, and defective adipose architecture. Strikingly, CTPS specifically binds with Integrin subunit α2, which influences Integrin function and Collagen IV deposition. cytoophidia promote Collagen IV mRNA expression and thus its extracellular deposition to strengthen adipocyte adhesion. Remarkably, Collagen IV-Integrin signaling reciprocally regulates cytoophidium formation at a post-translational level. Together, we demonstrate that a positive feedback signaling loop containing both cytoophidia and Integrin adhesion complex couples tissue architecture and metabolism in the fly adipose.

The Mechanosensing and Global DNA Methylation of Human Osteoblasts on MEW Fibers

Cells interact with 3D fibrous platform topography via a nano-scaled focal adhesion complex, and more research is required on how osteoblasts sense and respond to random and aligned fibers through nano-sized focal adhesions and their downstream events. The present study assessed human primary osteoblast cells’ sensing and response to random and aligned medical-grade polycaprolactone (PCL) fibrous 3D scaffolds fabricated via the melt electrowriting (MEW) technique. Cells cultured on a tissue culture plate (TCP) were used as 2D controls. Compared to 2D TCP, 3D MEW fibrous substrates led to immature vinculin focal adhesion formation and significantly reduced nuclear localization of the mechanosensor-yes-associated protein (YAP). Notably, aligned MEW fibers induced elongated cell and nucleus shape and highly activated global DNA methylation of 5-methylcytosine, 5-hydroxymethylcytosine, and N-6 methylated deoxyadenosine compared to the random fibers. Furthermore, although osteogenic markers (osterix-OSX and bone sialoprotein-BSP) were significantly enhanced in PCL-R and PCL-A groups at seven days post-osteogenic differentiation, calcium deposits on all seeded samples did not show a difference after normalizing for DNA content after three weeks of osteogenic induction. Overall, our study linked 3D extracellular fiber alignment to nano-focal adhesion complex, nuclear mechanosensing, DNA epigenetics at an early point (24 h), and longer-term changes in osteoblast osteogenic differentiation.

Specific N-glycans regulate an extracellular adhesion complex during somatosensory dendrite patterning

N-glycans are molecularly diverse sugars borne by over 70% of proteins transiting the secretory pathway and have been implicated in protein folding, stability, and localization. Mutations in genes important for N-glycosylation result in congenital disorders of glycosylation that are often associated with intellectual disability. Here, we show that structurally distinct N-glycans regulate the activity of an extracellular protein complex involved in patterning of somatosensory dendrites in Caenorhabditis elegans. Specifically, aman-2/Golgi alpha-mannosidase II, a conserved key enzyme in the biosynthesis of specific N-glycans regulates the activity of the Menorin adhesion complex without obviously affecting protein stability and localization of its components. AMAN-2 functions cell-autonomously to ensure decoration of the neuronal transmembrane receptor DMA-1/LRR-TM with high-mannose/hybrid N-glycans. Moreover, distinct types of N-glycans on specific N-glycosylation sites regulate the DMA-1/LRR-TM receptor, which together with three other extracellular proteins forms the Menorin adhesion complex. In summary, specific N-glycan structures regulate dendrite patterning by coordinating the activity of an extracellular adhesion complex suggesting that the molecular diversity of N-glycans can contribute to developmental specificity in the nervous system.

Synergistic Phase Separation of Two Pathways Promotes Integrin Clustering and Integrin Adhesion Complex Formation

Integrin adhesion complexes (IACs) are integrin-based plasma membrane-associated comp iartments where cells sense environmental cues. The physical mechanisms and molecular interactions that mediate nascent IAC formation are unclear. We found that both p130Cas ("Cas") and Focal adhesion kinase ("FAK") undergo liquid-liquid phase separation in vitro under physiologic conditions. Cas- and FAK- driven phase separation is sufficient to reconstitute kindlindependent integrin clustering in vitro. In vitro condensates and cellular IACs exhibit similar sensitivities to environmental perturbations including changes in temperature and pH. Furthermore, mutations that inhibit or enhance phase separation in vitro reduce or increase the number of IACs in cells, respectively. Finally, we find that the Cas and FAK pathways act synergistically to promote phase separation, integrin clustering and IAC formation in vitro and in cells. We propose that Cas- and FAK- driven phase separation provides an intracellular trigger for integrin clustering and nascent IAC formation.

Quantification of fast molecular adhesion by fluorescence footprinting

Rolling adhesion is a unique process in which the adhesion events are short-lived and operate under highly nonequilibrium conditions. These characteristics pose a challenge in molecular force quantification, where in situ measurement of these forces cannot be achieved with molecular force sensors that probe near equilibrium. Here, we demonstrated a quantitative adhesion footprint assay combining DNA-based nonequilibrium force probes and modeling to measure the molecular force involved in fast rolling adhesion. We were able to directly profile the ensemble molecular force distribution in our system during rolling adhesion with a dynamic range between 0 and 18 pN. Our results showed that the shear stress driving bead rolling motility directly controls the molecular tension on the probe-conjugated adhesion complex. Furthermore, the shear stress can steer the dissociation bias of components within the molecular force probe complex, favoring either DNA probe dissociation or receptor-ligand dissociation.

Integrin Adhesion Complex Organization in Sheep Myometrium Reflects Changing Mechanical Forces during Pregnancy and Postpartum

Cells respond to extracellular mechanical forces through the assembly of integrin adhesion complexes (IACs) that provide a scaffold through which cells sense and transduce responses to those forces. IACs are composed of transmembrane integrin receptors that bind to extracellular matrix (ECM) proteins externally and connect with the actomyosin cytoskeleton internally. Myometrial smooth muscle cells respond to forces that arise due to increases in fetal growth/weight, placental fluid volumes, and blood flow. As a result, the uterus transforms into an organ that can forcefully expel the fetus and placental membranes during parturition. While earlier studies focused on IAC expression in the myometrial compartment of rodents and humans to explore pregnancy-associated responses, the present study examines IAC assembly in ovine myometrium where mechanical forces are expected to be amplified in a manner similar to humans. Results indicate that the ITGA5 and ITGB1 heterodimers associate with the ECM protein FN1 externally, and with VCL and TLN1 internally, to form IACs in myometrial cells during the first trimester of pregnancy. These IACs become increasingly ordered until parturition. This ordered structure is lost by one day postpartum; however, the abundance of the integrin proteins remains elevated for at least two weeks postpartum. Implications of the present study are that sheep are similar to humans regarding the assembly of IACs in the pregnant myometrium and suggest that IACs may form much earlier in human gestation than was previously implied by the rat model. Results highlight the continued value of the sheep model as a flagship gynecological model for understanding parturition in humans.

Reactive Oxygen Species Generated by Polycyclic Aromatic Hydrocarbons From Ambient Particulate Matter Enhance Vascular Smooth Muscle Cell Migration Through MMP Upregulation and Actin Reorganization

BackgroundEpidemiological studies have suggested that elevated concentrations of particulate matter (PM) are strongly associated with the incidence of atherosclerosis; however, the underlying cellular and molecular mechanisms of atherosclerosis by PM exposure and the components that are mainly responsible for this adverse effect remain to be established. In this investigation, we evaluated the effects of ambient PM on vascular smooth muscle cell (VSMC) behavior. In addition, the effects of polycyclic aromatic hydrocarbons (PAHs), including oxygenated PAHs (oxy-PAHs), on VSMC migration and the underlying mechanisms were examined.ResultsVSMC migration was significantly increased by treatment with ambient PM. The total amount of PAHs contained in WPM was higher than that in SPM, leading to higher ROS generation and VSMC migration. The increased migration was successfully inhibited by treatment with the ROS scavenger, N-acetyl-cysteine (NAC). The levels of matrix metalloproteinase (MMP) 2 and 9 were significantly increased in ambient PM-treated VSMCs, with MMP9 levels being significantly higher in WPM-treated VSMCs than in those treated with SPM. As expected, migration was significantly increased in all tested PAHs (anthracene, ANT; benz(a)anthracene, BaA) and their oxygenated derivatives (9,10-Anthraquinone, AQ; 7,12-benz(a)anthraquinone, BAQ, respectively). The phosphorylated levels of focal adhesion kinase (FAK) and formation of the focal adhesion complex were significantly increased in ambient PM or PAH-treated VSMCs, and these effects were blocked by administration of NAC or a-NF, an inhibitor of AhR, the receptor that allows PAH uptake. Subsequently, the levels of phosphorylated Src and NRF, the downstream targets of FAK, were altered with a pattern similar to that of p-FAK.ConclusionsPAHs, including oxy-PAHs, in ambient PM may have dual effects that lead to an increase in VSMC migration. One is the generation of ROS followed by MMP upregulation, and the other is actin reorganization that results from the activation of the focal adhesion complex.

Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells

Glioblastomas are the most common and aggressive primary brain tumors in adults, and patients with glioblastoma have a median survival of 15 months. Some alternative therapies, such as Src family kinase inhibitors, have failed presumably because other signaling pathways compensate for their effects. In the last ten years, it has been proven that sex hormones such as progesterone (P4) can induce growth, migration, and invasion of glioblastoma cells through its intracellular progesterone receptor (PR), which is mostly known for its role as a transcription factor, but it can also induce non-genomic actions. These non-classic actions are, in part, a consequence of its interaction with cSrc, which plays a significant role in the progression of glioblastomas. We studied the relation between PR and cSrc, and its effects in human glioblastoma cells. Our results showed that P4 and R5020 (specific PR agonist) activated cSrc protein since both progestins increased the p-cSrc (Y416)/cSrc ratio in U251 and U87 human glioblastoma derived cell lines. When siRNA against the PR gene was used, the activation of cSrc by P4 was abolished. The co-immunoprecipitation assay showed that cSrc and PR interact in U251 cells. P4 treatment also promoted the increase in the p-Fak (Y397) (Y576/577)/Fak and the decrease in p-Paxillin (Y118)/Paxillin ratio, which are significant components of the focal adhesion complex and essential for migration and invasion processes. A siRNA against cSrc gene blocked the increase in the p-Fak (Y576/Y577)/Fak ratio and the migration induced by P4, but not the decrease in p-Paxillin (Y118)/Paxillin ratio. We analyzed the potential role of cSrc over PR phosphorylation in three databases, and one putative tyrosine residue in the amino acid 87 of PR was found. Our results showed that P4 induces the activation of cSrc protein through its PR. The latter and cSrc could interact in a bidirectional mode for regulating the activity of proteins involved in migration and invasion of glioblastomas.

Altered Actin Dynamics in Cell Migration of GNE Mutant Cells

Cell migration is an essential cellular process that requires coordination of cytoskeletal dynamics, reorganization, and signal transduction. The actin cytoskeleton is central in maintaining the cellular structure as well as regulating the mechanisms of cell motility. Glycosylation, particularly sialylation of cell surface proteins like integrins, regulates signal transduction from the extracellular matrix to the cytoskeletal network. The activation of integrin by extracellular cues leads to recruitment of different focal adhesion complex proteins (Src, FAK, paxillin, etc.) and activates the signal including Rho GTPases for the regulation of actin assembly and disassembly. During cell migration, the assembly and disassembly of actin filament provides the essential force for the cell to move. Abnormal sialylation can lead to actin signaling dysfunction leading to aberrant cell migration, one of the main characteristics of cancer and myopathies. In the present study, we have reported altered F-actin to G-actin ratios in GNE mutated cells. These cells exhibit pathologically relevant mutations of GNE (UDP N-acetylneuraminic 2-epimerase/N-acetylmannosamine kinase), a key sialic acid biosynthetic enzyme. It was found that GNE neither affects the actin polymerization nor binds directly to actin. However, mutation in GNE resulted in increased binding of α-actinin to actin filaments. Further, through confocal imaging, GNE was found to be localized in focal adhesion complex along with paxillin. We further elucidated that mutation in GNE resulted in upregulation of RhoA protein and Cofilin activity is downregulated, which could be rescued with Rhosin and chlorogenic acid, respectively. Lastly, mutant in GNE reduced cell migration as implicated from wound healing assay. Our study indicates that molecules altering Cofilin function could significantly revert the cell migration defect due to GNE mutation in sialic acid-deficient cells. We propose cytoskeletal proteins to be alternate drug targets for disorders associated with GNE such as GNE myopathy.

A novel adhesive complex at the base of intestinal microvilli

Intestinal epithelial cells form dense arrays of microvilli at the apical membrane to enhance their functional capacity. Microvilli contain a protocadherin-based intermicrovillar adhesion complex localized at their tips which regulates microvillar length and packaging. Here, we identify a second adhesive complex in microvilli of intestinal epithelial cells. This complex is localized at the basal region of microvilli and consists of the adhesion molecule TMIGD1, the phosphoprotein EBP50 and the F-actin – plasma membrane cross-linking protein ezrin. Ternary complex formation requires unmasking of the EBP50 PDZ domains by ezrin binding and is strongly enhanced upon mutating Ser162 located in PDZ domain 2 of EBP50. Dephosphorylation of EBP50 at S162 is mediated by PP1α, a serine/threonine phosphatase localized at the microvillar base and involved in ezrin phosphocycling. Importantly, the binding of EBP50 to TMIGD1 enhances the dynamic turnover of EBP50 at microvilli in a Ser162 phosphorylation-dependent manner. We identify an adhesive complex at the microvillar base and propose a potential mechanism that regulates microvillar dynamics in enterocytes.