Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling.

Signaling via the T cell antigen receptor (TCR) is initiated by Src-family kinases (SFKs). To understand how the kinase Csk, a negative regulator of SFKs, controls the basal state and the initiation of TCR signaling, we generated mice that express a Csk variant sensitive to an analog of the common kinase inhibitor PP1 (Csk(AS)). Inhibition of Csk(AS) in thymocytes, without engagement of the TCR, induced potent activation of SFKs and proximal TCR signaling up to phospholipase C-γ1 (PLC-γ1). Unexpectedly, increases in inositol phosphates, intracellular calcium and phosphorylation of the kinase Erk were impaired. Altering the actin cytoskeleton pharmacologically or providing costimulation via CD28 'rescued' those defects. Thus, Csk has a critical role in preventing TCR signaling. However, our studies also revealed a requirement for actin remodeling, initiated by costimulation, for full TCR signaling.

The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Active superelasticity in three-dimensional epithelia of controlled shape.

Fundamental biological processes are carried out by curved epithelial sheets that enclose a pressurized lumen. How these sheets develop and withstand three-dimensional deformations has remained unclear. Here we combine measurements of epithelial tension and shape with theoretical modelling to show that epithelial sheets are active superelastic materials. We produce arrays of epithelial domes with controlled geometry. Quantification of luminal pressure and epithelial tension reveals a tensional plateau over several-fold areal strains. These extreme strains in the tissue are accommodated by highly heterogeneous strains at a cellular level, in seeming contradiction to the measured tensional uniformity. This phenomenon is reminiscent of superelasticity, a behaviour that is generally attributed to microscopic material instabilities in metal alloys. We show that in epithelial cells this instability is triggered by a stretch-induced dilution of the actin cortex, and is rescued by the intermediate filament network. Our study reveals a type of mechanical behaviour-which we term active superelasticity-that enables epithelial sheets to sustain extreme stretching under constant tension.

CD8αα⁺ innate-type lymphocytes in the intestinal epithelium mediate mucosal immunity.

Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.

Upregulation of girdin delays endothelial cell apoptosis via promoting engulfment of platelets.

BACKGROUND: Endothelial cells are crucial in maintaining the homeostasis of the blood-brain barrier. Girders of actin filament (Girdin) and phosphor (p)-Girdin are essential for the engulfment of human brain microvascular endothelial cells (HBMECs) into platelets (PLTs), but the potential mechanism remains unclear and requires further study. METHODS: Following PLT and cytochalasin D treatment, Hoechst 33,342 detected apoptosis. The transfection efficiency of the short hairpin RNA targeting Girdin (sh-Girdin) or overexpressing Girdin (OE-Girdin) was determined using western blotting. Sh-Girdin, OE-Girdin, mutated Girdin (m-Girdin), and microfilament binding region deleted Girdin (Del-Girdin) were transfected into HBMECs under PLT conditions. Subsequently, the engulfment of HBMECs by PLTs was detected by flow cytometry and transmission electron microscopy. Girdin and phosphorylated (p)-Girdin levels were quantified by western blot. The positive expression of Girdin was measured by immunohistochemistry (IHC). The localization of PLT, Girdin, and p-Girdin and the engulfment of HBMECs in PLTs were analyzed by confocal microscopy. RESULT: Cytochalasin D overturned the inhibitory effect of PLT on cell apoptosis. OE-Girdin enhanced the fluorescent intensity of PLT-labelling and the engulfment of HBMECs by PLTs, while sh-Girdin, m-Girdin, and Del-Girdin ran reversely. OE-Girdin elevated the Girdin and p-Girdin levels, while sh-Girdin and Del-Girdin were the opposite, but m-Girdin did not affect the p-Girdin and Girdin levels. CONCLUSION: Girdin and p-Girdin were co-located with PLTs in HBMECs. The over-expression of Girdin was identified as being associated with the increasing engulfment of PTLs. Girdin may be an effective target to alleviate endothelial cell apoptosis.

Actin cytoskeleton aggregation involves the water channel protein aquaporin 1-mediated human chondrocyte degeneration.

Water metabolism and actin cytoskeleton remoulding act as essential characters in the process of osteoarthritis (OA). However, the relation between water channel protein aquaporin 1 (AQP1) and actin filament during chondrocytes (CHs) degeneration is not evident. Therefore, the present study aimed to evaluate the role of actin remoulding in the AQP1 mediated CHs degeneration. Primary CHs were collected from human hip cartilage and were degenerated from long-time monolayer culture or IL-1ß stimulation. Besides, the CHs were transfected with AQP1­specific siRNA or vectors to mediate the AQP1 gene expression. The potent inhibitor of actin polymerization Cytochalasin D was also supplemented during culture. RT-PCR was performed to determine the relative gene expression. AQP1 and F-actin fluorescence staining were performed to determine the AQP1 and F-actin organization. Moreover, the cell area and viability were also analyzed. AQP1 and F-actin organization were both increased during seven days' CHs culture or three days' IL-1ß stimulation. Silencing of AQP1 prevented the cell area spreading and degenerated phenotype of CHs with suppression of F-actin aggregation in both natural or IL-1ß-caused inflammatory-related degeneration. Besides, upregulating the AQP1 in the CHs via gene editing promoted the cell area spreading, and F-actin accumulation, and accelerated the CHs degeneration, which can be alleviated by Cytochalasin D treatment. These findings suggested that AQP1-mediated human CHs degeneration is related to F-actin aggregation.

Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

OBJECTIVES: During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. RESULTS: A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. CONCLUSIONS: Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

Cytoskeletal organization in isolated plant cells under geometry control.

The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be tested in cellulo Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells of Arabidopsis thaliana We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.

Cancer cell viscoelasticity measurement by quantitative phase and flow stress induction.

Cell viscoelastic properties are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotyping are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative phase imaging (QPI) is used for image acquisition because this technique enables one to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live-cell experiments and even through a time lapse, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, whereby an increase of stiffness corresponded to actin repolymerization in time.

Integrity of the Actin Cytoskeleton of Host Macrophages is Necessary for Mycobacterial Entry.

Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.

A look into hemostatic characteristics during pediatric liver transplantation using the thromboelastometry (ROTEM® ) test.

There is a paucity of evidence about the coagulation profile regarding the complexity of children undergoing liver transplantation (LT). This study aimed to investigate intraoperative hemostatic changes during pediatric LT according to the etiology for LT and examine the ability of rotational thromboelastometry (ROTEM® , TEM International GmbH, Munich, Germany) as a point-of-care monitoring method. We evaluated 106 patients aged 3 months to 17 years undergoing LT for acute liver failure (ALF) and chronic liver disease, which consists of patients with cholestatic disease, metabolic/genetic disease, and cancer. A total of 731 ROTEM® measurements, including 301 ellagic acid to initiate clotting via the intrinsic pathway, 172 tissue factor to initiate the extrinsic clotting cascade (EXTEM), and 258 cytochalasin D to inhibit platelet activity reflecting fibrinogen (FIBTEM), were analyzed at predetermined time points (the preanhepatic, anhepatic, and postreperfusion phases). We simultaneously conducted conventional coagulation tests. In children with ALF, preanhepatic measurements of conventional coagulation tests and ROTEM® showed a more hypocoagulable state than other diseases. During LT, the coagulation profile was deranged, with a prolonged clotting time and reduced clot firmness, changes that were more profound in the cholestatic disease group. Maximum clot firmness (MCF) on EXTEM and FIBTEM were well correlated with the platelet count and fibrinogen concentration (r = 0.830, p < 0.001 and r = 0.739, p < 0.001, respectively). On the EXTEM, MCF with 30 mm predicted a platelet count <30,000/mm3 (area under the curve, 0.985), and 6 mm predicted a fibrinogen concentration <100 mg/dl on the FIBTEM (area under the curve, 0.876). However, the activated partial thromboplastin time and prothrombin time were significant but only weakly correlated with the clotting time on the ROTEM® . In children undergoing LT, coagulation profiles depend on the etiology for LT. During LT, ROTEM® parameters could help detect thrombocytopenia and hypofibrinogenemia and guide transfusion therapy as a point-of-care monitoring method.

Spatial organization and crosstalk of vimentin and actin stress fibers regulate the osteogenic differentiation of human adipose-derived stem cells.

Human adipose-derived stem cells (hASCs) are ideal seed cells for tissue engineering due to their multidirectional differentiation potential. Microfilaments, microtubules, and intermediate filaments are responsible for supporting the intracellular space. Vimentin, a type III intermediate filament protein that is specifically expressed in cells of mesenchymal origin, can function as a scaffold and endow cells with tension and shear stress resistance. Actin stress fibers (ASF) act as an important physical device in stress signal transduction, providing stiffness for cells, and promoting osteogenesis. Through direct physical contact, cross-linkers, and spatial interactions, vimentin and actin networks exist as intersecting entities. Spatial interactions occur in the overlapping area of cytoskeleton subsystems, which could affect cell morphology, cell mechanics, and cell fate. However, how does the spatial organization between the cytoskeletal subsystems changed during osteogenesis, especially between vimentin and ASF, is still not understood, and its mechanism effect on cell fate remains unclear. In our study, WB experiment was used to detect the expression changes in Vimentin, ASF, and other proteins. Cells were reconstructed by three-dimensional scanning with fluorescence microscope, and the spatial thickness of vimentin and ASF cytoskeletons and the thickness of the overlapping area between them were calculated, respectively, so as to observe the spatial reorganization of vimentin and ASF in cells. Cytochalasin D (an inhibitor of actin polymerization) and vimentin upregulated/downregulated cells were used to verify the change in the spatial organization between vimentin and ASF and its influence on osteogenesis. Then, heat shock protein 27 (HSP27) was downregulated to illuminate the regulatory mechanisms of spatial organization between vimentin and ASF during osteogenesis. The amounts and the spatial positions of vimentin and actin stress fiber exhibited opposite trends during osteogenesis. Through controlling the anchor sites on the nucleus, intermediate filaments vimentin can reduce the spatial proportion of actin stress fibers, which can be regulated by HSP27. In addition, depolymerization of actin stress fibers lead to lower osteogenic differentiation ability, resulting in osteogenesis and lipogenesis existed simultaneously, that can be resisted by vimentin. Our data indicate that the spatial reorganization of vimentin and actin stress fibers is a key factor in the regulation of the differentiation state of hASCs. And their spatial overlapping area is detrimental to hASCs osteogenesis, providing a new perspective for further exploring the mechanism underlying hASCs osteogenesis.

Transcytosis mechanisms of cell-penetrating peptides: Cation-independent CC12 and cationic penetratin.

Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the development and further application of CPPs. Flow cytometry and laser confocal fluorescence microscopy were performed to detect the effects of different endocytosis inhibitors on the internalization of CC12 and penetratin in ARPE-19 cells. The co-localization of CPPs with the lysosome and macropinosome was detected via an endocytosis tracing experiment. The flow cytometry results showed that chlorpromazine, wortmannin, cytochalasin D, and the ATP inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. The laser confocal fluorescence results showed that oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. For penetratin, cytochalasin D and oligomycin had obvious inhibitory effects. The laser confocal fluorescence results indicated that oligomycin had the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, and they are typically dependent on ATP. The transport of penetratin could be partly achieved through the direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway.

Role of actin cytoskeleton in cargo delivery mediated by vertically aligned silicon nanotubes.

Nanofabrication technologies have been recently applied to the development of engineered nano-bio interfaces for manipulating complex cellular processes. In particular, vertically configurated nanostructures such as nanoneedles (NNs) have been adopted for a variety of biological applications such as mechanotransduction, biosensing, and intracellular delivery. Despite their success in delivering a diverse range of biomolecules into cells, the mechanisms for NN-mediated cargo transport remain to be elucidated. Recent studies have suggested that cytoskeletal elements are involved in generating a tight and functional cell-NN interface that can influence cargo delivery. In this study, by inhibiting actin dynamics using two drugs-cytochalasin D (Cyto D) and jasplakinolide (Jas), we demonstrate that the actin cytoskeleton plays an important role in mRNA delivery mediated by silicon nanotubes (SiNTs). Specifically, actin inhibition 12 h before SiNT-cellular interfacing (pre-interface treatment) significantly dampens mRNA delivery (with efficiencies dropping to 17.2% for Cyto D and 33.1% for Jas) into mouse fibroblast GPE86 cells, compared to that of untreated controls (86.9%). However, actin inhibition initiated 2 h after the establishment of GPE86 cell-SiNT interface (post-interface treatment), has negligible impact on mRNA transfection, maintaining > 80% efficiency for both Cyto D and Jas treatment groups. The results contribute to understanding potential mechanisms involved in NN-mediated intracellular delivery, providing insights into strategic design of cell-nano interfacing under temporal control for improved effectiveness.

Intervention time decides the status of autophagy, NLRP3 activity and apoptosis in macrophages induced by ox-LDL.

BACKGROUND: It has been determined through extensive studies that autophagy, the Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and apoptotic responses in macrophages jointly contribute to atherogenesis and its development in the presence of lipid abnormalities. Few studies have investigated in full-scale if the intervention time for lipids abnormality or NLRP3 activation have a significant effect on autophagy, NLRP3 or the apoptotic status in macrophages. METHODS: Human THP-1 monocyte-derived macrophages were established by challenging THP-1 monocytes with 80 µg/ml oxidized low-density lipoprotein (ox-LDL) for specific durations. Foam cell formation was observed by Oil Red O (ORO) staining. Western blots were employed to determine protein expression. Transmission electron microscope (TEM) and immunofluorescence microscopy were applied to observe the autophagic status of cells. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). RESULTS: The cells were treated with ox-LDL for 12 h and 36 h, which were considered to represent early and advanced stages of atherogenesis for this study. The results showed that inhibition of ox-LDL phagocytosis by cytochalasin D in the early stage improved autophagic status, reduced NLRP3 activation and the apoptotic response significantly. In contrast, cytochalasin D had little effect on blocking the detrimental effect of ox-LDL at the advanced stage. Moreover, the changes in autophagy, apoptosis and NLRP3 expression after treatment with small interfering (si) RNA targeting NLRP3 in the early and advanced stages of atherogenesis were consistent with the above data. CONCLUSIONS: Interventions against lipid disorders or inflammatory reactions in the early or advanced stages of atherogenesis may have different results depending on when they are applied during the process of atherosclerotic pathogenesis. These results may help improve therapeutic strategies for atherosclerosis prevention. Furthermore, a healthy lifestyle should still be recommended as the most important and inexpensive measure to prevent atherogenesis.

The Role of Cytoskeleton Revealed by Quartz Crystal Microbalance and Digital Holographic Microscopy.

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which was altered by treatment with cytochalasin D (Cyt-D) and nocodazole (Noc), respectively. We used two complementary techniques: quartz crystal microbalance (QCM) and digital holographic microscopy (DHM). Qualitative data on cell viscoelasticity and adhesion changes at the cell-substrate near-interface layer were obtained with QCM, while DHM allowed the measurement of morphological changes due to the cytoskeletal alterations. A rapid effect of Cyt-D was observed, leading to a reduction in cell viscosity, loss of adhesion, and cell rounding, often followed by detachment from the surface. Noc treatment, instead, induced slower but continuous variations in the rheological behavior for four hours of treatment. The higher vibrational energy dissipation reflected the cell's ability to maintain a stable attachment to the substrate, while a cytoskeletal rearrangement occurs. In fact, along with the complete disaggregation of microtubules at prolonged drug exposure, a compensatory effect of actin polymerization emerged, with increased stress fiber formation.

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air-liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

Class A scavenger receptor-1/2 facilitates the uptake of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice.

Bovine milk exosomes (BMEs) are being explored in drug delivery despite their rapid elimination by macrophages. We aimed at identifying the BME transporter in murine bone marrow-derived macrophages (BMDMs). Fluorophore-labeled BMEs were used in transport studies in BMDMs from C57BL/6J and class A scavenger receptor type 1/2 (CASR-1/2) knockout mice and tissue accumulation in macrophage-depleted C57BL/6J mice. Parametric and nonparametric statistics tests for pairwise and multiple comparisons were used. Chemical inhibitors of phagocytosis by cytochalasin D led to a 69 ± 18% decrease in BME uptake compared with controls (P < 0.05), whereas inhibitors of endocytic pathways other than phagocytosis had a modest effect on uptake (P > 0.05). Inhibitors of class A scavenger receptors (CASRs) including CASR-1/2 caused a 70% decrease in BME uptake (P < 0.05). The uptake of BMEs by BMDMs from CASR-1/2 knockout mice was smaller by 58 ± 23% compared with wild-type controls (P < 0.05). Macrophage depletion by clodronate caused a more than 44% decrease in BME uptake in the spleen and lungs (P < 0.05), whereas the decrease observed in liver was not statistically significant. In conclusion, CASR-1/2 facilitates the uptake of BMEs in BMDMs and C57BL/6J mice.

Matrix hardness regulates the cancer cell malignant progression through cytoskeletal network.

The tumor microenvironment is a complex microenvironment that combines the biochemical and biophysical factors. When the cells are exposed to the microenvironment, the direct biophysical factor is the matrix hardness. As an auxiliary indicator of clinical disease diagnosis, it is still not clear how the matrix hardness induces cell malignant changes and the regulation mechanisms. In this study, we identified that hard matrix significantly promoted cancer cell migratory behaviors. Cell shape was closely associated with cancer cell malignancy, the high malignant cells were associated with high ratios of length/width and low circularity. F-actin networks were also linked with extracellular matrix, it was not regularly distributed when cells were in non-malignant tumor phases or under F-actin inhibition. F-actin might play the key role that transmitted the signal from extracellular matrix to the intracellular organelles. Further study confirmed that active YAP was translocated to nucleus on hard matrix. Cells on hard matrix with cytochalasin D reversed the cancer cell malignancy, meanwhile F-actin re-distributed to the membrane and YAP nucleus translocations were hindered. This work confirmed that F-actin and YAP were upstream-downstream cascade for the cellular and nucleus outside-in signal transductions. The above results demonstrated that hard matrix promoted breast cancer cell malignant behaviors through F-actin network and YAP activation. These results not only described the signal transductions from extracellular to intracellular that was initiated by the biophysical tumor microenvironment, but provided clinical intervention ideas for cancer treatments.

Receptor dynamics regulates actin polymerization state through phosphorylation of cofilin in mast cells.

Aggregation of IgE bound to the high-affinity IgE receptor (FcεRI) by a multivalent antigen induces mast cell activation, while disaggregation of aggregated FcεRI by monomer hapten immediately terminates degranulation mediated by dephosphorylation of Syk and mediates a decrease in intracellular Ca2+ concentration ([Ca2+]i). The actin polymerization state is intimately involved in mast cell activation mediated by FcεRI aggregation. However, the relation between aggregation-disaggregation of FcεRI and actin rearrangement in mast cells is not well understood. The addition of a multivalent antigen rapidly depolymerized actin filaments, while the subsequent addition of monomer hapten rapidly recovered actin polymerization. Whereas cofilin, an actin-severing protein, was temporally dephosphorylated several minutes after a multivalent antigen stimulation and the addition of monomer hapten rapidly increased cofilin phosphorylation level within 30 s. The removal of extracellular Ca2+ instead of monomer hapten addition did not restore cofilin phosphorylation, suggesting that the significant decrease in [Ca2+]i by monovalent hapten was not a critical reason for the actin rearrangement. Additionally, monovalent hapten did not completely reduce [Ca2+]i in mast cells pretreated with jasplakinolide, an inhibitor of actin depolymerization. These results suggest that the multivalent antigen-induced actin depolymerization mediated by cofilin dephosphorylation, and the subsequent addition of monovalent hapten in the F-actin severing state efficiently elicited actin re-polymerization by cofilin phosphorylation.