Cellular and Transcriptional Response of Human Astrocytes to Hybrid Protein Materials.

Collagen is a major component of the tissue matrix, and soybean can regulate the tissue immune response. Both materials have been used to fabricate biomaterials for tissue repair. In this study, adult and fetal human astrocytes were grown in a soy protein isolate (SPI)-collagen hybrid gel or on the surface of a cross-linked SPI-collagen membrane. Hybrid materials reduced the cell proliferation rate compared to materials generated by collagen alone. However, the hybrid materials did not significantly change the cell motility compared to the control collagen material. RNA-sequencing (RNA-Seq) analysis showed downregulated genes in the cell cycle pathway, including CCNA2, CCNB1, CCNB2, CCND1, CCND2, and CDK1, which may explain lower cell proliferation in the hybrid material. This study also revealed the downregulation of genes encoding extracellular matrix (ECM) components, including HSPG2, LUM, SDC2, COL4A1, COL4A5, COL4A6, and FN1, as well as genes encoding chemokines, including CCL2, CXCL1, CXCL2, CX3CL1, CXCL3, and LIF, for adult human astrocytes grown on the hybrid membrane compared with those grown on the control collagen membrane. The study explored the cellular and transcriptional responses of human astrocytes to the hybrid material and indicated a potential beneficial function of the material in the application of neural repair.

Flagellar beating forces of human spermatozoa with different motility behaviors.

BACKGROUND: One of the causes of male infertility is associated with altered spermatozoa motility. These sperm features are frequently analyzed by image-based approaches, which, despite allowing the acquisition of crucial parameters to assess sperm motility, they are unable to provide details regarding the flagellar beating forces, which have been neglected until now. RESULTS: In this work we exploit Fluidic Force Microscopy to investigate and quantify the forces associated with the flagellar beating frequencies of human spermatozoa. The analysis is performed on two groups divided according to the progressive motility of semen samples, as identified by standard clinical protocols. In the first group, 100% of the spermatozoa swim linearly (100% progressive motility), while, in the other, spermatozoa show both linear and circular motility (identified as 80 - 20% progressive motility). Significant differences in flagellar beating forces between spermatozoa from semen sample with different progressive motility are observed. Particularly, linear motile spermatozoa exhibit forces higher than those with a circular movement. CONCLUSIONS: This research can increase our understanding of sperm motility and the role of mechanics in fertilization, which could help us unveil some of the causes of idiopathic male infertility.

Integrin α5 regulates motility of human monocyte-derived Langerhans cells during immune response.

Langerhans cells (LCs) are mainly present in the epidermis and mucosa, and have important roles during skin infection. Migration of LCs to lymph nodes is essential for antigen presentation. However, due to the difficulties in isolating and culturing human LCs, it is not fully understood how LCs move and interact with the extracellular matrix (ECM) through their adhesion molecules such as integrin, during the immune responses. In this study, we aimed to investigate LC motility, cell shape and the role of integrin under inflammatory conditions using monocyte-derived Langerhans cells (moLCs) as a model. As a result, lipopolysaccharide (LPS) stimulation increased adhesion on fibronectin coated substrate and integrin α5 expression in moLCs. Time-lapse imaging of moLCs revealed that stimulation with LPS elongated cell shape, whilst decreasing their motility. Additionally, this decrease in motility was not observed when pre-treated with a neutralising antibody targeting integrin α5. Together, our data suggested that activation of LCs decreases their motility by promoting integrin α5 expression to enhance their affinity to the fibronectin, which may contribute to their migration during inflammation.

CTC-Race: Single-Cell Motility Assay of Circulating Tumor Cells from Metastatic Lung Cancer Patients.

Distinctive subpopulations of circulating tumor cells (CTCs) with increased motility are considered to possess enhanced tumor-initiating potential and contribute to metastasis. Single-cell analysis of the migratory CTCs may increase our understanding of the metastatic process, yet most studies are limited by technical challenges associated with the isolation and characterization of these cells due to their extreme scarcity and heterogeneity. We report a microfluidic method based on CTCs' chemotactic motility, termed as CTC-Race assay, that can analyze migrating CTCs from metastatic non-small-cell lung cancer (NSCLC) patients with advanced tumor stages and enable concurrent biophysical and biochemical characterization of them with single-cell resolution. Analyses of motile CTCs in the CTC-Race assay, in synergy with other single cell characterization techniques, could provide insights into cancer metastasis.

LSP1 promotes the progression of acute myelogenous leukemia by regulating KSR/ERK signaling pathway and cell migration.

We aimed to investigate the role and mechanism of LSP1 in the progression of acute myelogenous leukemia. In this study, we established shLSP1 cell line to analyze the function of LSP1 in AML. We observed high expression of LSP1 in AML patients, whereas it showed no expression in normal adults. Furthermore, we found that LSP1 expression was associated with disease prognosis. Our results indicate that LSP1 plays a crucial role in mediating proliferation and survival of leukemia cells through the KSR/ERK signaling pathway. Additionally, LSP1 promotes cell chemotaxis and homing by enhancing cell adhesion and migration. We also discovered that LSP1 confers chemotactic ability to leukemia cells in vivo. Finally, our study identified 12 genes related to LSP1 in AML, which indicated poor survival outcome in AML patients and were enriched in Ras and cell adhesion signaling pathways. Our results revealed that the overexpression of LSP1 is related to the activation of the KSR/ERK signaling pathway, as well as cell adhesion and migration in AML patients. Reducing LSP1 expression impair AML progression, suggesting that LSP1 may serve as a potential drug therapy target for more effective treatment of AML.

A pyrazolopyridine as a novel AhR signaling activator with anti-breast cancer properties in vitro and in vivo.

Breast cancer is one of the main causes of malignancy-related deaths globally and has a significant impact on women's quality of life. Despite significant therapeutic advances, there is a medical need for targeted therapies in breast cancer. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor mediates responses to environment stimuli, is emerging as a unique pleiotropic target. Herein, a combined molecular simulation and in vitro investigations identified 3-(3-fluorophenyl)-1H-pyrazolo[3,4-b]pyridine (3FPP) as a novel AhR ligand in T47D and MDA-MB-231 breast cancer cells. Its agonistic effects induced formation of the AhR-AhR nuclear translocator (Arnt) heterodimer and prompted its binding to the penta-nucleotide sequence, called xenobiotic-responsive element (XRE) motif. Moreover, 3FPP augmented the promoter-driven luciferase activities and expression of AhR-regulated genes encoding cytochrome P450 1A1 (CYP1A1) and microRNA (miR)-212/132 cluster. It reduced cell viability, migration, and invasion of both cell lines through AhR signaling. These anticancer properties were concomitant with reduced levels of B-cell lymphoma 2 (BCL-2), SRY-related HMG-box4 (SOX4), snail family zinc finger 2 (SNAI2), and cadherin 2 (CDH2). In vivo, 3FPP suppressed tumor growth and activated AhR signaling in an orthotopic mouse model. In conclusion, our results introduce the fused pyrazolopyridine 3FPP as a novel AhR agonist with AhR-specific anti-breast cancer potential in vitro and in vivo.

Metagenomic insights into phenanthrene biodegradation in electrical field-governed biofilms for groundwater bioremediation.

Electrical fields (EFs)-assisted in-situ bioremediation of petroleum-contaminated groundwater, such as polycyclic aromatic hydrocarbons, has drawn increasing attention. However, the long-term stability, the EFs influence, and metabolic pathways are still poorly understood, hindering the further development of robust technology design. Herein, a series of EFs was applied to the phenanthrene-contaminated groundwater, and the corresponding system performance was investigated. The highest removal capacity of phenanthrene (phe) (7.63 g/(m3·d)) was achieved with EF_0.8 V biofilm at a hydrolytic retention time of 0.5 d. All the biofilms with four EFs exhibited a high removal efficiency of phe over 80% during a 100-d continuous-flow operation. Intermediates analysis revealed the main pathways of phe degradation: phthalate and salicylate via hydroxylation, methylation, carboxylation, and ring cleavage steps. Synergistic effects between phe-degraders (Dechloromonas), fermentative bacteria (Delftia), and electroactive microorganisms (Geobacter) were the main contributors to the complete phe mineralization. Genes encoding various proteins of methyl-accepting (mcp), response regulator (cheABDRY), and type IV pilus (pilABCMQV) were dominant, revealing the importance of cell motility and extracellular electron transfer. Metagenomics analysis unveiled phe-degrading genes, including ring reduction enzymes (bamBCDE), carboxylase of aromatics (ubiD), and methyltransferase protein (ubiE, pcm). These findings offered a molecular understanding of refractory organics' decompositions in EFs-governed biotechnology.

Biofabrication of Composite Bioink-Nanofiber Constructs: Effect of Rheological Properties of Bioinks on 3D (Bio)Printing and Cells Interaction with Aligned Touch Spun Nanofibers.

This paper reports on a novel approach for the fabrication of composite multilayered bioink-nanofibers construct. This work achieves this by using a hands-free 3D (bio)printing integrated touch-spinning approach. Additionally, this work investigates the interaction of fibroblasts in different bioinks with the highly aligned touch-spun nanofibers. This work conducts a comprehensive characterization of the rheological properties of the inks, starting with low-strain oscillatory rheology to analyze the viscoelastic behavior, when the material structure remains intact. Moreover, this work performs amplitude sweeps to investigate the stability of the inks under large deformations, rotational rheology to examine the shear thinning profile, and a three-step creep experiment to study time-dependent rheological behavior. The obtained rheological results are correlated to visual observation of the flow behavior of inks. These behaviors span from an ink with zero-shear viscosity, very weak shear thinning, and no thixotropic behavior to inks exhibiting flow stress, pronounced shear thinning, and thixotropy. It is demonstrated that inks have an essential effect on cell behavior. While all bioinks allow a preferred directionality of the fibroblasts along the fiber direction, cells tend to form aggregates in bioinks with higher viscosity, and a considerable number of agglomerates are observed in the presence of laponite-RD.

Migrate3D: Software for simplified post-tracking analysis of 3D and 2D cell migration data.

Migrate3D is a cell migration analysis tool whose purpose is to computationally process positional cell tracking data generated via other image acquisition/analysis software and generate biologically meaningful results. The functionalities of Migrate3D include step-based calculations of each cell track, single-cell-level summary statistics, mean squared displacement analysis, and machine learning-based evaluation of the entire dataset and subpopulations of cells found within it. The parameters calculated within Migrate3D have been previously developed and validated by other groups, and were selected to facilitate extraction of the maximum depth of information possible from input datasets. Variables are user-adjustable to enable customized analyses of diverse motility patterns and cell types, both in three-and two-dimensional timelapse data. Independent of any particular upstream image analysis or cell tracking software, Migrate3D only needs positional data over time to execute the suite of calculations. This presents a unique opportunity to standardize and streamline cell migration analysis.

The Snail signaling branch downstream of the TGF-ß/Smad3 pathway mediates Rho activation and subsequent stress fiber formation.

Cancer cells acquire malignant phenotypes through an epithelial-mesenchymal transition, which is induced by environmental factors or extracellular signaling molecules, including transforming growth factor-ß (TGF-ß). Among epithelial-mesenchymal transition-associated cell responses, cell morphological changes and cell motility are closely associated with remodeling of the actin stress fibers. Here, we examined the TGF-ß signaling pathways leading to these cell responses. Through knockdown experiments in A549 lung adenocarcinoma cells, we found that Smad3-mediated induction of Snail, but not that of Slug, is indispensable for morphological changes, stress fiber formation, and enhanced motility in cells stimulated with TGF-ß. Ectopic expression of Snail in SMAD3-knockout cells rescued the defect in morphological changes and stress fiber formation by TGF-ß, indicating that the role of Smad3 in these responses is to upregulate Snail expression. Mechanistically, Snail is required for TGF-ß-induced upregulation of Wnt5b, which in turn activates RhoA and subsequent stress fiber formation in cooperation with phosphoinositide 3-kinase. However, ectopic expression of Snail in SMAD3-knockout cells failed to rescue the defect in cell motility enhancement by TGF-ß, indicating that activation of the Smad3/Snail/Wnt5b axis is indispensable but not sufficient for enhancing cell motility; a Smad3-dependent but Snail-independent pathway to activate Rac1 is additionally required. Therefore, the Smad3-dependent pathway leading to enhanced cell motility has two branches: a Snail-dependent branch to activate RhoA and a Snail-independent branch to activate Rac1. Coordinated activation of these branches, together with activation of non-Smad signaling pathways, mediates enhanced cell motility induced by TGF-ß.

Pulses of RhoA signaling stimulate actin polymerization and flow in protrusions to drive collective cell migration.

In animals, cells often move as collectives to shape organs, close wounds, or-in the case of disease-metastasize. To accomplish this, cells need to generate force to propel themselves forward. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network. Whether cells migrating as collectives use the same machinery for motility is unclear. Using the zebrafish posterior lateral line primordium as a model for collective cell migration, we find that active RhoA and myosin II cluster on the basal sides of the primordium cells and are required for primordium motility. Positive and negative feedbacks cause RhoA and myosin II activities to pulse. These pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions and myosin-II-dependent actin flow and protrusion retraction at the base of the protrusions and deform the basement membrane underneath the migrating primordium. This suggests that RhoA-induced actin flow on the basal sides of the cells constitutes the motor that pulls the primordium forward, a scenario that likely underlies collective migration in other contexts.

Establishment of an immortalized cell line derived from human adenomyosis ectopic lesions.

Because adenomyosis (AM) ectopic primary cells are hard to come by, have a short lifespan, and the characteristics that alter over time, their utility in AM research is constrained. This study aimed to establish a line of immortalized human adenomyosis ectopic cell (ihAMEC) to change this situation. Primary cells were obtained from AM ectopic lesion tissue and then infected with Simian Vacuolating Virus 40 Tag (SV40 T) lentivirus and screened to establish immortalized cells. We verified the main features and found that the ihAMEC could be cultured for more than 50 generations and the proliferation ability of ihAMEC was more active than that of primary cells. The cytoskeleton and cell types of ihAMEC were similar to primary cells and maintained a normal karyotype. The expression of epithelial-mesenchymal transition (EMT) markers, estrogen-metabolizing proteins, and estrogen/progesterone receptors in ihAMEC was similar to the expression seen in primary cells. In addition, the response of ihAMEC under estrogen treatment and Lipopolysaccharide intervention is similar to primary cells. The clonogenic ability of ihAMEC was lower than tumor cells and did not form tumors in tumorigenicity assays. Thus, ihAMEC can be used as in vitro cellular model for pathogenesis and drug development studies regarding AM.

Unexpected Differences in the Speed of Non-Malignant versus Malignant Cell Migration Reveal Differential Basal Intracellular ATP Levels.

Cellular locomotion is required for survival, fertility, proper embryonic development, regeneration, and wound healing. Cell migration is a major component of metastasis, which accounts for two-thirds of all solid tumor deaths. While many studies have demonstrated increased energy requirements, metabolic rates, and migration of cancer cells compared with normal cells, few have systematically compared normal and cancer cell migration as well as energy requirements side by side. Thus, we investigated how non-malignant and malignant cells migrate, utilizing several cell lines from the breast and lung. Initial screening was performed in an unbiased high-throughput manner for the ability to migrate/invade on collagen and/or Matrigel. We unexpectedly observed that all the non-malignant lung cells moved significantly faster than cells derived from lung tumors regardless of the growth media used. Given the paradigm-shifting nature of our discovery, we pursued the mechanisms that could be responsible. Neither mass, cell doubling, nor volume accounted for the individual speed and track length of the normal cells. Non-malignant cells had higher levels of intracellular ATP at premigratory-wound induction stages. Meanwhile, cancer cells also increased intracellular ATP at premigratory-wound induction, but not to the levels of the normal cells, indicating the possibility for further therapeutic investigation.

A semi-automated cell tracking protocol for quantitative analyses of neutrophil swarming to sterile and S. aureus contaminated bone implants in a mouse femur model.

Implant-associated osteomyelitis remains a major orthopaedic problem. As neutrophil swarming to the surgical site is a critical host response to prevent infection, visualization and quantification of this dynamic behavior at the native microenvironment of infection will elucidate previously unrecognized mechanisms central to understanding the host response. We recently developed longitudinal intravital imaging of the bone marrow (LIMB) to visualize fluorescent S. aureus on a contaminated transfemoral implant and host cells in live mice, which allows for direct visualization of bacteria colonization of the implant and host cellular responses using two-photon laser scanning microscopy. To the end of rigorous and reproducible quantitative outcomes of neutrophil swarming kinetics in this model, we developed a protocol for robust segmentation, tracking, and quantifications of neutrophil dynamics adapted from Trainable Weka Segmentation and TrackMate, two readily available Fiji/ImageJ plugins. In this work, Catchup mice with tdTomato expressing neutrophils received a transfemoral pin with or without ECFP-expressing USA300 methicillin-resistant Staphylococcus aureus (MRSA) to obtain 30-minute LIMB videos at 2-, 4-, and 6-hours post-implantation. The developed semi-automated neutrophil tracking protocol was executed independently by two users to quantify the distance, displacement, speed, velocity, and directionality of the target cells. The results revealed high inter-reader reliability for all outcomes (ICC > 0.98; p > 0.05). Consistent with the established paradigm on increased neutrophil swarming during active infection, the results also demonstrated increased neutrophil speed and velocity at all measured time points, and increased displacement at later time points (6 hours) in infected versus uninfected mice (p < 0.05). Neutrophils and bacteria also exhibit directionality during migration in the infected mice. The semi-automated cell tracking protocol provides a streamlined approach to robustly identify and track individual cells across diverse experimental settings and eliminates inter-observer variability.

Interaction of Integrin αvß8 With Type I Collagen Promotes Squamous Cell Carcinoma Cell Motility via RAC1 Activation.

BACKGROUND/AIM: The interaction of integrin αvß8 with type I collagen was shown to promote oral squamous cell carcinoma (SCC) cell proliferation via the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. However, the role of integrin αvß8 in SCC progression remains poorly understood. In this study, the role of integrin αvß8 in oral SCC progression was therefore investigated. MATERIALS AND METHODS: Integrin αv and ß8 protein expression in oral SCC cells was examined by western blotting. Oral SCC cell motility was investigated using modified Boyden chamber assays. Behavior of oral SCC cells was examined in three-dimensional culture using type I collagen gel. Ras homolog family member A (RHOA), Ras-related C3 botulinum toxin substrate 1 (RAC1), and cell division control protein 42 homolog (CDC42) activity of oral SCC cells was analyzed by pull-down assays. RESULTS: SCC cells with high integrin αvß8 expression levels had a high ability to migrate on type I collagen and exhibited enhanced invasion into type I collagen gel. In SCC cells with high integrin αvß8 expression level, cultivation on type I collagen induced RAC1 activation. Treatment with RAC1 inhibitor reduced type I collagen-induced motility of SCC cells. Down-regulation of integrin ß8 by specific antisense oligonucleotide reduced type I collagen-induced RAC1 activation and suppressed cell motility and invasion into type I collagen gel. CONCLUSION: The interaction of integrin αvß8 with type I collagen facilitates SCC cell motility and invasion via RAC1 activation. Therefore, integrin αvß8 and RAC1 may represent new targets for inhibiting metastasis and invasion in patients with oral SCC.

Maladaptive T-Cell Metabolic Fitness in Autoimmune Diseases.

Immune surveillance and adaptive immune responses, involving continuously circulating and tissue-resident T-lymphocytes, provide host defense against infectious agents and possible malignant transformation while avoiding autoimmune tissue damage. Activation, migration, and deployment of T-cells to affected tissue sites are crucial for mounting an adaptive immune response. An effective adaptive immune defense depends on the ability of T-cells to dynamically reprogram their metabolic requirements in response to environmental cues. Inability of the T-cells to adapt to specific metabolic demands may skew cells to become either hyporesponsive (creating immunocompromised conditions) or hyperactive (causing autoimmune tissue destruction). Here, we review maladaptive T-cell metabolic fitness that can cause autoimmune diseases and discuss how T-cell metabolic programs can potentially be modulated to achieve therapeutic benefits.

Menthol induces apoptosis and inhibits proliferation and migration of nonsmall cell lung carcinoma in vitro and in vivo through Akt pathway.

BACKGROUND: About 40% of nonsmall cell lung cancers (NSCLCs) have already progressed in an advanced stage at the time of diagnosis. Development of effective prevention and therapy approaches against NSCLC is critical for reducing mortality. As a fundamental ingredient of peppermint oil, menthol has been demonstrated to possess an antitumor activity in several types of carcinomas. However, the potential role of menthol on NSCLC has not been reported. The present study aims to investigate the effect and underlying mechanism of menthol on proliferation, apoptosis, and mobility of human lung adenocarcinoma. METHODS: Cell apoptosis was examined by MTT and flow cytometry. The motility of cells was determined by Transwell assay. Western blot analysis was performed to determine expression level of proteins. In vivo model of nude mice was established for evaluating the influence of menthol on tumorigenicity of A549 cells. The expression lentiviral vector of Akt was established in NSCLC cells for further verifying the inhibiting effect of menthol on survival and mobility of NSCLC cells via Akt pathway. RESULTS: The results showed that menthol promoted A549 cell apoptosis, suppressed cell proliferation, and motility by altering the phosphorylated protein level of Akt. Menthol enhanced the expression level of Bax while decreasing expression of Bcl-2, Caspase-3, and MMPs proteins. In vivo experiments suggested that menthol exhibited an inhibitory effect in tumor growth on xenografts. These results were further validated in Akt over-expressed A549 and H1299 cells. CONCLUSIONS: Menthol could display an inhibitory effect on NSCLC cells through Akt signaling pathway, making it a potential target for NSCLC treatment.

Motility of Mytilus galloprovincialis hemocytes: Sensitivity to paracetamol in vitro exposure.

Pharmaceuticals released into the environment (PiEs) represent an environmental problem of growing concern for the health of ecosystems and humans. An increasing number of studies show that PiEs pose a risk to aquatic organisms. The aim of the present work was to contribute to increasing the knowledge of the effects of PiE on marine biota focusing on the effect of paracetamol on the motility of hemocytes in Mytilus galloprovincialis, a bivalve mollusk species widely utilized as bioindicator organism. Hemocytes are the immunocompetent cells of bivalve mollusks. An early and key stage of mollusk immune response is represented by the recruitment and migration of these cells to the site of infection. Therefore, motility is an intrinsic characteristic of these cells. Here, we first characterized the spontaneous cell movement of M. galloprovincialis hemocytes when plated in a TC-treated polystyrene 96-well microplate. Two different cellular morphotypes were distinguished based on their appearance and motility behavior: spread cells and round-star-shaped cells. The two motility morphotypes were characterized by different velocities as well as movement directness, which were significantly lower in round-star-shaped cells with respect to spread cells. The sensitivity of the motility of M. galloprovincialis hemocytes to paracetamol at different concentrations (0.02, 0.2 and 2 mg/L) was investigated in vitro after 1h and 24h exposure. Paracetamol induced alterations in the motility behavior (both velocity and trajectories) of the hemocytes and the effects were cell-type specific. The study of hemocyte movements at the single cell level by cell tracking and velocimetric parameters analysis provides new sensitive tools for assessing the effects of emerging pollutants at the cellular levels in non-target organisms.

NG2/CSPG4 attenuates motility in mandibular fibrochondrocytes under serum starvation conditions.

The migration of mandibular fibrochondrocytes is important for the development of the mandible, the homeostasis of the mandibular cartilage, and for the capacity of the tissue to respond to injury. Mandibular fibrochondrocytes have to overcome formidable obstacles during migration including a dense and heterogeneous three-dimensional matrix. Guiding the direction of cell migration and commitment to a migratory phenotype in this microenvironment necessitates a multivalent response to chemotactic and extracellular matrix-mediated stimuli. One of the key matrix components in the cartilage of the temporomandibular joint is type VI collagen. Neuron/glial antigen 2 (NG2/CSPG4) is a transmembrane proteoglycan that binds with collagen VI and has been implicated in a wide range of cell behaviors including cell migration, motility, adhesion, and proliferation. While NG2/CSPG4 has been shown to be a key regulator of mandibular cartilage homeostasis, its role in the migration of mandibular fibrochondrocytes during normal and cell stress conditions has yet to be resolved. Here, we address this gap in knowledge by characterizing NG2/CSPG4-dependent migration in mandibular fibrochondrocytes using primary mandibular fibrochondrocytes isolated from control and full length NG2/CSPG4 knockout mice, in primary mandibular fibrochondrocytes isolated from NG2|DsRed reporter mice and in an immortalized mandibular fibrochondrocyte cell line with a mutated NG2/CSPG4 ectodomain. All three cells demonstrate similar results, with loss of the full length or truncated NG2/CSPG4 increasing the rate of cell migration in serum starvation/cell stress conditions. These findings clearly implicate NG2/CSPG4 as a key molecule in the regulation of cell migration in mandibular fibrochondrocytes in normal and cell stress conditions, underscoring the role of NG2/CSPG4 as a mechanosensitive signaling hub in the mandibular cartilage.

SPIN-4/Spinster supports sperm activation in C. elegans via sphingosine-1-phosphate transport.

Spermiogenesis, a sperm-activation step, is crucial for the transformation of immotile spermatids into motile sperm. Though membrane transport of ions and molecules across the sperm plasma membrane has been implicated in this process, the full repertoire of transporters involved, and their respective substrates, is unclear. Here, we report that the major facilitator superfamily transporter SPIN-4/Spinster governs efficient spermiogenesis and fertility in the hermaphrodite nematode Caenorhabditis elegans. Unlike other C. elegans Spinster paralogs, SPIN-4 is germline-expressed. Moreover, SPIN-4 expression is gamete-specific; it is strongly expressed in developing sperm, where it localizes to the plasma membrane, but it is absent from oocytes. Consistent with these expression data, we demonstrate that knocking out spin-4 impairs sperm development, leading to the formation of non-motile sperm that lack pseudopodia. Consequently, hermaphrodites homozygous for the spin-4(knu1099) knockout allele show extensive sperm wasting and reduced self-progeny. We observe similar defects when we genetically inhibit production of sphingosine-1-phosphate, a lipid molecule that stimulates cell motility when exported extracellularly by Spinster homologs in other contexts. Remarkably, extracellular supplementation with sphingosine-1-phosphate rescues sperm activation and motility in the absence of SPIN-4, suggesting that Spinster-dependent efflux of sphingosine-1-phosphate plays a key role in sperm mobilization. These findings identify a new signaling mechanism in C. elegans spermiogenesis entailing Spinster and sphingosine-1-phosphate.