Deposited footprints let cells switch between confined, oscillatory, and exploratory migration.

For eukaryotic cells to heal wounds, respond to immune signals, or metastasize, they must migrate, often by adhering to extracellular matrix (ECM). Cells may also deposit ECM components, leaving behind a footprint that influences their crawling. Recent experiments showed that some epithelial cell lines on micropatterned adhesive stripes move persistently in regions they have previously crawled over, where footprints have been formed, but barely advance into unexplored regions, creating an oscillatory migration of increasing amplitude. Here, we explore through mathematical modeling how footprint deposition and cell responses to footprint combine to allow cells to develop oscillation and other complex migratory motions. We simulate cell crawling with a phase field model coupled to a biochemical model of cell polarity, assuming local contact with the deposited footprint activates Rac1, a protein that establishes the cell's front. Depending on footprint deposition rate and response to the footprint, cells on micropatterned lines can display many types of motility, including confined, oscillatory, and persistent motion. On two-dimensional (2D) substrates, we predict a transition between cells undergoing circular motion and cells developing an exploratory phenotype. Small quantitative changes in a cell's interaction with its footprint can completely alter exploration, allowing cells to tightly regulate their motion, leading to different motility phenotypes (confined vs. exploratory) in different cells when deposition or sensing is variable from cell to cell. Consistent with our computational predictions, we find in earlier experimental data evidence of cells undergoing both circular and exploratory motion.

A geometrical theory of gliding motility based on cell shape and surface flow.

Gliding motility proceeds with little changes in cell shape and often results from actively driven surface flows of adhesins binding to the extracellular environment. It allows for fast movement over surfaces or through tissue, especially for the eukaryotic parasites from the phylum apicomplexa, which includes the causative agents of the widespread diseases malaria and toxoplasmosis. We have developed a fully three-dimensional active particle theory which connects the self-organized, actively driven surface flow over a fixed cell shape to the resulting global motility patterns. Our analytical solutions and numerical simulations show that straight motion without rotation is unstable for simple shapes and that straight cell shapes tend to lead to pure rotations. This suggests that the curved shapes of Plasmodium sporozoites and Toxoplasma tachyzoites are evolutionary adaptations to avoid rotations without translation. Gliding motility is also used by certain myxo- or flavobacteria, which predominantly move on flat external surfaces and with higher control of cell surface flow through internal tracks. We extend our theory for these cases. We again find a competition between rotation and translation and predict the effect of internal track geometry on overall forward speed. While specific mechanisms might vary across species, in general, our geometrical theory predicts and explains the rotational, circular, and helical trajectories which are commonly observed for microgliders. Our theory could also be used to design synthetic microgliders.

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.

Endogenous oncogenic KRAS expression increases cell proliferation and motility in near-diploid hTERT RPE-1 cells.

About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anticancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. It also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have used the near-diploid hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with WT KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. Epidermal growth factor -induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than WT cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signaling and developing effective anti-KRAS reagents with minimum cytotoxicity on WT cells.

I told you to stop: obscurin's role in epithelial cell migration.

The giant cytoskeletal protein obscurin contains multiple cell signaling domains that influence cell migration. Here, we follow each of these pathways, examine how these pathways modulate epithelial cell migration, and discuss the cross-talk between these pathways. Specifically, obscurin uses its PH domain to inhibit phosphoinositide-3-kinase (PI3K)-dependent migration and its RhoGEF domain to activate RhoA and slow cell migration. While obscurin's effect on the PI3K pathway agrees with the literature, obscurin's effect on the RhoA pathway runs counter to most other RhoA effectors, whose activation tends to lead to enhanced motility. Obscurin also phosphorylates cadherins, and this may also influence cell motility. When taken together, obscurin's ability to modulate three independent cell migration pathways is likely why obscurin knockout cells experience enhanced epithelial to mesenchymal transition, and why obscurin is a frequently mutated gene in several types of cancer.

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.

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.

Modelling the effect of cell motility on mixing and invasion in epithelial monolayers.

Collective cell invasion underlies several biological processes such as wound healing, embryonic development, and cancerous invasion. Here, we investigate the impact of cell motility on invasion in epithelial monolayers and its coupling to cellular mechanical properties, such as cell-cell adhesion and cortex contractility. We develop a two-dimensional computational model for cells with active motility based on the cellular Potts model, which predicts that the cellular invasion speed is mainly determined by active cell motility and is independent of the biological and mechanical properties of the cells. We also find that, in general, motile cells out-compete and invade non-motile cells, however, this can be reversed by differential cell proliferation. Stable coexistence of motile and static cell types is also possible for certain parameter regimes.

The dysadherin/FAK axis promotes individual cell migration in colon cancer.

Dysregulation of cancer cell motility is a key driver of invasion and metastasis. High dysadherin expression in cancer cells is correlated with invasion and metastasis. Here, we found the molecular mechanism by which dysadherin regulates the migration and invasion of colon cancer (CC). Comprehensive analysis using single-cell RNA sequencing data from CC patients revealed that high dysadherin expression in cells is linked to cell migration-related gene signatures. We confirmed that the deletion of dysadherin in tumor cells hindered local invasion and distant migration using in vivo tumor models. In this context, by performing cell morphological analysis, we found that aberrant cell migration resulted from impaired actin dynamics, focal adhesion turnover and protrusive structure formation upon dysadherin expression. Mechanistically, the activation of focal adhesion kinase (FAK) was observed in dysadherin-enriched cells. The dysadherin/FAK axis enhanced cell migration and invasion by activating the FAK downstream cascade, which includes the Rho family of small GTPases. Overall, this study illuminates the role of dysadherin in modulating cancer cell migration by forcing actin dynamics and protrusive structure formation via FAK signaling, indicating that targeting dysadherin may be a potential therapeutic strategy for CC patients.

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.

Profiling Dynamic Patterns of Single-Cell Motility.

Cell motility plays an essential role in many biological processes as cells move and interact within their local microenvironments. Current methods for quantifying cell motility typically involve tracking individual cells over time, but the results are often presented as averaged values across cell populations. While informative, these ensemble approaches have limitations in assessing cellular heterogeneity and identifying generalizable patterns of single-cell behaviors, at baseline and in response to perturbations. In this study, CaMI is introduced, a computational framework designed to leverage the single-cell nature of motility data. CaMI identifies and classifies distinct spatio-temporal behaviors of individual cells, enabling robust classification of single-cell motility patterns in a large dataset (n = 74 253 cells). This framework allows quantification of spatial and temporal heterogeneities, determination of single-cell motility behaviors across various biological conditions and provides a visualization scheme for direct interpretation of dynamic cell behaviors. Importantly, CaMI reveals insights that conventional cell motility analyses may overlook, showcasing its utility in uncovering robust biological insights. Together, a multivariate framework is presented to classify emergent patterns of single-cell motility, emphasizing the critical role of cellular heterogeneity in shaping cell behaviors across populations.

Clustering of RNA co-expression network identifies novel long non-coding RNA biomarkers in squamous cell carcinoma.

Long non-coding RNAs (lncRNAs) have emerged as important players in cancer progression. Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer with increasing incidence worldwide. The prognosis of the metastatic cSCC is poor, and currently there are no established biomarkers to predict metastasis risk or specific therapeutic targets for advanced or metastatic cSCC. To elucidate the role of lncRNAs in cSCC, RNA sequencing of patient derived cSCC cell lines and normal human epidermal keratinocytes was performed. The correlation analysis of differentially expressed lncRNAs and protein-coding genes revealed six distinct gene clusters with one of the upregulated clusters featuring genes associated with cell motility. Upregulation of the expression of lncRNAs linked to cSCC cell motility in cSCC and head and neck SCC (HNSCC) cells was confirmed using qRT-PCR. Elevated expression of HOTTIP and LINC00543 was also noted in SCC tumors in vivo and was associated with poorer prognosis in HNSCC and lung SCC cohorts within TCGA data, respectively. Altogether, these findings uncover a novel set of lncRNAs implicated in cSCC cell locomotion. These lncRNAs may serve as potential novel biomarkers and as putative therapeutic targets for locally advanced and metastatic cSCC.

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.

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.

Filopodia: integrating cellular functions with theoretical models.

Filopodia, widely distributed on cell surfaces, are distinguished by their dynamic extensions, playing pivotal roles in a myriad of biological processes. Their functions span from mechanosensing and guidance to cell-cell communication during cellular organization in the early embryo. Filopodia have significant roles in pathogenic processes, such as cancer invasion and viral dissemination. Molecular mapping of the filopodome has revealed generic components essential for filopodia functions. In parallel, recent insights into biophysical mechanisms governing filopodia dynamics have provided the foundation for broader investigations of filopodia's biological functions. We highlight recent discoveries of engagement of filopodia in various stages of development and pathogenesis and present an overview of intricate molecular and physical features of these cellular structures across a spectrum of cellular activities.

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.

Protein tyrosine phosphatase 1B is a regulator of alpha-actinin4 in the glomerular podocyte.

Glomerular podocytes are instrumental for the barrier function of the kidney, and podocyte injury contributes to proteinuria and the deterioration of renal function. Protein tyrosine phosphatase 1B (PTP1B) is an established metabolic regulator, and the inactivation of this phosphatase mitigates podocyte injury. However, there is a paucity of data regarding the substrates that mediate PTP1B actions in podocytes. This study aims to uncover novel substrates of PTP1B in podocytes and validate a leading candidate. To this end, using substrate-trapping and mass spectroscopy, we identified putative substrates of this phosphatase and investigated the actin cross-linking cytoskeletal protein alpha-actinin4. PTP1B and alpha-actinin4 co-localized in murine and human glomeruli and transiently transfected E11 podocyte cells. Additionally, podocyte PTP1B deficiency in vivo and culture was associated with elevated tyrosine phosphorylation of alpha-actinin4. Conversely, reconstitution of the knockdown cells with PTP1B attenuated alpha-actinin4 tyrosine phosphorylation. We demonstrated co-association between alpha-actinin4 and the PTP1B substrate-trapping mutant, which was enhanced upon insulin stimulation and disrupted by vanadate, consistent with an enzyme-substrate interaction. Moreover, we identified alpha-actinin4 tandem tyrosine residues 486/487 as mediators of its interaction with PTP1B. Furthermore, knockdown studies in E11 cells suggest that PTP1B and alpha-actinin4 are modulators of podocyte motility. These observations indicate that PTP1B and alpha-actinin4 are likely interacting partners in a signaling node that modulates podocyte function. Targeting PTP1B and plausibly this one of its substrates may represent a new therapeutic approach for podocyte injury that warrants additional investigation.

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.

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice.

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.