The role of cell-matrix adhesion and cell migration in breast tumor growth and progression.

During breast cancer progression, there is typically increased collagen deposition resulting in elevated extracellular matrix rigidity. This results in changes to cell-matrix adhesion and cell migration, impacting processes such as the epithelial-mesenchymal transition (EMT) and metastasis. We aim to investigate the roles of cell-matrix adhesion and cell migration on breast tumor growth and progression by studying the impacts of different types of extracellular matrices and their rigidities. We embedded MCF7 spheroids within three-dimensional (3D) collagen matrices and agarose matrices. MCF7 cells adhere to collagen but not agarose. Contrasting the results between these two matrices allows us to infer the role of cell-matrix adhesion. We found that MCF7 spheroids exhibited the fastest growth rate when embedded in a collagen matrix with a rigidity of 5.1 kPa (0.5 mg/mL collagen), whereas, for the agarose matrix, the rigidity for the fastest growth rate is 15 kPa (1.0% agarose) instead. This discrepancy is attributable to the presence of cell adhesion molecules in the collagen matrix, which initiates collagen matrix remodeling and facilitates cell migration from the tumor through the EMT. As breast tumors do not adhere to agarose matrices, it is suitable to simulate the cell-cell interactions during the early stage of breast tumor growth. We conducted further analysis to characterize the stresses exerted by the expanding spheroid on the agarose matrix. We identified two distinct MCF7 cell populations, namely, those that are non-dividing and those that are dividing, which exerted low and high expansion stresses on the agarose matrix, respectively. We confirmed this using Western blot which showed the upregulation of proliferating cell nuclear antigen, a proliferation marker, in spheroids grown in the 1.0% agarose (≈13 kPa). By treating the embedded MCF7 spheroids with an inhibitor or activator of myosin contractility, we showed that the optimum spheroids' growth can be increased or decreased, respectively. This finding suggests that tumor growth in the early stage, where cell-cell interaction is more prominent, is determined by actomyosin tension, which alters cell rounding pressure during cell division. However, when breast tumors begin generating collagen into the surrounding matrix, collagen remodeling triggers EMT to promote cell migration and invasion, ultimately leading to metastasis.

ContrastivePose: A contrastive learning approach for self-supervised feature engineering for pose estimation and behavorial classification of interacting animals.

In recent years, supervised machine learning models trained on videos of animals with pose estimation data and behavior labels have been used for automated behavior classification. Applications include, for example, automated detection of neurological diseases in animal models. However, we identify two potential problems of such supervised learning approach. First, such models require a large amount of labeled data but the labeling of behaviors frame by frame is a laborious manual process that is not easily scalable. Second, such methods rely on handcrafted features obtained from pose estimation data that are usually designed empirically. In this paper, we propose to overcome these two problems using contrastive learning for self-supervised feature engineering on pose estimation data. Our approach allows the use of unlabeled videos to learn feature representations and reduce the need for handcrafting of higher-level features from pose positions. We show that this approach to feature representation can achieve better classification performance compared to handcrafted features alone, and that the performance improvement is due to contrastive learning on unlabeled data rather than the neural network architecture. The method has the potential to reduce the bottleneck of scarce labeled videos for training and improve performance of supervised behavioral classification models for the study of interaction behaviors in animals.

Subcellular Force Imbalance in Actin Bundles Induces Nuclear Repositioning and Durotaxis.

Durotaxis is a phenomenon in which cells migrate toward substrates of increasing stiffness. However, how cells assimilate substrate stiffness as a directional cue remains poorly understood. In this study, we experimentally show that mouse embryonic fibroblasts can discriminate between different substrate stiffnesses and develop higher traction forces at regions of the cell adhering to the stiffer pillars. In this way, the cells generate a force imbalance between adhesion sites. It is this traction force imbalance that drives durotaxis by providing directionality for cell migration. Significantly, we found that traction forces are transmitted via LINC complexes to the cell nucleus, which serves to maintain the global force imbalance. In this way, LINC complexes play an essential role in anterograde nuclear movement and durotaxis. This conclusion is supported by the fact that LINC complex-deficient cells are incapable of durotaxis and instead migrate randomly on substrates featuring a stiffness gradient.

Fungus-derived protein particles as cell-adhesive matrices for cell-cultivated food.

Cell-adhesive factors mediate adhesion of cells to substrates via peptide motifs such as the Arg-Gly-Asp (RGD) sequence. With the onset of sustainability issues, there is a pressing need to find alternatives to animal-derived cell-adhesive factors, especially for cell-cultivated food applications. In this paper, we show how data mining can be a powerful approach toward identifying fungal-derived cell-adhesive proteins and present a method to isolate and utilize these proteins as extracellular matrices (ECM) to support cell adhesion and culture in 3D. Screening of a protein database for fungal and plant proteins uncovered that ~5.5% of the unique reported proteins contain RGD sequences. A plot of fungi species vs RGD percentage revealed that 98% of the species exhibited an RGD percentage > = 1%. We observed the formation of protein particles in crude extracts isolated from basidiomycete fungi, which could be correlated to their stability towards particle aggregation at different temperatures. These protein particles were incorporated in 3D fiber matrices encapsulating mouse myoblast cells, showing a positive effect on cell alignment. We demonstrated a cell traction stress on the protein particles (from Flammulina velutipes) that was comparable to cells on fibronectin. A snapshot of the RGD-containing proteins in the fungal extracts was obtained by combining SDS-PAGE and mass spectrometry of the peptide fragments obtained by enzymatic cleavage. Therefore, a sustainable source of cell-adhesive proteins is widely available in the fungi kingdom. A method has been developed to identify candidate species and produce cell-adhesive matrices, applicable to the cell-cultivated food and healthcare industries.

A review of algorithmic approaches for cell culture media optimization.

Cell culture media composition and culture conditions play a crucial role in product yield, quality and cost of production. Culture media optimization is the technique of improving media composition and culture conditions to achieve desired product outcomes. To achieve this, there have been many algorithmic methods proposed and used for culture media optimization in the literature. To help readers evaluate and decide on a method that best suits their specific application, we carried out a systematic review of the different methods from an algorithmic perspective that classifies, explains and compares the available methods. We also examine the trends and new developments in the area. This review provides recommendations to researchers regarding the suitable media optimization algorithm for their applications and we hope to also promote the development of new cell culture media optimization methods that are better suited to existing and upcoming challenges in this biotechnology field, which will be essential for more efficient production of various cell culture products.

Zyxin regulates embryonic stem cell fate by modulating mechanical and biochemical signaling interface.

Biochemical signaling and mechano-transduction are both critical in regulating stem cell fate. How crosstalk between mechanical and biochemical cues influences embryonic development, however, is not extensively investigated. Using a comparative study of focal adhesion constituents between mouse embryonic stem cell (mESC) and their differentiated counterparts, we find while zyxin is lowly expressed in mESCs, its levels increase dramatically during early differentiation. Interestingly, overexpression of zyxin in mESCs suppresses Oct4 and Nanog. Using an integrative biochemical and biophysical approach, we demonstrate involvement of zyxin in regulating pluripotency through actin stress fibres and focal adhesions which are known to modulate cellular traction stress and facilitate substrate rigidity-sensing. YAP signaling is identified as an important biochemical effector of zyxin-induced mechanotransduction. These results provide insights into the role of zyxin in the integration of mechanical and biochemical cues for the regulation of embryonic stem cell fate.

Transcriptomics indicate nuclear division and cell adhesion not recapitulated in MCF7 and MCF10A compared to luminal A breast tumours.

Breast cancer (BC) cell lines are useful experimental models to understand cancer biology. Yet, their relevance to modelling cancer remains unclear. To better understand the tumour-modelling efficacy of cell lines, we performed RNA-seq analyses on a combined dataset of 2D and 3D cultures of tumourigenic MCF7 and non-tumourigenic MCF10A. To our knowledge, this was the first RNA-seq dataset comprising of 2D and 3D cultures of MCF7 and MCF10A within the same experiment, which facilitates the elucidation of differences between MCF7 and MCF10A across culture types. We compared the genes and gene sets distinguishing MCF7 from MCF10A against separate RNA-seq analyses of clinical luminal A (LumA) and normal samples from the TCGA-BRCA dataset. Among the 1031 cancer-related genes distinguishing LumA from normal samples, only 5.1% and 15.7% of these genes also distinguished MCF7 from MCF10A in 2D and 3D cultures respectively, suggesting that different genes drive cancer-related differences in cell lines compared to clinical BC. Unlike LumA tumours which showed increased nuclear division-related gene expression compared to normal tissue, nuclear division-related gene expression in MCF7 was similar to MCF10A. Moreover, although LumA tumours had similar cell adhesion-related gene expression compared to normal tissues, MCF7 showed reduced cell adhesion-related gene expression compared to MCF10A. These findings suggest that MCF7 and MCF10A cell lines were limited in their ability to model cancer-related processes in clinical LumA tumours.

Alzheimer's Disease Blood Biomarkers Associated With Neuroinflammation as Therapeutic Targets for Early Personalized Intervention.

The definitive diagnosis of Alzheimer's Disease (AD) without the need for neuropathological confirmation remains a challenge in AD research today, despite efforts to uncover the molecular and biological underpinnings of the disease process. Furthermore, the potential for therapeutic intervention is limited upon the onset of symptoms, providing motivation for studying and treating the AD precursor mild cognitive impairment (MCI), the prodromal stage of AD instead. Applying machine learning classification to transcriptomic data of MCI, AD, and cognitively normal (CN) control patients, we identified differentially expressed genes that serve as biomarkers for the characterization and classification of subjects into MCI or AD groups. Predictive models employing these biomarker genes exhibited good classification performances for CN, MCI, and AD, significantly above random chance. The PI3K-Akt, IL-17, JAK-STAT, TNF, and Ras signaling pathways were also enriched in these biomarker genes, indicating their diagnostic potential and pathophysiological roles in MCI and AD. These findings could aid in the recognition of MCI and AD risk in clinical settings, allow for the tracking of disease progression over time in individuals as part of a therapeutic approach, and provide possible personalized drug targets for early intervention of MCI and AD.

Integration of a microfluidic multicellular coculture array with machine learning analysis to predict adverse cutaneous drug reactions.

Adverse cutaneous reactions are potentially life-threatening skin side effects caused by drugs administered into the human body. The availability of a human-specific in vitro platform that can prospectively screen drugs and predict this risk is therefore of great importance to drug safety. However, since adverse cutaneous drug reactions are mediated by at least 2 distinct mechanisms, both involving systemic interactions between liver, immune and dermal tissues, existing in vitro skin models have not been able to comprehensively recapitulate these complex, multi-cellular interactions to predict the skin-sensitization potential of drugs. Here, we report a novel in vitro drug screening platform, which comprises a microfluidic multicellular coculture array (MCA) to model different mechanisms-of-action using a collection of simplistic cellular assays. The resultant readouts are then integrated with a machine-learning algorithm to predict the skin sensitizing potential of systemic drugs. The MCA consists of 4 cell culture compartments connected by diffusion microchannels to enable crosstalk between hepatocytes that generate drug metabolites, antigen-presenting cells (APCs) that detect the immunogenicity of the drug metabolites, and keratinocytes and dermal fibroblasts, which collectively determine drug metabolite-induced FasL-mediated apoptosis. A single drug screen using the MCA can simultaneously generate 5 readouts, which are integrated using support vector machine (SVM) and principal component analysis (PCA) to classify and visualize the drugs as skin sensitizers or non-skin sensitizers. The predictive performance of the MCA and SVM classification algorithm is then validated through a pilot screen of 11 drugs labelled by the US Food and Drug Administration (FDA), including 7 skin-sensitizing and 4 non-skin sensitizing drugs, using stratified 4-fold cross-validation (CV) on SVM. The predictive performance of our in vitro model achieves an average of 87.5% accuracy (correct prediction rate), 75% specificity (prediction rate of true negative drugs), and 100% sensitivity (prediction rate of true positive drugs). We then employ the MCA and the SVM training algorithm to prospectively identify the skin-sensitizing likelihood and mechanism-of-action for obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist which has undergone clinical trials for non-alcoholic steatohepatitis (NASH) with well-documented cutaneous side effects.

Compressive forces driven by lateral actin fibers are a key to the nuclear deformation under uniaxial cell-substrate stretching.

Cells sense the direction of mechanical stimuli including substrate stretching and show morphological and functional responses. The nuclear deformation with respect to the direction of mechanical stimuli is thought of as a vital factor in mechanosensitive intracellular signaling and gene transcription, but the detailed relationship between the direction of stimuli and nuclear deformation behavior is not fully solved yet. Here, we assessed the role of actin cytoskeletons in nuclear deformation caused by cell substrate stretching with different directions. Cells on a PDMS stretching chamber were subjected to a step-strain and changes of long- and short-axes of nucleus before and after stretching were evaluated in terms of nuclear orientation against the direction of stretching. Nuclei oriented parallel to the stretching direction showed elongation and shrinkage in the long and short axes, respectively, and vice versa. However, calculation of the aspect ratio (ratio of long- and short-axes) changes revealed orientation-depend nuclear deformation: The nucleus oriented parallel to the stretching direction showed a greater aspect ratio change than it aligned in the perpendicular direction of the stretching. A decrease in actin cytoskeletal tension significantly changed the nuclear deformation only in the short axis direction, thereby abolishing the orientation-depend deformation of the nucleus. These results suggest that lateral compressive forces exerted by the actin cytoskeleton is a key factor of orientation-depend deformation in short axis of the nucleus under the cell-substrate stretching condition, and may be crucial for mechano-sensing and responses to the cell-substrate stretching direction.

Zyxin Is Involved in Fibroblast Rigidity Sensing and Durotaxis.

Focal adhesions (FAs) are specialized structures that enable cells to sense their extracellular matrix rigidity and transmit these signals to the interior of the cells, bringing about actin cytoskeleton reorganization, FA maturation, and cell migration. It is known that cells migrate towards regions of higher substrate rigidity, a phenomenon known as durotaxis. However, the underlying molecular mechanism of durotaxis and how different proteins in the FA are involved remain unclear. Zyxin is a component of the FA that has been implicated in connecting the actin cytoskeleton to the FA. We have found that knocking down zyxin impaired NIH3T3 fibroblast's ability to sense and respond to changes in extracellular matrix in terms of their FA sizes, cell traction stress magnitudes and F-actin organization. Cell migration speed of zyxin knockdown fibroblasts was also independent of the underlying substrate rigidity, unlike wild type fibroblasts which migrated fastest at an intermediate substrate rigidity of 14 kPa. Wild type fibroblasts exhibited durotaxis by migrating toward regions of increasing substrate rigidity on polyacrylamide gels with substrate rigidity gradient, while zyxin knockdown fibroblasts did not exhibit durotaxis. Therefore, we propose zyxin as an essential protein that is required for rigidity sensing and durotaxis through modulating FA sizes, cell traction stress and F-actin organization.

Dynamic swimming pattern of Pseudomonas aeruginosa near a vertical wall during initial attachment stages of biofilm formation.

Studying the swimming behaviour of bacteria in 3 dimensions (3D) allows us to understand critical biological processes, such as biofilm formation. It is still unclear how near wall swimming behaviour may regulate the initial attachment and biofilm formation. It is challenging to address this as visualizing the movement of bacteria with reasonable spatial and temporal resolution in a high-throughput manner is technically difficult. Here, we compared the near wall (vertical) swimming behaviour of P. aeruginosa (PAO1) and its mutants ΔdipA (reduced in swarming motility and increased in biofilm formation) and ΔfimX (deficient in twitching motility and reduced in biofilm formation) using our new imaging technique based on light sheet microscopy. We found that P. aeruginosa (PAO1) increases its speed and changes its swimming angle drastically when it gets closer to a wall. In contrast, ΔdipA mutant moves toward the wall with steady speed without changing of swimming angle. The near wall behavior of ΔdipA allows it to be more effective to interact with the wall or wall-attached cells, thus leading to more adhesion events and a larger biofilm volume during initial attachment when compared with PAO1. Furthermore, we found that ΔfimX has a similar near wall swimming behavior as PAO1. However, it has a higher dispersal frequency and smaller biofilm formation when compared with PAO1 which can be explained by its poor twitching motility. Together, we propose that near wall swimming behavior of P. aeruginosa plays an important role in the regulation of initial attachment and biofilm formation.

Cellular ageing of oral fibroblasts differentially modulates extracellular matrix organization.

BACKGROUND AND OBJECTIVES: Ageing is associated with an impaired cellular function that can affect tissue architecture and wound healing in gingival and periodontal tissues. However, the impact of oral fibroblast ageing on the structural organization of the extracellular matrix (ECM) proteins is poorly understood. Hence, in this study, we investigated the impact of cellular ageing of oral fibroblasts on the production and structural organization of collagen and other ECM proteins. METHODS: Oral fibroblasts were serially subcultured, and replicative cellular senescence was assessed using population doubling time, Ki67 counts and expression of P21WAFI . The production and structural organization of ECM proteins were assessed at early (young-oFB) and late (aged-oFB) passages. The thickness and pattern of collagen produced by live cultures of young- and aged-oFB were assessed using a label-free and non-invasive second harmonic generation (SHG)-based multiphoton imaging. Expression of other ECM proteins (fibronectin, fibrillin, collagen-IV and laminins) was evaluated using immunocytochemistry and confocal microscopy-based depth profile analysis. RESULTS: Aged-oFB displayed a higher population doubling time, lower Ki67+ cells and higher expression of P21WAFI indicative of slower proliferation rate and senescence phenotype. SHG imaging demonstrated that young-oFB produced a thick, interwoven network of collagen fibres, while the aged-oFB produced thin and linearly organized collagen fibres. Similarly, analysis of immunostained cultures showed that young-oFB produced a rich, interwoven mesh of fibronectin, fibrillin and collagen-IV fibres. In contrast, the aged-oFB produced linearly organized fibronectin, fibrillin and collagen-IV fibres. Lastly, there was no observable difference in production and organization of laminins among the young- and aged-oFB. CONCLUSION: Our results suggest that oral fibroblast ageing impairs ECM production and more importantly the organization of ECM fibres, which could potentially impair wound healing in the elderly.

Phosphatase POPX2 interferes with cell cycle by interacting with Chk1.

Protein-protein interaction network analysis plays critical roles in predicting the functions of target proteins. In this study, we used a combination of SILAC-MS proteomics and bioinformatic approaches to identify Checkpoint Kinase 1 (Chk1) as a possible POPX2 phosphatase interacting protein. POPX2 is a PP2C phosphatase that has been implicated in cancer cell invasion and migration. From the Domain-Domain Interaction (DDI) database, we first determined that the PP2C phosphatase domain interacts with Pkinase domain. Subsequently, 46 proteins with Pkinase domain were identified from POPX2 SILAC-MS data. We then narrowed down the leads and confirmed the biological interaction between Chk1 and POPX2. We also found that Chk1 is a substrate of POPX2. Chk1 is a key regulator of the cell cycle and is activated when the cell suffers DNA damage. Our approach has led us to identify POPX2 as a regulator of Chk1 and can interfere with the normal function of Chk1 at G1-S transition of the cell cycle in response to DNA damage.

Regulation of flagellar motor switching by c-di-GMP phosphodiesterases in Pseudomonas aeruginosa.

The second messenger cyclic diguanylate (c-di-GMP) plays a prominent role in regulating flagellum-dependent motility in the single-flagellated pathogenic bacterium Pseudomonas aeruginosa The c-di-GMP-mediated signaling pathways and mechanisms that control flagellar output remain to be fully unveiled. Studying surface-tethered and free-swimming P. aeruginosa PAO1 cells, we found that the overexpression of an exogenous diguanylate cyclase (DGC) raises the global cellular c-di-GMP concentration and thereby inhibits flagellar motor switching and decreases motor speed, reducing swimming speed and reversal frequency, respectively. We noted that the inhibiting effect of c-di-GMP on flagellar motor switching, but not motor speed, is exerted through the c-di-GMP-binding adaptor protein MapZ and associated chemotactic pathways. Among the 22 putative c-di-GMP phosphodiesterases, we found that three of them (DipA, NbdA, and RbdA) can significantly inhibit flagellar motor switching and swimming directional reversal in a MapZ-dependent manner. These results disclose a network of c-di-GMP-signaling proteins that regulate chemotactic responses and flagellar motor switching in P. aeruginosa and establish MapZ as a key signaling hub that integrates inputs from different c-di-GMP-signaling pathways to control flagellar output and bacterial motility. We rationalized these experimental findings by invoking a model that postulates the regulation of flagellar motor switching by subcellular c-di-GMP pools.

Superresolution microscopy reveals distinct localisation of full length IRSp53 and its I-BAR domain protein within filopodia.

Superresolution microscopy offers the advantage of imaging biological structures within cells at the nano-scale. Here we apply two superresolution microscopy techniques, specifically 3D structured illumination microscopy (3D-SIM) and direct stochastic optical reconstruction microscopy (dSTORM), a type of single molecule localisation microscopy, to localise IRSp53 protein and its I-BAR domain in relation to F-actin within filopodia. IRSp53 generates dynamic (extending and retracting) filopodia 300 nm wide with a distinct gap between IRSp53 and F-actin. By contrast, protrusions induced by the I-BAR domain alone are non-dynamic measuring between 100-200 nm in width and exhibit a comparatively closer localisation of the I-BAR domain with the F-actin. The data suggest that IRSp53 membrane localisation is spatially segregated to the lateral edges of filopodia, in contrast to the I-BAR domain is uniformly distributed throughout the membranes of protrusions. Modeling of fluorescence recovery after photobleaching (FRAP) data suggests that a greater proportion of I-BAR domain is associated with membranes when compared to full length IRSp53. The significance of this new data relates to the role filopodia play in cell migration and its importance to cancer.

A chemotaxis model to explain WHIM neutrophil accumulation in the bone marrow of WHIM mouse model.

Neutrophils are essential immune cells that defend the host against pathogenic microbial agents. Neutrophils are produced in the bone marrow and are retained there through CXCR4-CXCL12 signaling. However, patients with the Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome are prone to infections due to increased accumulation of neutrophils in the bone marrow leading to low numbers of circulating neutrophils. How neutrophils accumulate in the bone marrow in this condition is poorly understood. To better understand factors involved in neutrophil accumulation in the bone marrow, neutrophils from wildtype and WHIM mouse models were characterized in their response to CXCL12 stimulation. WHIM neutrophils were found to exert stronger traction forces, formed significantly more lamellipodia-type protrusions and migrated with increased speed and displacement upon CXCL12 stimulation as compared to wildtype cells. Migration speed of WHIM neutrophils showed a larger initial increase upon CXCL12 stimulation, which decayed over a longer time period as compared to wildtype cells. We proposed a computational model based on the chemotactic behavior of neutrophils that indicated increased CXCL12 sensitivity and prolonged CXCR4 internalization adaptation time in WHIM neutrophils as being responsible for increased accumulation in the bone marrow. These findings provide a mechanistic understanding of bone marrow neutrophil accumulation in WHIM condition and novel insights into restoring neutrophil regulation in WHIM patients.

Enrichment and Identification of Neural Stem Cells in Neurospheres Using Rigidity-Tunable Gels.

IMPACT STATEMENT: Neural stem cells (NCSs) are integral to establishing in vitro models and regenerative medicine. To this day, there is an unmet need to enrich these cells from a heterogeneous cell population for clinical applications without irreversible manipulation. We identified a method to propagate human NCSs via computational analysis of their mechanical signature. In this study, we report a novel analytical method for mechanical forces in three-dimensional cultures. Further, our results revealed that stemness may, in part, be mediated by physical properties of the extracellular matrix. In conclusion, our findings have potential implications in understanding stem cell mechanobiology for enrichment or differentiation.

Anisotropic traction stresses and focal adhesion polarization mediates topography-induced cell elongation.

Cell elongation and differentiation has been shown to be modulated by topographical cues provided by grating substratum. However, little is known about the mechanisms and forces involved in the grating-induced cell elongation, due to the difficulty in fabricating soft elastic gels that allow 3-dimensional (3D) cell traction stress measurements. In this paper, we present a method to fabricate soft elastic polyacrylamide grating substrates, using an imprinted polyethylene terephthalate mould, for 3D cell traction stress measurements. Fibroblasts were observed to form protrusions in the grating grooves, and elongate and align parallel to the grating direction on the soft polyacrylamide grating substrates. Focal adhesions were also found to be aligned parallel to the grating direction as compared to cells on flat substrates, suggesting that grating grooves restrict focal adhesion growth perpendicular to the grating direction. The 3D traction stress measurements revealed that highly elongated fibroblasts on grating substrates exert anisotropic traction stresses, in the direction parallel to the grating direction. We propose that focal adhesion alignment along the grating direction may result in increased actin stress fibre formation in the direction parallel to the grating, leading to polarized traction stresses which drive cell elongation.

Enhanced Delta-Notch Lateral Inhibition Model Incorporating Intracellular Notch Heterogeneity and Tension-Dependent Rate of Delta-Notch Binding that Reproduces Sprouting Angiogenesis Patterns.

Endothelial cells adopt unique cell fates during sprouting angiogenesis, differentiating into tip or stalk cells. The fate selection process is directed by Delta-Notch lateral inhibition pathway. Classical Delta-Notch models produce a spatial pattern of tip cells separated by a single stalk cell, or the salt-and-pepper pattern. However, classical models cannot explain alternative tip-stalk patterning, such as tip cells that are separated by two or more stalk cells. We show that lateral inhibition models involving only Delta and Notch proteins can also recapitulate experimental tip-stalk patterns by invoking two mechanisms, specifically, intracellular Notch heterogeneity and tension-dependent rate of Delta-Notch binding. We introduce our computational model and analysis where we establish that our enhanced Delta-Notch lateral inhibition model can recapitulate a greater variety of tip-stalk patterning which is previously not possible using classical lateral inhibition models. In our enhanced Delta-Notch lateral inhibition model, we observe the existence of a hybrid cell type displaying intermediate tip and stalk cells' characteristics. We validate the existence of such hybrid cells by immuno-staining of endothelial cells with tip cell markers, Delta and CD34, which substantiates our enhanced model.