CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization.

BACKGROUND: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. METHODS: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. FINDINGS: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. INTERPRETATION: CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Tumor Characteristics and Treatment Responsiveness in Pembrolizumab-Treated Non-Small Cell Lung Carcinoma.

Pembrolizumab, a widely used immune checkpoint inhibitor (ICI), has revolutionized the treatment of non-small cell lung cancer (NSCLC). Identifying unique tumor characteristics in patients likely to respond to pembrolizumab could help the clinical adjudication and development of a personalized therapeutic strategy. In this retrospective study, we reviewed the clinical data and pathological features of 84 NSCLC patients treated with pembrolizumab. We examined the correlation between the clinical and demographic characteristics and the tumor histopathologic features obtained before immunotherapy. The response to pembrolizumab therapy was evaluated via the Response Evaluation Criteria in Solid Tumors (RECIST). The clinical data and cancer tissue characteristics were assessed and compared among three groups according to the following RECIST: the responsive group (RG), the stable disease group (SD), and the progressive disease group (PD), where the RG comprised patients with either a complete response (CR) or a partial response (PR). The overall survival rate of the RG group was significantly higher than the SD and PD groups. In addition, the percentage of pre-treatment viable tumor cell content in the RG and SD groups was significantly higher. At the same time, the extracellular stroma proportion was significantly lower than that of the PD group. The number of tumor-infiltrating lymphocytes (TILs) in the RG group was significantly higher than in the PD group. There were no significant differences in tumor necrosis, the stroma composition, PD-L1 expression level (TPS 1-49% vs. ≥50%), and treatment response. In conclusion, our population of NSCLC patients who experienced positive treatment responses to pembrolizumab therapy had a better prognosis compared to patients with either SD or PD. Moreover, the relative proportions of viable tumor cells to tumor-associated lymphocytes were associated with responsiveness to treatment. It is expected that larger prospective clinical studies will further validate these findings.

FAK and p130Cas modulate stiffness-mediated early transcription and cellular metabolism.

Cellular metabolism is influenced by the stiffness of the extracellular matrix. Focal adhesion kinase (FAK) and its binding partner, p130Cas, transmit biomechanical signals about substrate stiffness to the cell to regulate a variety of cellular responses, but their roles in early transcriptional and metabolic responses remain largely unexplored. We cultured mouse embryonic fibroblasts with or without siRNA-mediated FAK or p130Cas knockdown and assessed the early transcriptional responses of these cells to placement on soft and stiff substrates by RNA sequencing and bioinformatics analyses. Exposure to the stiff ECM altered the expression of genes important for metabolic and biosynthetic processes, and these responses were influenced by knockdown of FAK and p130Cas. Our findings reveal that FAK-p130Cas signaling mechanotransduces ECM stiffness to early transcriptional changes that alter cellular metabolism and biosynthesis.

Actin-binding protein profilin1 is an important determinant of cellular phosphoinositide control.

Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.

Survivin as a mediator of stiffness-induced cell cycle progression and proliferation of vascular smooth muscle cells.

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers a phenotypic switch, hypermigration, and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation. However, the mechanism underlying this trigger remains unknown. Our analyses of whole-transcriptome microarray data from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology identified 623 genes that were significantly and differentially expressed (360 upregulated and 263 downregulated) relative to expression in VSMCs cultured on soft hydrogels. Functional enrichment and gene network analyses revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Importantly, we found that survivin, an inhibitor of apoptosis protein, mediates stiffness-dependent cell cycle progression and proliferation as determined by gene network and pathway analyses, RT-qPCR, immunoblotting, and cell proliferation assays. Furthermore, we found that inhibition of cell cycle progression did not reduce survivin expression, suggesting that survivin functions as an upstream regulator of cell cycle progression and proliferation in response to ECM stiffness. Mechanistically, we found that the stiffness signal is mechanotransduced via the FAK-E2F1 signaling axis to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Overall, our findings indicate that survivin is necessary for VSMC cycling and proliferation and plays a role in regulating stiffness-responsive phenotypes.

Survivin regulates intracellular stiffness and extracellular matrix production in vascular smooth muscle cells.

Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs significantly decreased the stiffness-mediated expression of ECM components related to arterial stiffening, such as collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these ECM proteins was rescued by ectopic expression of survivin in human VSMCs cultured on soft hydrogels. Interestingly, atomic force microscopy analysis showed that suppressed or ectopic expression of survivin decreases or increases intracellular stiffness, respectively. Furthermore, we observed that inhibiting Rac and Rho reduces survivin expression, elucidating a mechanical pathway connecting intracellular tension, mediated by Rac and Rho, to survivin induction. Finally, we found that survivin inhibition decreases FAK phosphorylation, indicating that survivin-dependent intracellular tension feeds back to maintain signaling through FAK. These findings suggest a novel mechanism by which survivin potentially modulates arterial stiffness.

Key role for Rac in the early transcriptional response to extracellular matrix stiffness and stiffness-dependent repression of ATF3.

The Rho family GTPases Rac and Rho play critical roles in transmitting mechanical information contained within the extracellular matrix (ECM) to the cell. Rac and Rho have well-described roles in regulating stiffness-dependent actin remodeling, proliferation and motility. However, much less is known about the relative roles of these GTPases in stiffness-dependent transcription, particularly at the genome-wide level. Here, we selectively inhibited Rac and Rho in mouse embryonic fibroblasts cultured on deformable substrata and used RNA sequencing to elucidate and compare the contribution of these GTPases to the early transcriptional response to ECM stiffness. Surprisingly, we found that the stiffness-dependent activation of Rac was dominant over Rho in the initial transcriptional response to ECM stiffness. We also identified activating transcription factor 3 (ATF3) as a major target of stiffness- and Rac-mediated signaling and show that ATF3 repression by ECM stiffness helps to explain how the stiffness-dependent activation of Rac results in the induction of cyclin D1.

Neuronal actin cytoskeleton gain of function in the human brain.

BACKGROUND: While advancements in imaging techniques have led to major strides in deciphering the human brain, successful interventions are elusive and represent some of the most persistent translational gaps in medicine. Human restricted CHRFAM7A has been associated with neuropsychiatric disorders. METHODS: The physiological role of CHRFAM7A in human brain is explored using multiomics approach on 600 post mortem human brain tissue samples. The emerging pathways and mechanistic hypotheses are tested and validated in an isogenic hiPSC model of CHRFAM7A knock-in medial ganglionic eminence progenitors and neurons. FINDINGS: CHRFAM7A is identified as a modulator of intracellular calcium dynamics and an upstream regulator of Rac1. Rac1 activation re-designs the actin cytoskeleton leading to dynamic actin driven remodeling of membrane protrusion and a switch from filopodia to lamellipodia. The reinforced cytoskeleton leads to an advantage to tolerate stiffer mechanical properties of the extracellular environment. INTERPRETATION: CHRFAM7A modifies the actin cytoskeleton to a more dynamic and stiffness resistant state in an α7nAChR dependent manner. CHRFAM7A may facilitate neuronal adaptation to changes in the brain environment in physiological and pathological conditions contributing to risk or recovery. Understanding how CHRFAM7A affects human brain requires human studies in the areas of memory formation and erasure, cognitive reserve, and neuronal plasticity. FUNDING: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti). Also, in part by the International Society for Neurochemistry (ISN) and The Company of Biologists (Nicolas Rosas). ROSMAP is supported by NIA grants P30AG10161, P30AG72975, R01AG15819, R01AG17917. U01AG46152, and U01AG61356.

Model-based investigation of elasticity and spectral exponent from atomic force microscopy and electrophysiology in normal versus Schizophrenia human cerebral organoids.

The physiological origin of the aperiodic signal present in the electrophysiological recordings, called l/f neural noise, is unknown; nevertheless, it has been associated with health and disease. The power spectrum slope, -α in 1/fα, has been postulated to be related to the dynamic balance between excitation (E) and inhibition (I). Our study found that human cerebral organoids grown from induced pluripotent stem cells (iPSCs) from Schizophrenia patients (SCZ) showed structural changes associated with altered elasticity compared to that of the normal cerebral organoids. Furthermore, mitochondrial drugs modulated the elasticity in SCZ that was found related to the changes in the spectral exponent. Therefore, we developed an electro-mechanical model that related the microtubular-actin tensegrity structure to the elasticity and the 1/fα noise. Model-based analysis showed that a decrease in the number and length of the constitutive elements in the tensegrity structure decreased its elasticity and made the spectral exponent more negative while thermal white noise will make α = 0.. Based on the microtubularactin model and the cross-talk in structural (elasticity) and functional (electrophysiology) response, aberrant mitochondrial dynamics in SCZ are postulated to be related to the deficits in mitochondrial-cytoskeletal interactions for long-range transport of mitochondria to support synaptic activity for E/I balance. Clinical Relevance-Our experimental data and modeling present a structure-function relationship between mechanical elasticity and electrophysiology of human cerebral organoids that differentiated SCZ patients from normal controls.

A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation.

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis.

Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation.

Cell cycle control is a key aspect of numerous physiological and pathological processes. The contribution of biophysical cues, such as stiffness or elasticity of the underlying extracellular matrix (ECM), is critically important in regulating cell cycle progression and proliferation. Indeed, increased ECM stiffness causes aberrant cell cycle progression and proliferation. However, the molecular mechanisms that control these stiffness-mediated cellular responses remain unclear. Here, we address this gap and show good evidence that lamellipodin (symbol RAPH1), previously known as a critical regulator of cell migration, stimulates ECM stiffness-mediated cyclin expression and intracellular stiffening in mouse embryonic fibroblasts. We observed that increased ECM stiffness upregulates lamellipodin expression. This is mediated by an integrin-dependent FAK-Cas-Rac signaling module and supports stiffness-mediated lamellipodin induction. Mechanistically, we find that lamellipodin overexpression increased, and lamellipodin knockdown reduced, stiffness-induced cell cyclin expression and cell proliferation, and intracellular stiffness. Overall, these results suggest that lamellipodin levels may be critical for regulating cell proliferation. This article has an associated First Person interview with the first author of the paper.

Emerging machine learning approaches to phenotyping cellular motility and morphodynamics.

Cells respond heterogeneously to molecular and environmental perturbations. Phenotypic heterogeneity, wherein multiple phenotypes coexist in the same conditions, presents challenges when interpreting the observed heterogeneity. Advances in live cell microscopy allow researchers to acquire an unprecedented amount of live cell image data at high spatiotemporal resolutions. Phenotyping cellular dynamics, however, is a nontrivial task and requires machine learning (ML) approaches to discern phenotypic heterogeneity from live cell images. In recent years, ML has proven instrumental in biomedical research, allowing scientists to implement sophisticated computation in which computers learn and effectively perform specific analyses with minimal human instruction or intervention. In this review, we discuss how ML has been recently employed in the study of cell motility and morphodynamics to identify phenotypes from computer vision analysis. We focus on new approaches to extract and learn meaningful spatiotemporal features from complex live cell images for cellular and subcellular phenotyping.

Development of a decellularized meniscus matrix-based nanofibrous scaffold for meniscus tissue engineering.

The meniscus plays a critical role in knee mechanical function but is commonly injured given its central load bearing role. In the adult, meniscus repair is limited, given the low number of endogenous cells, the density of the matrix, and the limited vascularity. Menisci are fibrocartilaginous tissues composed of a micro-/nano- fibrous extracellular matrix (ECM) and a mixture of chondrocyte-like and fibroblast-like cells. Here, we developed a fibrous scaffold system that consists of bioactive components (decellularized meniscus ECM (dME) within a poly(e-caprolactone) material) fashioned into a biomimetic morphology (via electrospinning) to support and enhance meniscus cell function and matrix production. This work supports that the incorporation of dME into synthetic nanofibers increased hydrophilicity of the scaffold, leading to enhanced meniscus cell spreading, proliferation, and fibrochondrogenic gene expression. This work identifies a new biomimetic scaffold for therapeutic strategies to substitute or replace injured meniscus tissue. STATEMENT OF SIGNIFICANCE: In this study, we show that a scaffold electrospun from a combination of synthetic materials and bovine decellularized meniscus ECM provides appropriate signals and a suitable template for meniscus fibrochondrocyte spreading, proliferation, and secretion of collagen and proteoglycans. Material characterization and in vitro cell studies support that this new bioactive material is susceptible to enzymatic digestion and supports meniscus-like tissue formation.

Phosphoinositide Signaling and Mechanotransduction in Cardiovascular Biology and Disease.

Phosphoinositides, which are membrane-bound phospholipids, are critical signaling molecules located at the interface between the extracellular matrix, cell membrane, and cytoskeleton. Phosphoinositides are essential regulators of many biological and cellular processes, including but not limited to cell migration, proliferation, survival, and differentiation, as well as cytoskeletal rearrangements and actin dynamics. Over the years, a multitude of studies have uniquely implicated phosphoinositide signaling as being crucial in cardiovascular biology and a dominant force in the development of cardiovascular disease and its progression. Independently, the cellular transduction of mechanical forces or mechanotransduction in cardiovascular cells is widely accepted to be critical to their homeostasis and can drive aberrant cellular phenotypes and resultant cardiovascular disease. Given the versatility and diversity of phosphoinositide signaling in the cardiovascular system and the dominant regulation of cardiovascular cell functions by mechanotransduction, the molecular mechanistic overlap and extent to which these two major signaling modalities converge in cardiovascular cells remain unclear. In this review, we discuss and synthesize recent findings that rightfully connect phosphoinositide signaling to cellular mechanotransduction in the context of cardiovascular biology and disease, and we specifically focus on phosphatidylinositol-4,5-phosphate, phosphatidylinositol-4-phosphate 5-kinase, phosphatidylinositol-3,4,5-phosphate, and phosphatidylinositol 3-kinase. Throughout the review, we discuss how specific phosphoinositide subspecies have been shown to mediate biomechanically sensitive cytoskeletal remodeling in cardiovascular cells. Additionally, we discuss the direct interaction of phosphoinositides with mechanically sensitive membrane-bound ion channels in response to mechanical stimuli. Furthermore, we explore the role of phosphoinositide subspecies in association with critical downstream effectors of mechanical signaling in cardiovascular biology and disease.

Global Genome Conformational Programming during Neuronal Development Is Associated with CTCF and Nuclear FGFR1-The Genome Archipelago Model.

During the development of mouse embryonic stem cells (ESC) to neuronal committed cells (NCC), coordinated changes in the expression of 2851 genes take place, mediated by the nuclear form of FGFR1. In this paper, widespread differences are demonstrated in the ESC and NCC inter- and intra-chromosomal interactions, chromatin looping, the formation of CTCF- and nFGFR1-linked Topologically Associating Domains (TADs) on a genome-wide scale and in exemplary HoxA-D loci. The analysis centered on HoxA cluster shows that blocking FGFR1 disrupts the loop formation. FGFR1 binding and genome locales are predictive of the genome interactions; likewise, chromatin interactions along with nFGFR1 binding are predictive of the genome function and correlate with genome regulatory attributes and gene expression. This study advances a topologically integrated genome archipelago model that undergoes structural transformations through the formation of nFGFR1-associated TADs. The makeover of the TAD islands serves to recruit distinct ontogenic programs during the development of the ESC to NCC.

Cardiovascular protection in females linked to estrogen-dependent inhibition of arterial stiffening and macrophage MMP12.

Arterial stiffening is a consequence of aging and a cholesterol-independent risk factor for cardiovascular disease (CVD). Arterial stiffening and CVD show a sex bias, with men more susceptible than premenopausal women. How arterial stiffness and sex interact at a molecular level to confer risk of CVD is not well understood. Here, we used the sexual dimorphism in LDLR-null mice to show that the protective effect of female sex on atherosclerosis is linked to reduced aortic stiffness and reduced expression of matrix metalloproteinase-12 (MMP12) by lesional macrophages. Deletion of MMP12 in LDLR-null mice attenuated the male sex bias for both arterial stiffness and atherosclerosis, and these effects occurred despite high serum cholesterol. Mechanistically, we found that oxidized LDL stimulates secretion of MMP12 in human as well as mouse macrophages. Estrogen antagonizes this effect by downregulating MMP12 expression. Our data support cholesterol-independent causal relationships between estrogen, oxidized LDL-induced secretion of macrophage MMP12, and arterial stiffness that protect against atherosclerosis in females and emphasize that reduced MMP12 functionality can confer atheroprotection to males.

Analysis of Light Propagation on Physiological Properties of Neurons for Nanoscale Optogenetics.

Miniaturization of implantable devices is an important challenge for future brain-computer interface applications, and in particular for achieving precise neuron stimulation. For stimulation that utilizes light, i.e., optogenetics, the light propagation behavior and interaction at the nanoscale with elements within the neuron is an important factor that needs to be considered when designing the device. This paper analyzes the effect of light behavior for a single neuron stimulation and focuses on the impact from different cell shapes. Based on the Mie scattering theory, the paper analyzes how the shape of the soma and the nucleus contributes to the focusing effect resulting in an intensity increase, which ensures that neurons can assist in transferring light through the tissue toward the target cells. At the same time, this intensity increase can in turn also stimulate neighboring cells leading to interference within the neural circuits. This paper also analyzes the ideal placements of the device with respect to the angle and position within the cortex that can enable axonal biophoton communications, which can contain light within the cell to avoid the interference.

Predicting Ligand-Free Cell Attachment on Next-Generation Cellulose-Chitosan Hydrogels.

There is a growing appreciation that engineered biointerfaces can regulate cell behaviors, or functions. Most systems aim to mimic the cell-friendly extracellular matrix environment and incorporate protein ligands; however, the understanding of how a ligand-free system can achieve this is limited. Cell scaffold materials comprised of interfused chitosan-cellulose hydrogels promote cell attachment in ligand-free systems, and we demonstrate the role of cellulose molecular weight, MW, and chitosan content and MW in controlling material properties and thus regulating cell attachment. Semi-interpenetrating network (SIPN) gels, generated from cellulose/ionic liquid/cosolvent solutions, using chitosan solutions as phase inversion solvents, were stable and obviated the need for chemical coupling. Interface properties, including surface zeta-potential, dielectric constant, surface roughness, and shear modulus, were modified by varying the chitosan degree of polymerization and solution concentration, as well as the source of cellulose, creating a family of cellulose-chitosan SIPN materials. These features, in turn, affect cell attachment onto the hydrogels and the utility of this ligand-free approach is extended by forecasting cell attachment using regression modeling to isolate the effects of individual parameters in an initially complex system. We demonstrate that increasing the charge density, and/or shear modulus, of the hydrogel results in increased cell attachment.

Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging.

Cell protrusion is morphodynamically heterogeneous at the subcellular level. However, the mechanism of cell protrusion has been understood based on the ensemble average of actin regulator dynamics. Here, we establish a computational framework called HACKS (deconvolution of heterogeneous activity in coordination of cytoskeleton at the subcellular level) to deconvolve the subcellular heterogeneity of lamellipodial protrusion from live cell imaging. HACKS identifies distinct subcellular protrusion phenotypes based on machine-learning algorithms and reveals their underlying actin regulator dynamics at the leading edge. Using our method, we discover "accelerating protrusion", which is driven by the temporally ordered coordination of Arp2/3 and VASP activities. We validate our finding by pharmacological perturbations and further identify the fine regulation of Arp2/3 and VASP recruitment associated with accelerating protrusion. Our study suggests HACKS can identify specific subcellular protrusion phenotypes susceptible to pharmacological perturbation and reveal how actin regulator dynamics are changed by the perturbation.

Modulating cell response on cellulose surfaces; tunable attachment and scaffold mechanics.

Combining surface chemical modification of cellulose to introduce positively charged trimethylammonium groups by reaction with glycidyltrimethylammonium chloride (GTMAC) allowed for direct attachment of mammalian MG-63 cells, without addition of protein modifiers, or ligands. Very small increases in the surface charge resulted in significant increases in cell attachment: at a degree of substitution (DS) of only 1.4%, MG-63 cell attachment was > 90% compared to tissue culture plastic, whereas minimal attachment occurred on unmodified cellulose. Cell attachment plateaued above DS of ca. 1.85% reflecting a similar trend in surface charge, as determined from ζ-potential measurements and capacitance coupling (electric force microscopy). Cellulose film stiffness was modulated by cross linking with glyoxal (0.3-2.6% degree of crosslinking) to produce a range of materials with surface shear moduli from 76 to 448 kPa (measured using atomic force microscopy). Cell morphology on these materials could be regulated by tuning the stiffness of the scaffolds. Thus, we report tailored functionalised biomaterials based on cationic cellulose that can be tuned through surface reaction and glyoxal crosslinkin+g, to influence the attachment and morphology of cells. These scaffolds are the first steps towards materials designed to support cells and to regulate cell morphology on implanted biomaterials using only scaffold and cells, i.e. without added adhesion promoters.