Directed Cell Migration Research Articles

Multiomics of Bohring-Opitz syndrome truncating ASXL1 mutations identify canonical and noncanonical Wnt signaling dysregulation.

ASXL1 (additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo protein-truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS; OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, distinctive facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies, including heart defects and severe skeletal defects giving rise to a typical BOS posture. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia. We used primary cells from individuals with BOS (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multiomics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data show that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, which encodes a planar cell polarity pathway protein that acts through noncanonical Wnt signaling to direct tissue patterning and cell migration. This multiomics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.

Actin-binding protein filamin B regulates the cell-surface retention of endothelial sphingosine 1-phosphate receptor 1

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor essential for vascular development and postnatal vascular homeostasis. When exposed to sphingosine 1-phosphate (S1P) in the blood of ∼1μM, S1PR1 in endothelial cells retains cell-surface localization, while lymphocyte S1PR1 shows almost complete internalization, suggesting the cell-surface retention of S1PR1 is endothelial cell specific. To identify regulating factors that function to retain S1PR1 on the endothelial cell surface, here we utilized an enzyme-catalyzed proximity labeling technique followed by proteomic analyses. We identified Filamin B (FLNB), an actin-binding protein involved in F-actin cross-linking, as a candidate regulating protein. We show FLNB knockdown by RNA interference induced massive internalization of S1PR1 into early endosomes, which was partially ligand dependent and required receptor phosphorylation. Further investigation showed FLNB was also important for the recycling of internalized S1PR1 back to the cell surface. FLNB knockdown did not affect the localization of S1PR3, another S1P receptor subtype expressed in endothelial cells, nor did it affect localization of ectopically expressed β2-adrenergic receptor. Functionally, we show FLNB knockdown in endothelial cells impaired S1P-induced intracellular phosphorylation events and directed cell migration and enhancement of the vascular barrier. Taken together, our results demonstrate that FLNB is a novel regulator critical for S1PR1 cell-surface localization and thereby proper endothelial cell function.

A Yap-dependent mechanoregulatory program sustains cell migration for embryo axis assembly

The assembly of the embryo’s primary axis is a fundamental landmark for the establishment of the vertebrate body plan. Although the morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are well-known transcriptional mechanotransducers, yet their role in gastrulation remains elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure, due to reduced displacement and migratory persistence in mutant cells. Accordingly, we identified genes involved in cytoskeletal organization and cell-ECM adhesion as potentially direct Yap targets. Dynamic analysis of live sensors and downstream targets reveal that Yap is acting in migratory cells, promoting cortical actin and focal adhesions recruitment. Our results indicate that Yap coordinates a mechanoregulatory program to sustain intracellular tension and maintain the directed cell migration for embryo axis development.

Reactive oxygen species produced by myeloid cells in psoriasis as a potential biofactor contributing to the development of vascular inflammation.

Psoriasis is an immune-mediated inflammatory skin disease driven by interleukin-17A (IL-17A) and associated with cardiovascular dysfunction. We used a severe psoriasis mouse model of keratinocyte IL-17A overexpression (K14-IL-17Aind/+ , IL-17Aind/+ control mice) to investigate the activity of neutrophils and a potential cellular interconnection between skin and vasculature. Levels of dermal reactive oxygen species (ROS) and their release by neutrophils were measured by lucigenin-/luminol-based assays, respectively. Quantitative RT-PCR determined neutrophilic activity and inflammation-related markers in skin and aorta. To track skin-derived immune cells, we used PhAM-K14-IL-17Aind/+ mice allowing us to mark all cells in the skin by photoconversion of a fluorescent protein to analyze their migration into spleen, aorta, and lymph nodes by flow cytometry. Compared to controls, K14-IL-17Aind/+ mice exhibited elevated ROS levels in the skin and a higher neutrophilic oxidative burst accompanied by the upregulation of several activation markers. In line with these results psoriatic mice displayed elevated expression of genes involved in neutrophil migration (e.g., Cxcl2 and S100a9) in skin and aorta. However, no direct immune cell migration from the psoriatic skin into the aortic vessel wall was observed. Neutrophils of psoriatic mice showed an activated phenotype, but no direct cellular migration from the skin to the vasculature was observed. This suggests that highly active vasculature-invading neutrophils must originate directly from the bone marrow. Hence, the skin-vasculature crosstalk in psoriasis is most likely based on the systemic effects of the autoimmune skin disease, emphasizing the importance of a systemic therapeutic approach for psoriasis patients.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

Investigating the role of Mesp1-mediated mechanisms in directing cell migration and cell fate of distinct cardiac progenitor subpopulations

The heart is composed of four cardiac chambers built by distinct progenitor populations. Precise positioning of these subpopulations is critical for normal heart development. These progenitor subpopulations are thus implicated in congenital heart diseases. Elucidation of the molecular mechanisms involved in the specification and migration of these subpopulations will therefore contribute to a better understanding of the etiology of congenital heart diseases and towards improving therapies for cardiac diseases. Cardiac progenitors arise in two waves during gastrulation, both expressing key cardiac transcription factor Mesp1. Subsequently, they migrate to their defined destinations in the embryo. These Mesp1-expressing cells are heterogeneous, and differentially express key transcriptional regulators and signalling pathway components. We wish to uncover the Mesp1-mediated mechanisms that drive the migration of the two distinct subpopulations. We use a combination of mouse embryos (in vivo) and embryonic stem cell-derived (in vitro) models to address our objective. We have uncovered that the heterogeneity in the cardiac progenitor populations extends to the activation of intracellular signalling pathways. This heterogeneity is correlated with their cell fate as progenitors of specific cardiac chambers. Whether this heterogeneity affects their migratory cell behaviour was unknown. We reveal that loss-of-function of the individual downstream pathways leads to distinct migratory phenotypes. We are therefore poised to ask if the priming of a cardiac progenitor population towards a specific cell fate instructs their migratory behaviour, or whether the route they take may restrict their cell fate. In summary, we have begun to reveal differences in the signalling requirements for the migration and cell fate of distinct Mesp1-expressing cardiac progenitor populations.

TIPE proteins control directional migration of human T cells via GPCR and lipid second messenger signaling

Abstract Infiltration of specific tissues by circulating leukocytes is a common pathogenic mechanism of inflammatory diseases. G-protein-coupled receptors (GPCR) are essential for sensing chemokine gradients by leukocytes during immune responses. However, how leukocytes integrate site-specific directional cues such as chemokine gradients and make directional decisions in human immune cells is largely unknown. The TNFa-induced protein 8-Like (TIPE or TNFAIP8) family proteins are newly described pilot proteins that control directional migration (chemotaxis) of leukocytes to targeted tissues in mice. Without TIPE proteins, leukocytes become directionless, but the underlying mechanism remains elusive. Using siRNA and biochemical methods, we demonstrated here that two TIPE family members, TNFAIP8 and TIPE2, synergistically controlled directional migration (chemotaxis) in human CD4 T cells: TNFAIP8, through interacting with the Ga subunit of heterotrimeric G-proteins, and TIPE2, through utilizing lipid second messenger signaling by binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3). Using site-directed mutagenesis, we identified the region of Ga that interacts with TNFAIP8, and the amino acids of TIPE2 protein crucial for its interaction with PIP2 and PIP3. We also discovered that TIPE protein membrane translocation was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm of human T cells integrating GPCR and phospholipid signaling pathways to control directional migration of human immune cells. Additionally, our findings have implications for therapeutic targeting of TIPE proteins in human inflammatory and autoimmune diseases. NIH 5R01AI136945

A compound PCP scheme underlies sequential rosettes-based cell intercalation.

The formation of sequential rosettes is a type of collective cell behavior recently discovered in the Caenorhabditis elegans embryo that mediates directional cell migration through sequential formation and resolution of multicellular rosettes involving the migrating cell and its neighboring cells along the way. Here, we show that a planar cell polarity (PCP)-based polarity scheme regulates sequential rosettes, which is distinct from the known mode of PCP regulation in multicellular rosettes during the process of convergent extension. Specifically, non-muscle myosin (NMY) localization and edge contraction are perpendicular to that of Van Gogh as opposed to colocalizing with Van Gogh. Further analyses suggest a two-component polarity scheme: one being the canonical PCP pathway with MIG-1/Frizzled and VANG-1/Van Gogh localized to the vertical edges, the other being MIG-1/Frizzled and NMY-2 localized to the midline/contracting edges. The NMY-2 localization and contraction of the midline edges also required LAT-1/Latrophilin, an adhesion G protein-coupled receptor that has not been shown to regulate multicellular rosettes. Our results establish a distinct mode of PCP-mediated cell intercalation and shed light on the versatile nature of the PCP pathway.

Soft topographical patterns trigger a stiffness-dependent cellular response to contact guidance.

Topographical patterns are a powerful tool to study directional migration. Grooved substrates have been extensively used as in vitro models of aligned extracellular matrix fibers because they induce cell elongation, alignment, and migration through a phenomenon known as contact guidance. This process, which involves the orientation of focal adhesions, F-actin, and microtubule cytoskeleton along the direction of the grooves, has been primarily studied on hard materials of non-physiological stiffness. But how it unfolds when the stiffness of the grooves varies within the physiological range is less known. Here we show that substrate stiffness modulates the cellular response to topographical contact guidance. We find that for fibroblasts, while focal adhesions and actin respond to topography independently of the stiffness, microtubules show a stiffness-dependent response that regulates contact guidance. On the other hand, both clusters and single breast carcinoma epithelial cells display stiffness-dependent contact guidance, leading to more directional and efficient migration when increasing substrate stiffness. These results suggest that both matrix stiffening and alignment of extracellular matrix fibers cooperate during directional cell migration, and that the outcome differs between cell types depending on how they organize their cytoskeletons.

Matrix Anisotropy Promotes a Transition of Collective to Disseminated Cell Migration via a Collective Vortex Motion.

Cells detached and disseminated away from collectively migrating cells are frequently found during tumor invasion at the invasion front, where extracellular matrix (ECM) fibers are parallel to the cell migration direction. However, it remains unclear how anisotropic topography promotes the transition of collective to disseminated cell migration. This study applies a collective cell migration model with and without 800 nm wide aligned nanogrooves parallel, perpendicular, or diagonal to the cell migration direction. After 120 hour migration, MCF7-GFP-H2B-mCherry breast cancer cells display more disseminated cells at the migration front on parallel topography than on other topographies. Notably, a fluid-like collective motion with high vorticity is enhanced at the migration front on parallel topography. Furthermore, high vorticity but not velocity is correlated with disseminated cell numbers on parallel topography. Enhanced collective vortex motion colocalizes with cell monolayer defects where cells extend protrusions into the free space, suggesting that topography-driven cell crawling for defect closure promotes the collective vortex motion. In addition, elongated cell morphology and frequent protrusions induced by topography may further contribute to the collective vortex motion. Overall, a high-vorticity collective motion at the migration front promoted by parallel topography suggests a cause of the transition of collective to disseminated cell migration.

The Formation of NETs and Their Mechanism of Promoting Tumor Metastasis.

Neutrophil extracellular traps (NETs) are network structures comprised of decondensed DNA strands coated with granule proteins. There have been three types of NETs recorded. NETs have been discovered concerning the progression of some malignancies, including gastric cancer, breast cancer, ovarian cancer, hepatocellular carcinoma, colorectal cancer, glioblastoma, diffuse large B cell lymphoma (DLBCL), and lung cancer, among others. In various methods, tumors encourage the formation of NETs, and NETs, in turn, promote tumor growth. NETs can stimulate primary tumor cell proliferation, suppress immune cells to create a tumor-friendly immune microenvironment, and stimulate epithelial-mesenchymal transition (EMT). NETs significantly promote liver and lung metastasis, possibly by altering vascular permeability, inducing cytoskeleton rearrangement and directional cell migration, and reawakening dormant cancer cells. NETs are therapeutically promising targets for cancer patients. Cancer patients may benefit from anti-NETs therapy, especially when combined with immune checkpoint inhibitors.

Multi-omics analysis reveals focal adhesion characteristic associated tumor immune microenvironment in colon adenocarcinoma.

Colon adenocarcinoma (COAD) is one of the most frequent malignant lesions of the digestive system in humans, with an insidious onset. At the time of diagnosis, most of them have developed to the middle and late stages, and cancer cells have metastasized, and the prognosis is poor. Treatment options for progressive COAD are limited, and despite the promise of immunotherapy, immunotherapy response rates are low. The assembly and disaggregation of focal adhesion are critical for the directional migration of tumor cells to different sites, and it is unclear whether focal adhesion-related genes are involved in the development and prognosis of colon adenocarcinoma. This study aimed to investigate the role of focal adhesion genes in the occurrence and prognosis of COAD. We obtained datasets of COAD patients, including RNA-sequencing data and clinical information, from the TCGA and GEO databases (GSE17538 and GSE39582). Through CNMF clustering, two molecular subtypes with different expression patterns of focal adhesion genes were identified, and it was found that the molecular subtype with low expression of focal adhesion genes had better prognosis. Then the prediction signature was constructed by LASSO-Cox regression model, and the receiver operating characteristic (ROC) curve showed that the 4-gene signature had a good prediction effect on COAD 1-, 2-, and 3-year OS. Gene function enrichment analysis showed that the high-risk group was mainly enriched in immune and adhesion-related signaling pathways, suggesting that focal adhesion genes may affect the development and prognosis of COAD by regulating the immune microenvironment and tumor metastasis. The interaction between focal adhesion genes and immunity during the occurrence of COAD may help improve the response rate of immunotherapy, which also provides new ideas for the molecular mechanism and targeted therapy in COAD.

Abstract P6-14-07: Genetic ablation of ACF7, a member of the +TIP family of microtubule-binding proteins, impairs metastasis of HER2-positive breast cancer

Abstract Breast cancer is the leading cause of cancer death in women worldwide. Among the different subtypes of this disease, HER2-positive breast cancer is known to be one of the most aggressive and is linked to poor prognosis. This subtype is prone to develop metastases, reflecting a significant cellular plasticity. While the molecular machinery controlling actin dynamics is well implicated in cell invasion, the contribution of the microtubules is ill-defined. We recently conducted a functional screen to identify such regulators, and characterized ACF7, a microtubule plus-end binding protein (+TIP), as a new promoter of invasion. Here, we aim to determine the in vivo contribution of ACF7 to tumor progression and to reveal the cellular and molecular mechanisms allowing this +TIP to promote metastasis. We analyzed the expression of ACF7 at the protein level across a panel of tumor microarrays and found that it is detectable in all breast cancer subtypes with the highest levels seen in HER2-positive. To study the role of ACF7 during tumor progression, we generated an ACF7 genetically engineered mouse model of breast cancer. We exploited a HER2-positive breast cancer mouse model (MMTV-NIC) that we bred with ACF7Flox mice. This approach allows for conditional deletion of ACF7 in mammary glands and represents a powerful model to define the roles of ACF7 in HER2-driven breast cancer. Our results show that ACF7 is not involved in tumor initiation and tumor growth as no difference between number of nodules per mouse, tumor mass or number of mammary intraepithelial neoplastic lesions was shown. However, our results show a significant decrease in lung metastasis in the ACF7cKO mice. To gain mechanistic insights into the roles of ACF7 in this specific process, we derived primary cell lines from tumors isolated from MMTV-NIC-ACF7WT and MMTV-NIC-ACF7cKO mice. We explored if ACF7 contributes to migration and invasion in a HER2 context. By using live-cell imaging, we demonstrated that ACF7-null cells display defects in both random and directed cell migration, as they show a delay in wound closure. We determined that ACF7KO cells show an increase in focal adhesions size, and exhibit defects in reorienting their actin and microtubules cytoskeletons parallel to the direction of migration. ACF7 was also found to promote cell invasion since ACF7KO cells were less efficient to cross a Matrigel membrane. To investigate the metastatic potential of ACF7-null tumors, we performed RNA-sequencing using RNA isolated from MMTV-NIC-ACF7WT and MMTV-NIC-ACF7cKO tumors. We identified a striking change in expression of genes involved in epithelial to mesenchymal transition (EMT), such as for example E-cadherin, Vimentin or the transcription factor Twist. These results suggest that ACF7 promotes metastasis through maintaining an EMT state in HER2 breast cancer cells. Collectively, our work demonstrates ACF7 as an important player in HER2 positive breast cancer progression via its functions in both cell migration and cell invasion. This works also suggests that developing approaches to therapeutically target regulators of microtubule dynamics may reveal new opportunities to decrease tumor progression and metastatic expansion. Citation Format: Rebecca Cusseddu, Jean-François Côté. Genetic ablation of ACF7, a member of the +TIP family of microtubule-binding proteins, impairs metastasis of HER2-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-07.

Localization of Kif1c mRNA to cell protrusions dictates binding partner specificity of the encoded protein.

Subcellular localization of messenger RNA (mRNA) is a widespread phenomenon that can impact the regulation and function of the encoded protein. In nonneuronal cells, specific mRNAs localize to cell protrusions, and proper mRNA localization is required for cell migration. However, the mechanisms by which mRNA localization regulates protein function in this setting remain unclear. Here, we examined the functional consequences of localization of the mRNA encoding KIF1C. KIF1C is a kinesin motor protein required for cell migration and mRNA trafficking, including trafficking of its own mRNA. We show that Kif1c mRNA localization does not regulate KIF1C's protein abundance, distribution, or ability to traffic other mRNAs. Conversely, Kif1c mRNA localization to protrusions is required for directed cell migration. We used mass spectrometry to identify binding partners of endogenous KIF1C, which revealed dramatic dysregulation of the number and identity of KIF1C interactors in response to Kif1c mRNA mislocalization. These results therefore uncovered a mechanistic connection between mRNA localization to cell protrusions and the specificity of protein-protein interactions. We anticipate that this mechanism is not limited to Kif1c and is likely to be a general principle that impacts the functions of proteins encoded by protrusion-enriched mRNAs in nonneuronal cells.

Coordinated regulation of Cdc42ep1, actin, and septin filaments during neural crest cell migration.

The septin cytoskeleton has been demonstrated to interact with other cytoskeletal components to regulate various cellular processes, including cell migration. However, the mechanisms of how septin regulates cell migration are not fully understood. In this study, we use the highly migratory neural crest cells of frog embryos to examine the role of septin filaments in cell migration. We found that septin filaments are required for the proper migration of neural crest cells by controlling both the speed and the direction of cell migration. We further determined that septin filaments regulate these features of cell migration by interacting with actin stress fibers. In neural crest cells, septin filaments co-align with actin stress fibers, and the loss of septin filaments leads to impaired stability and contractility of actin stress fibers. In addition, we showed that a partial loss of septin filaments leads to drastic changes in the orientations of newly formed actin stress fibers, suggesting that septin filaments help maintain the persistent orientation of actin stress fibers during directed cell migration. Lastly, our study revealed that these activities of septin filaments depend on Cdc42ep1, which colocalizes with septin filaments in the center of neural crest cells. Cdc42ep1 interacts with septin filaments in a reciprocal manner, with septin filaments recruiting Cdc42ep1 to the cell center and Cdc42ep1 supporting the formation of septin filaments.

Effect of Nanosecond Pulsed Currents on Directions of Cell Elongation and Migration through Time-Lapse Analysis.

It is generally known that cells elongate perpendicularly to an electric field and move in the direction of the field when an electric field is applied. We have shown that irradiation of plasma-simulated nanosecond pulsed currents elongates cells, but the direction of cell elongation and migration has not been elucidated. In this study, a new time-lapse observation device that can apply nanosecond pulsed currents to cells was constructed, and software to analyze cell migration was created to develop a device that can sequentially observe cell behavior. The results showed nanosecond pulsed currents elongate cells but do not affect the direction of elongation and migration. It was also found the behavior of cells changes depending on the conditions of the current application.

The androgen receptor as a potential regulator of prostate cancer contractility.

Prostate cancer (PCa) is a common health hazard for men that displays androgen-dependent growth at early stages through signalling mediated by the Androgen Receptor (AR). This has inspired androgen deprivation therapy (ADT) to be the gold standard treatment; however, in later stages, PCa displays a transition to androgen-independence and loss of AR, accompanied by a drastic increase in invasiveness. Critically, both the loss of AR and ADT have been shown to induce PCa invasiveness by promoting the epithelial-to-mesenchymal transition (EMT) which has been associated with biophysical changes in other cell models. Indeed, during metastasis cancers cells change their biophysical properties (reduced cell stiffness, increased contractility) facilitating migration through crowded microenvironments. Given AR's essential role in mediating PCa progression, we suspect that AR signalling may also regulate mechanical transitions required for PC metastasis and directed cell migration. In this study we characterize PCa biophysics in response to changes in AR signalling and expression. First, we applied Traction Force Microscopy on the metastatic PCa cell line LNCaP (AR-positive) to quantify contractile changes from androgen deprivation and supplementation, observing a non-monotonic contractile response with increasing androgen dose. These results suggest that the androgen-induced biophysical response shifts depending on the androgen availability, a finding consistent with past reports demonstrating that distinct pathways depend on both androgen concentration and AR expression. Regarding the latter, we then evaluated how AR expression and activity (nuclear localization) affects PCa biophysics by quantifying the contractility of highly metastatic PC3 cells with and without AR expression under varying androgen concentrations. We find that cell contractility is positively correlated with AR nuclear localization in androgen-containing media, yet negatively correlated in androgen-deprived media. These results complement emerging trends suggesting AR may have an important mechanobiological role in PCa migration and metastasis.

External bioelectric control of tissue hydrostatic pressure and ion pumping.

Direct-Current (DC) currents exist throughout the body and regulate cellular responses spanning directed cell migration (electrotaxis) to transcriptional regulation. New approaches using electrical cues to control biological systems are, therefore, quite exciting, and we have previously demonstrated electrically programmable collective migration in epithelia. Here, we demonstrate those same cues reprogramming 3D growth in lumenized tissues such as hollow kidney spheroids and intestinal organoids. We found that electrical stimulation induces a rapid, powerful, and reversible swelling in these tissues, and we discuss how external electrical stimulation can control the inflation of lumenized structures and cellular mechanisms mediating the response. We first studied hollow kidney spheroids within a hydrogel integrated into our SCHEEPDOG electrobioreactor. This allowed 3D, timelapse imaging during electrical stimulation. Hollow spheroids inflated nearly 3X by volume relative to control spheroids within 4 hrs of stimulation. The rate and magnitude of inflation increased with increasing electric field strength, and overly strong field gradients induced ‘popping’. We hypothesized that the dense actomyosin network at the apical surface of the structures provided a physical limit to the inflation response, and showed that inhibiting actomyosin contractility prevented popping and increased the maximum swelling volume. We used ion channel inhibitors to determine that the majority of the response can be attributed to the CFTR ion channel (apical chloride channel) and NKCC (basal sodium/potassium channel). Our computational model relating direct-current fields, electrochemistry, and hydrostatic pressure capture many of these effects, as well as predicting asymmetric ion distributions around spheroids. Interestingly, 3D cross-sections of inflated tissues reveal asymmetric cell shapes on the anode/cathode cysts of the spheroids. We have recently confirmed that mouse intestinal organoids also inflate upon field stimulation in a CFTR-mediated fashion, and are exploring other lumenized tissues to assess the generality of this phenomenon.

The Actin Crosslinker Fascin Regulates Cell Chirality.

The left-right (L-R) asymmetry of the cells, or cell chirality, is a well-known intrinsic property derived from the dynamic organization of the actin cytoskeleton. Cell chirality can be regulated by actin-binding proteins such as α-actinin-1 and can also be mediated by certain signaling pathways, such as protein kinase C (PKC) signaling. Fascin, an actin crosslinker known to mediate parallel bundling of actin filaments, appears as a prominent candidate in cell chirality regulation, given its role in facilitating cell migration as an important PKC substrate. Here, it is shown that the chirality of NIH/3T3 cells can be altered by PKC activation and fascin manipulation. With either small-molecule drug inhibition or genetic knockdown of fascin, the chirality of 3T3 cells is reversed from a clockwise (CW) bias to a counterclockwise (CCW) bias on ring-shaped micropatterns, accompanied by the reversal in cell directional migration. The Ser-39 fascin-actin binding sites are further explored in cell chirality regulation. The findings of this study reveal the critical role of fascin as an important intermediator in cell chirality, shedding novel insights into the mechanisms of L-R asymmetric cell migration and multicellular morphogenesis.

Heparin Specifically Interacts with Basic BBXB Motifs of the Chemokine CCL21 to Define CCR7 Signaling.

Chemokines are critically involved in controlling directed leukocyte migration. Spatiotemporal secretion together with local retention processes establish and maintain local chemokine gradients that guide directional cell migration. Extracellular matrix proteins, particularly glycosaminoglycans (GAGs), locally retain chemokines through electrochemical interactions. The two chemokines CCL19 and CCL21 guide CCR7-expressing leukocytes, such as antigen-bearing dendritic cells and T lymphocytes, to draining lymph nodes to initiate adaptive immune responses. CCL21-in contrast to CCL19-is characterized by a unique extended C-terminus composed of highly charged residues to facilitate interactions with GAGs. Notably, both chemokines can trigger common, but also ligand-biased signaling through the same receptor. The underlying molecular mechanism of ligand-biased CCR7 signaling is poorly understood. Using a series of naturally occurring chemokine variants in combination with newly designed site-specific chemokine mutants, we herein assessed CCR7 signaling, as well as GAG interactions. We demonstrate that the charged chemokine C-terminus does not fully confer CCL21-biased CCR7 signaling. Besides the positively charged C-terminus, CCL21 also possesses specific BBXB motifs comprising basic amino acids. We show that CCL21 variants where individual BBXB motifs are mutated retain their capability to trigger G-protein-dependent CCR7 signaling, but lose their ability to interact with heparin. Moreover, we show that heparin specifically interacts with CCL21, but not with CCL19, and thereby competes with ligand-binding to CCR7 and prevents signaling. Hence, we provide evidence that soluble heparin, but not the other GAGs, complexes with CCL21 to define CCR7 signaling in a ligand-dependent manner.