Lamellipodia-Mediated Osteoblast Haptotaxis Guided by Fibronectin Ligand Concentrations on a Multiplex Chip.

Skull morphogenesis is a complex, dynamic process involving two different germ layers and progressing to the coordinated, directional growth of individual bones. The mechanisms underlying directional growth toward the apex are not completely understood. Here, a microfluidic chip-based approach is utilized to test whether calvarial osteoblast progenitors undergo haptotaxis on a gradient of Fibronectin1 (FN1) via lamellipodia. Mimicking the embryonic cranial mesenchyme's FN1 pattern, FN1 gradients is established in the chip using computer modeling and fluorescent labeling. Primary mouse calvarial osteoblast progenitors are plated in the chip along an array of segmented gradients of adsorbed FN1. The study performs single-cell tracking and measures protrusive activity. Haptotaxis is observed at an intermediate FN1 concentration, with an average directional migration index (yFMI) of 0.07, showing a significant increase compared to the control average yFMI of -0.01. A significant increase in protrusive activity is observed during haptotaxis. Haptotaxis is an Arp2/3-dependent, lamellipodia-mediated process. Calvarial osteoblast progenitors treated with the Arp2/3 (Actin Related Protein 2/3 complex) inhibitor CK666 show significantly diminished haptotaxis, with an average yFMI of 0.01. Together, these results demonstrate haptotaxis on an FN1 gradient as a new mechanism in the apical expansion of calvarial osteoblast progenitors during development and shed light on the etiology of calvarial defects.

Adhesion-clutch between DCC and netrin-1 mediates netrin-1-induced axonal haptotaxis.

The growth cone, a motile structure located at the tip of growing axons, senses extracellular guidance cues and translates them into directional forces that drive axon outgrowth and guidance. Axon guidance directed by chemical cues on the extracellular adhesive substrate is termed haptotaxis. Recent studies reported that netrin-1 on the substrate functions as a haptotactic axon guidance cue. However, the mechanism mediating netrin-1-induced axonal haptotaxis remains unclear. Here, we demonstrate that substrate-bound netrin-1 induces axonal haptotaxis by facilitating physical interactions between the netrin-1 receptor, DCC, and the adhesive substrates. DCC serves as an adhesion receptor for netrin-1. The clutch-linker molecule shootin1a interacted with DCC, linking it to actin filament retrograde flow at the growth cone. Speckle imaging analyses showed that DCC underwent either grip (stop) or retrograde slip on the adhesive substrate. The grip state was more prevalent on netrin-1-coated substrate compared to the control substrate polylysine, thereby transmitting larger traction force on the netrin-1-coated substrate. Furthermore, disruption of the linkage between actin filament retrograde flow and DCC by shootin1 knockout impaired netrin-1-induced axonal haptotaxis. These results suggest that the directional force for netrin-1-induced haptotaxis is exerted on the substrates through the adhesion-clutch between DCC and netrin-1 which occurs asymmetrically within the growth cone.

Global solvability of a chemotaxis-haptotaxis model in the whole 2-d space.

This paper investigates a two-dimensional chemotaxis-haptotaxis model $ \begin{eqnarray*} \left\{\begin{array}{lll} u_t = \Delta u-\chi\nabla\cdot(u\nabla v)-\xi\nabla\cdot(u\nabla w),&{} x\in\mathbb{R}^2,\ t>0,\\ v_t = \Delta v-v+u,&{}x\in\mathbb{R}^2,\ t>0,\\ w_t = -vw,&{}x\in\mathbb{R}^2,\ t>0, \end{array}\right. \end{eqnarray*} $ where $ \chi $ and $ \xi $ are positive parameters. It is proved that, for any suitable smooth initial data $ (u_0, v_0, w_0) $, this model admits a unique global strong solution if $ \left\|u_0\right\|_{L^1} < \frac{8 \pi}{\chi} $. Compared to the result by Calvez and Corrias (Calvez and Corrias, 2008 [1]), we can see that the haptotaxis effect is almost negligible in terms of global existence, which is consistent with the result of bounded domain (Jin and Xiang, 2021 [2]). Moreover, to the best of our knowledge, this is the first analytical work for the well-posedness of chemotaxis-haptotaxis system in the whole space.

Proteins Secreted by Lung Cancer Cells Induce the Onset of Proteinuria via Focal Adhesion Kinase Signaling in Mice.

Paraneoplastic nephrotic syndrome (PNS) is a complication seen in cancer patients. Ultrastructural examination shows the accumulation of proteins and the presence of foot process (FP) effacement in the glomeruli of PNS patients. Previously, we reported that orthotopic xenografts of Lewis lung carcinoma 1 in C57BL/6 mice caused them to develop lung cancer with albuminuria. This implies that these mice can be used as a model of human disease and suggests that Lewis lung carcinoma 1 cell-secreted proteins (LCSePs) contain nephrotoxic molecules and cause inflammation in renal cells. As podocyte effacement was present in glomeruli in this model, such podocyte injury may be attributable to either soluble LCSeP or LCSeP deposits triggering pathological progression. LCSePs in conditioned media was concentrated for nephrotoxicity testing. Integrin-focal adhesion kinase (FAK) signaling and inflammatory responses were evaluated in podocytes either exposed to soluble LCSePs or seeded onto substrates with immobilized LCSePs. FAK phosphorylation and interleukin-6 expression were higher in podocytes attached to LCSePs substrates than in those exposed to soluble LCSePs. Notably, LCSeP-based haptotaxis gave rise to altered signaling in podocytes. When podocytes were stimulated by immobilized LCSePs, FAK accumulated at focal adhesions, synaptopodin dissociated from F-actin, and disrupting the interactions between synaptopodin and α-actinin was observed. When FAK was inhibited by PF-573228 in immobilized LCSePs, the association between synaptopodin and α-actinin was observed in the podocytes. The association of synaptopodin and α-actinin with F-actin allowed FP stretching, establishing a functional glomerular filtration barrier. Therefore, in this mouse model of lung cancer, FAK signaling prompts podocyte FP effacement and proteinuria, indicative of PNS.

ccl19 and ccl21 affect cell movements and differentiation in early Xenopus development.

We characterized Xenopus laevis C-C motif chemokine ligand 19.L (ccl19.L) and C-C motif chemokine ligand 21.L (ccl21.L) during early Xenopus embryogenesis. The temporal and spatial expression patterns of ccl19.L and ccl21.L tended to show an inverse correlation, except that the expression level was higher in the dorsal side at the gastrula stage. For example, even at the dorsal sector of the gastrulae, ccl19.L was expressed in the axial region and ccl21.L was expressed in the paraxial region. Dorsal overexpression of ccl19.L and ccl21.L and knockdown of Ccl19.L and Ccl21.L inhibited gastrulation, but their functions were different in cell behaviors during morphogenesis. Observation of Keller sandwich explants revealed that overexpression of both ccl19.L and ccl21.L and knockdown of Ccl21.L inhibited the convergent extension movements, while knockdown of Ccl19.L did not. ccl19.L-overexpressing explants attracted cells at a distance and ccl21.L-overexpressing explants attracted neighboring cells. Ventral overexpression of ccl19.L and ccl21.L induced secondary axis-like structures and chrd.1 expression at the ventral side. Upregulation of chrd.1 was induced by ligand mRNAs through ccr7.S. Knockdown of Ccl19.L and Ccl21.L inhibited gastrulation and downregulated chrd.1 expression at the dorsal side. The collective findings indicate that ccl19.L and ccl21.L might play important roles in morphogenesis and dorsal-ventral patterning during early embryogenesis in Xenopus.

Structure and Dynamics of Human Chemokine CCL16-Implications for Biological Activity.

Human C-C motif ligand 16 (CCL16) is a chemokine that is distinguished by a large cleavable C-terminal extension of unknown significance. Conflicting data have been reported concerning its tissue distribution and modulation of expression, rendering the biological function of CCL16 enigmatic. Here, we report an integrated approach to the characterisation of this chemokine, including a re-assessment of its expression characteristics as well as a biophysical investigation with respect to its structure and dynamics. Our data indicate that CCL16 is chiefly synthesised by hepatocytes, without an appreciable response to mediators of inflammation, and circulates in the blood as a full-length protein. While the crystal structure of CCL16 confirms the presence of a canonical chemokine domain, molecular dynamics simulations support the view that the C-terminal extension impairs the accessibility of the glycosaminoglycan binding sites and may thus serve as an intrinsic modulator of biological activity.

Localization of signaling receptors maximizes cellular information acquisition in spatially structured natural environments.

Cells in natural environments, such as tissue or soil, sense and respond to extracellular ligands with intricately structured and non-monotonic spatial distributions, sculpted by processes such as fluid flow and substrate adhesion. In this work, we show that spatial sensing and navigation can be optimized by adapting the spatial organization of signaling pathways to the spatial structure of the environment. We develop an information-theoretic framework for computing the optimal spatial organization of a sensing system for a given signaling environment. We find that receptor localization previously observed in cells maximizes information acquisition in simulated natural contexts, including tissue and soil. Specifically, information acquisition is maximized when receptors form localized patches at regions of maximal ligand concentration. Receptor localization extends naturally to produce a dynamic protocol for continuously redistributing signaling receptors, which when implemented using simple feedback, boosts cell navigation efficiency by 30-fold.

Durotaxis: the mechanical control of directed cell migration.

Directed cell migration is essential for cells to efficiently migrate in physiological and pathological processes. While migrating in their native environment, cells interact with multiple types of cues, such as mechanical and chemical signals. The role of chemical guidance via chemotaxis has been studied in the past, the understanding of mechanical guidance of cell migration via durotaxis remained unclear until very recently. Nonetheless, durotaxis has become a topic of intensive research and several advances have been made in the study of mechanically guided cell migration across multiple fields. Thus, in this article we provide a state of the art about durotaxis by discussing in silico, in vitro and in vivo data. We also present insights on the general mechanisms by which cells sense, transduce and respond to environmental mechanics, to then contextualize these mechanisms in the process of durotaxis and explain how cells bias their migration in anisotropic substrates. Furthermore, we discuss what is known about durotaxis in vivo and we comment on how haptotaxis could arise from integrating durotaxis and chemotaxis in native environments.

HIV-infection and cocaine use regulate semen extracellular vesicles proteome and miRNAome in a manner that mediates strategic monocyte haptotaxis governed by miR-128 network.

BACKGROUND: Extracellular vesicles (EVs) are regulators of cell-cell interactions and mediators of horizontal transfer of bioactive molecules between cells. EV-mediated cell-cell interactions play roles in physiological and pathophysiological processes, which maybe modulated by exposure to pathogens and cocaine use. However, the effect of pathogens and cocaine use on EV composition and function are not fully understood. RESULTS: Here, we used systems biology and multi-omics analysis to show that HIV infection (HIV +) and cocaine (COC) use (COC +) promote the release of semen-derived EVs (SEV) with dysregulated extracellular proteome (exProtein), miRNAome (exmiR), and exmiR networks. Integrating SEV proteome and miRNAome revealed a significant decrease in the enrichment of disease-associated, brain-enriched, and HIV-associated miR-128-3p (miR-128) in HIV + COC + SEV with a concomitant increase in miR-128 targets-PEAK1 and RND3/RhoE. Using two-dimensional-substrate single cell haptotaxis, we observed that in the presence of HIV + COC + SEV, contact guidance provided by the extracellular matrix (ECM, collagen type 1) network facilitated far-ranging haptotactic cues that guided monocytes over longer distances. Functionalizing SEV with a miR-128 mimic revealed that the strategic changes in monocyte haptotaxis are in large part the result of SEV-associated miR-128. CONCLUSIONS: We propose that compositionally and functionally distinct HIV + COC + and HIV-COC- SEVs and their exmiR networks may provide cells relevant but divergent haptotactic guidance in the absence of chemotactic cues, under both physiological and pathophysiological conditions.

Glycosylation in Axonal Guidance.

How millions of axons navigate accurately toward synaptic targets during development is a long-standing question. Over decades, multiple studies have enriched our understanding of axonal pathfinding with discoveries of guidance molecules and morphogens, their receptors, and downstream signalling mechanisms. Interestingly, classification of attractive and repulsive cues can be fluid, as single guidance cues can act as both. Similarly, guidance cues can be secreted, chemotactic cues or anchored, adhesive cues. How a limited set of guidance cues generate the diversity of axonal guidance responses is not completely understood. Differential expression and surface localization of receptors, as well as crosstalk and spatiotemporal patterning of guidance cues, are extensively studied mechanisms that diversify axon guidance pathways. Posttranslational modification is a common, yet understudied mechanism of diversifying protein functions. Many proteins in axonal guidance pathways are glycoproteins and how glycosylation modulates their function to regulate axonal motility and guidance is an emerging field. In this review, we discuss major classes of glycosylation and their functions in axonal pathfinding. The glycosylation of guidance cues and guidance receptors and their functional implications in axonal outgrowth and pathfinding are discussed. New insights into current challenges and future perspectives of glycosylation pathways in neuronal development are discussed.

Functional Mapping of Adhesiveness on Live Cells Reveals How Guidance Phenotypes Can Emerge From Complex Spatiotemporal Integrin Regulation.

Immune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially in vivo. Here, we developed a novel in vitro method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/µm2 in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance.

Dorsal commissural axon guidance in the developing spinal cord.

Commissural axons have been a key model system for identifying axon guidance signals in vertebrates. This review summarizes the current thinking about the molecular and cellular mechanisms that establish a specific commissural neural circuit: the dI1 neurons in the developing spinal cord. We assess the contribution of long- and short-range signaling while sequentially following the developmental timeline from the birth of dI1 neurons, to the extension of commissural axons first circumferentially and then contralaterally into the ventral funiculus.

Nonlocal and local models for taxis in cell migration: a rigorous limit procedure.

A rigorous limit procedure is presented which links nonlocal models involving adhesion or nonlocal chemotaxis to their local counterparts featuring haptotaxis and classical chemotaxis, respectively. It relies on a novel reformulation of the involved nonlocalities in terms of integral operators applied directly to the gradients of signal-dependent quantities. The proposed approach handles both model types in a unified way and extends the previous mathematical framework to settings that allow for general solution-dependent coefficient functions. The previous forms of nonlocal operators are compared with the new ones introduced in this paper and the advantages of the latter are highlighted by concrete examples. Numerical simulations in 1D provide an illustration of some of the theoretical findings.

Cell Migration Driven by Self-Generated Integrin Ligand Gradient on Ligand-Labile Surfaces.

Integrin-ligand interaction mediates the adhesion and migration of many metazoan cells. Here, we report a unique mode of cell migration elicited by the lability of integrin ligands. We found that stationary cells spontaneously turn migratory on substrates where integrin ligands are subject to depletion by cellular force. Using TGT, a rupturable molecular linker, we quantitatively tuned the rate of ligand rupture by cellular force and tested platelets (anucleate cells), CHO-K1 cells (nucleated cells), and other cell types on TGT surfaces. These originally stationary cells readily turn motile on the uniform TGT surface, and their motility is correlated with the ligand depletion rate caused by cells. We named this new migration mode ligand-depleting (LD) migration. Through both experiments and simulations, we revealed the biophysical mechanism of LD migration. We found that the cells create and maintain a gradient of ligand surface density underneath the cell body by constantly rupturing local ligands, and the gradient in turn drives and guides cell migration. This is reminiscent of the phenomenon that some liquid droplets or solid beads can spontaneously move on homogeneous surfaces by chemically forming and maintaining a local gradient of surface energy. Here, we showed that cells, as living systems, can harness a similar mechanism to migrate. LD migration is beneficial for cells to maintain adhesion on ligand-labile surfaces, and might also play a role in the migration of cancer cells, immune cells, and platelets that deplete adhesive ligands of the matrix.

All Roads Lead to Directional Cell Migration.

Directional cell migration normally relies on a variety of external signals, such as chemical, mechanical, or electrical, which instruct cells in which direction to move. Many of the major molecular and physical effects derived from these cues are now understood, leading to questions about whether directional cell migration is alike or distinct under these different signals, and how cells might be directed by multiple simultaneous cues, which would be expected in complex in vivo environments. In this review, we compare how different stimuli are spatially distributed, often as gradients, to direct cell movement and the mechanisms by which they steer cells. A comparison of the downstream effectors of directional cues suggests that different external signals regulate a common set of components: small GTPases and the actin cytoskeleton, which implies that the mechanisms downstream of different signals are likely to be closely related and underlies the idea that cell migration operates by a common set of physical principles, irrespective of the input.

Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins.

Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules. Moreover, lymphocytes orient towards increasing adhesion with VLA-4 integrins (also known as integrin α4ß1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLß4), which has not previously been observed. This counterintuitive 'reverse haptotaxis' cannot be explained by existing mechanisms of mesenchymal haptotaxis involving either competitive anchoring of cell edges under tension or differential integrin-activated growth of lamellipodia, because they both favor orientation towards increasing adhesion. The mechanisms and functions of amoeboid adhesive haptotaxis remain unclear; however, multidirectional integrin-mediated haptotaxis might operate around transmigration ports on endothelia, stromal cells in lymph nodes, and inflamed tissue where integrin ligands are spatially modulated.

Mathematical models for cell migration: a non-local perspective.

We provide a review of recent advancements in non-local continuous models for migration, mainly from the perspective of its involvement in embryonal development and cancer invasion. Particular emphasis is placed on spatial non-locality occurring in advection terms, used to characterize a cell's motility bias according to its interactions with other cellular and acellular components in its vicinity (e.g. cell-cell and cell-tissue adhesions, non-local chemotaxis), but we also briefly address spatially non-local source terms. Following a short introduction and description of applications, we give a systematic classification of available PDE models with respect to the type of featured non-localities and review some of the mathematical challenges arising from such models, with a focus on analytical aspects. This article is part of the theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'.

Netrin Synergizes Signaling and Adhesion through DCC.

Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans-adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function.

Cell Migration in Microfluidic Devices: Invadosomes Formation in Confined Environments.

The last 20 years have seen the blooming of microfluidics technologies applied to biological sciences. Microfluidics provides effective tools for biological analysis, allowing the experimentalists to extend their playground to single cells and single molecules, with high throughput and resolution which were inconceivable few decades ago. In particular, microfluidic devices are profoundly changing the conventional way of studying the cell motility and cell migratory dynamics. In this chapter we will furnish a comprehensive view of the advancements made in the research domain of confinement-induced cell migration, thanks to the use of microfluidic devices. The chapter is subdivided in three parts. Each section will be addressing one of the fundamental questions that the microfluidic technology is contributing to unravel: (i) where cell migration takes place, (ii) why cells migrate and, (iii) how the cells migrate. The first introductory part is devoted to a thumbnail, and partially historical, description of microfluidics and its impact in biological sciences. Stress will be put on two aspects of the devices fabrication process, which are crucial for biological applications: materials used and coating methods. The second paragraph concerns the cell migration induced by environmental cues: chemical, leading to chemotaxis, mechanical, at the basis of mechanotaxis, and electrical, which induces electrotaxis. Each of them will be addressed separately, highlighting the fundamental role of microfluidics in providing the well-controlled experimental conditions where cell migration can be induced, investigated and ultimately understood. The third part of the chapter is entirely dedicated to how the cells move in confined environments. Invadosomes (the joint name for podosomes and invadopodia) are cell protrusion that contribute actively to cell migration or invasion. The formation of invadosomes under confinement is a research topic that only recently has caught the attention of the scientific community: microfluidic design is helping shaping the future direction of this emerging field of research.

Enhancing Schwann cell migration using concentration gradients of laminin-derived peptides.

Neuroregeneration following peripheral nerve injury is largely mediated by Schwann cells (SC), the principal glial cell that supports neurons in the peripheral nervous system. Axonal regeneration in vivo is limited by the extent of SC migration into the gap between the proximal and distal nerve, however, little is known regarding the principal driving forces for SC migration. Engineered microenvironments, such as molecular and protein gradients, play a role in the migration of many cell types, including cancer cells and fibroblasts. However, haptotactic strategies have not been applied widely to SC. Herein, a series of tethered laminin-derived peptides were analyzed for their influence on SC adhesion, proliferation, and alignment. Concentration gradient substrates were fabricated using a controlled vapor deposition method, followed by covalent peptide attachment via a thiol-ene reaction, and characterized by X-ray photoelectron spectroscopy (XPS) and MALDI-MS imaging. While tethered RGD peptides supported SC adhesion and proliferation, concentration gradients of RGD had little influence on biased SC directional migration. In contrast, YIGSR promoted less SC attachment than RGD, yet YIGSR peptide gradients directed migration with a strong bias to the concentration profile. With YIGSR peptide, overall speed increased with the steepness of the peptide concentration profile. YIGSR gradients had no haptotactic effect on rat dermal fibroblast migration, in contrast to fibroblast migration on RGD gradients. The response of SC to these tethered peptide gradients will guide the development of translationally relevant constructs designed to facilitate endogenous SC infiltration into defects for nerve regeneration.