Protrusion Fluctuations Direct Cell Motion

Many physiological phenomena involve directional cell migration. It is usually attributed to chemical gradients in vivo. Recently, other cues have been shown to guide cells in vitro, including stiffness/adhesion gradients or micropatterned adhesive motifs. However, the cellular mechanism leading to these biased migrations remains unknown, and, often, even the direction of motion is unpredictable. In this study, we show the key role of fluctuating protrusions on ratchet-like structures in driving NIH3T3 cell migration. We identified the concept of efficient protrusion and an associated direction index. Our analysis of the protrusion statistics facilitated the quantitative prediction of cell trajectories in all investigated conditions. We varied the external cues by changing the adhesive patterns. We also modified the internal cues using drug treatments, which modified the protrusion activity. Stochasticity affects the short- and long-term steps. We developed a theoretical model showing that an asymmetry in the protrusion fluctuations is sufficient for predicting all measures associated with the long-term motion, which can be described as a biased persistent random walk.     

Cells as Active Particles in Asymmetric Potentials: Motility under External Gradients

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.     

Cells as Active Particles in Asymmetric Potentials: Motility under External Gradients

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.     

A Dynamic Model of Chemoattractant-Induced Cell Migration

Cell migration refers to a directional cell movement in response to chemoattractant stimulation. In this work, we developed a cell-migration model by mimicking in vivo migration using optically manipulated chemoattractant-loaded microsources. The model facilitates a quantitative characterization of the relationship among the protrusion force, cell motility, and chemoattractant gradient for the first time (to our knowledge). We verified the correctness of the model using migrating leukemia cancer Jurkat cells. The results show that one can achieve the ideal migrating capacity by choosing the appropriate chemoattractant gradient and concentration at the leading edge of the cell.     

Coordinates of Human Visual and Inertial Heading Perception

Heading estimation involves both inertial and visual cues. Inertial motion is sensed by the labyrinth, somatic sensation by the body, and optic flow by the retina. Because the eye and head are mobile these stimuli are sensed relative to different reference frames and it remains unclear if a perception occurs in a common reference frame. Recent neurophysiologic evidence has suggested the reference frames remain separate even at higher levels of processing but has not addressed the resulting perception. Seven human subjects experienced a 2s, 16 cm/s translation and/or a visual stimulus corresponding with this translation. For each condition 72 stimuli (360° in 5° increments) were delivered in random order. After each stimulus the subject identified the perceived heading using a mechanical dial. Some trial blocks included interleaved conditions in which the influence of ±28° of gaze and/or head position were examined. The observations were fit using a two degree-of-freedom population vector decoder (PVD) model which considered the relative sensitivity to lateral motion and coordinate system offset. For visual stimuli gaze shifts caused shifts in perceived head estimates in the direction opposite the gaze shift in all subjects. These perceptual shifts averaged 13 ± 2° for eye only gaze shifts and 17 ± 2° for eye-head gaze shifts. This finding indicates visual headings are biased towards retina coordinates. Similar gaze and head direction shifts prior to inertial headings had no significant influence on heading direction. Thus inertial headings are perceived in body-centered coordinates. Combined visual and inertial stimuli yielded intermediate results.     

Glycogen synthase kinase 3β inhibition enhanced proliferation,migration and functional re-endothelialization of endothelial progenitor cells in hypercholesterolemia microenvironment

Hypercholesterolemia impairs the quantity and function of endothelial progenitor cell. We hypothesized that glycogen synthase kinase 3β activity is involved in regulating biological function of endothelial progenitor cells in hypercholesterolemia microenvironment. For study, endothelial progenitor cells derived from apolipoprotein E-deficient mice fed with high-fat diet were used. Glycogen synthase kinase 3β activity was interfered with glycogen synthase kinase 3β inhibitor lithium chloride or transduced with replication defective adenovirus vector expressing catalytically inactive glycogen synthase kinase 3β (GSK3β-KM). Functions of endothelial progenitor cells, proliferation, migration, secretion and network formation of endothelial progenitor cells were assessed in vitro. The expression of phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 in endothelial progenitor cells was detected by Western blot. The in vivo function re-endothelialization and vasodilation were also analyzed by artery injury model transplanted with glycogen synthase kinase 3β-inhibited endothelial progenitor cells. We demonstrated that while the proliferation, migration, network formation as well as VEGF and NO secretion were impaired in apolipoprotein E-deficient endothelial progenitor cells, glycogen synthase kinase 3β inhibition significantly improved all these functions. Apolipoprotein E-deficient endothelial progenitor cells showed decreased phospho-glycogen synthase kinase 3β, β-catenin and cyclinD1 expression, whereas these signals were enhanced by glycogen synthase kinase 3β inhibition and accompanied with β-catenin nuclear translocation. Our in vivo model showed that glycogen synthase kinase 3β inhibition remarkably increased re-endothelial and vasodilation. Taken together, our data suggest that inhibition of glycogen synthase kinase 3β is associated with endothelial progenitor cell biological functions both in vitro and in vivo. It might be an important interference target in hypercholesterolemia microenvironment.     

The cell ratchet: Interplay between efficient protrusions and adhesion determines cell motion

Many physiological and pathological processes involve directed cell motion. In general, migrating cells are represented with a polarized morphology with extending and retracting protrusions at the leading edge. However, cell motion is a more complex phenomenon. Cells show heterogeneous morphologies and high protrusive dynamics is not always related to cell shape. This prevents the quantitative prediction of cell motion and the identification of cellular mechanisms setting directionality. Here we discuss the importance of protrusion fluctuations in directed cell motion. We show how their spatiotemporal distribution and dynamics determine the fluctuations and directions of cell motion for NIH3T3 fibroblasts plated on micro-patterned adhesive ratchets.¹ We introduce efficient protrusions and direction index which capture short-term cell motility over hours: these new read-outs allow the prediction of parameters characteristic for the long-term motion of cells over days. The results may have important implications for the study of biological phenomena where directed cell migration is involved, in morphogenesis and in cancer.     

Ordered,Random, Monotonic and Non-Monotonic Digital Nanodot Gradients

Cell navigation is directed by inhomogeneous distributions of extracellular cues. It is well known that noise plays a key role in biology and is present in naturally occurring gradients at the micro- and nanoscale, yet it has not been studied with gradients in vitro. Here, we introduce novel algorithms to produce ordered and random gradients of discrete nanodots – called digital nanodot gradients (DNGs) – according to monotonic and non-monotonic density functions. The algorithms generate continuous DNGs, with dot spacing changing in two dimensions along the gradient direction according to arbitrary mathematical functions, with densities ranging from 0.02% to 44.44%. The random gradient algorithm compensates for random nanodot overlap, and the randomness and spatial homogeneity of the DNGs were confirmed with Ripley''s K function. An array of 100 DNGs, each 400×400 µm², comprising a total of 57 million 200×200 nm² dots was designed and patterned into silicon using electron-beam lithography, then patterned as fluorescently labeled IgGs on glass using lift-off nanocontact printing. DNGs will facilitate the study of the effects of noise and randomness at the micro- and nanoscales on cell migration and growth.     

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.     

GlialCAM,a CLC-2 Cl- Channel Subunit,Activates the Slow Gate of CLC Chloride Channels

GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl^- channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.     

A novel tubulin inhibitor, 6h,suppresses tumor-associated angiogenesis and shows potent antitumor activity against non–small cell lung cancers

Tumor angiogenesis is closely associated with the metastasis and progression of non–small cell lung cancer (NSCLC), a highly vascularized solid tumor. However, novel therapeutics are lacking for the treatment of this cancer. Here, we developed a series of 2-aryl-4-(3,4,5-trimethoxy-benzoyl)-5-substituted-1,2,3-triazol analogs (6a–6x) as tubulin colchicine-binding site inhibitors, aiming to find a novel promising drug candidate for NSCLC treatment. We first identified 2-(2-fluorophenyl)-3-(3,4,5-trimethoxybenzoyl)-5-(3-hydroxyazetidin-1-yl)-2H-1,2,3-triazole (6h) as a hit compound, which inhibited angiogenesis induced by NSCLC cells both in vivo and in vitro. In addition, our data showed that 6h could tightly bind to the colchicine-binding site of tubulin and inhibit tubulin polymerization. We also found that 6h could effectively induce G2/M cell cycle arrest of A549 and H460 cells, inhibit cell proliferation, and induce apoptosis. Furthermore, we showed 6h had the potential to inhibit the migration and invasion of NSCLC cells, two basic characteristics of tumor metastasis. Finally, we found 6h could effectively inhibit tumor progression in A549 xenograft mouse models with minimal toxicity. Taken together, these findings provide strong evidence for the development of 6h as a promising microtubule colchicine-binding site inhibitor for NSCLC treatment.     

Engineering a genome‐reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.     

T Lymphocytes Orient against the Direction of Fluid Flow during LFA-1-Mediated Migration

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.cm⁻². T lymphocytes were shown to respond to shear stress application by a rapid (30 s) and fully reversible orientation of their migration against the fluid flow without a change in migration speed. Primary T lymphocytes migrating on ICAM-1 in the presence of uniformly applied SDF-1α were also found to migrate against the direction of shear flow. In sharp contrast, neutrophils migrating in the presence of uniformly applied fMLP and leukemic HSB2 T lymphocytes migrating on ICAM-1 alone oriented their migration downstream, with the direction of fluid flow. Our findings suggest that, in addition to biochemical cues, shear stress is a contributing factor to leukocyte migration directionality.     

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and β2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin).     

A Mechanistic Collective Cell Model for Epithelial Colony Growth and Contact Inhibition

We present a mechanistic hybrid continuum-discrete model to simulate the dynamics of epithelial cell colonies. Collective cell dynamics are modeled using continuum equations that capture plastic, viscoelastic, and elastic deformations in the clusters while providing single-cell resolution. The continuum equations can be viewed as a coarse-grained version of previously developed discrete models that treat epithelial clusters as a two-dimensional network of vertices or stochastic interacting particles and follow the framework of dynamic density functional theory appropriately modified to account for cell size and shape variability. The discrete component of the model implements cell division and thus influences cell size and shape that couple to the continuum component. The model is validated against recent in vitro studies of epithelial cell colonies using Madin-Darby canine kidney cells. In good agreement with experiments, we find that mechanical interactions and constraints on the local expansion of cell size cause inhibition of cell motion and reductive cell division. This leads to successively smaller cells and a transition from exponential to quadratic growth of the colony that is associated with a constant-thickness rim of growing cells at the cluster edge, as well as the emergence of short-range ordering and solid-like behavior. A detailed analysis of the model reveals a scale invariance of the growth and provides insight into the generation of stresses and their influence on the dynamics of the colonies. Compared to previous models, our approach has several advantages: it is independent of dimension, it can be parameterized using classical elastic properties (Poisson’s ratio and Young’s modulus), and it can easily be extended to incorporate multiple cell types and general substrate geometries.     

Regulation of the activity of the bacterial histidine kinase PleC by the scaffolding protein PodJ

Scaffolding proteins can customize the response of signaling networks to support cell development and behaviors. PleC is a bifunctional histidine kinase whose signaling activity coordinates asymmetric cell division to yield a motile swarmer cell and a stalked cell in the gram-negative bacterium Caulobacter crescentus. Past studies have shown that PleC’s switch in activity from kinase to phosphatase correlates with a change in its subcellular localization pattern from diffuse to localized at the new cell pole. Here we investigated how the bacterial scaffolding protein PodJ regulates the subcellular positioning and activity of PleC. We reconstituted the PleC-PodJ signaling complex through both heterologous expressions in Escherichia coli and in vitro studies. In vitro, PodJ phase separates as a biomolecular condensate that recruits PleC and inhibits its kinase activity. We also constructed an in vivo PleC-CcaS chimeric histidine kinase reporter assay and demonstrated using this method that PodJ leverages its intrinsically disordered region to bind to PleC’s PAS sensory domain and regulate PleC-CcaS signaling. Regulation of the PleC-CcaS was most robust when PodJ was concentrated at the cell poles and was dependent on the allosteric coupling between PleC-CcaS’s PAS sensory domain and its downstream histidine kinase domain. In conclusion, our in vitro biochemical studies suggest that PodJ phase separation may be coupled to changes in PleC enzymatic function. We propose that this coupling of phase separation and allosteric regulation may be a generalizable phenomenon among enzymes associated with biomolecular condensates.     

Recurrent De Novo Mutations in PACS1 Cause Defective Cranial-Neural-Crest Migration and Define a Recognizable Intellectual-Disability Syndrome

We studied two unrelated boys with intellectual disability (ID) and a striking facial resemblance suggestive of a hitherto unappreciated syndrome. Exome sequencing in both families identified identical de novo mutations in PACS1, suggestive of causality. To support these genetic findings and to understand the pathomechanism of the mutation, we studied the protein in vitro and in vivo. Altered PACS1 forms cytoplasmic aggregates in vitro with concomitant increased protein stability and shows impaired binding to an isoform-specific variant of TRPV4, but not the full-length protein. Furthermore, consistent with the human pathology, expression of mutant PACS1 mRNA in zebrafish embryos induces craniofacial defects most likely in a dominant-negative fashion. This phenotype is driven by aberrant specification and migration of SOX10-positive cranial, but not enteric, neural-crest cells. Our findings suggest that PACS1 is necessary for the formation of craniofacial structures and that perturbation of its functions results in a specific syndromic ID phenotype.