Directed Cell Movement Research Articles

1D micro-nanopatterned integrin ligand surfaces for directed cell movement.

Cell-extracellular matrix (ECM) adhesion mediated by integrins is a highly regulated process involved in many vital cellular functions such as motility, proliferation and survival. However, the influence of lateral integrin clustering in the coordination of cell front and rear dynamics during cell migration remains unresolved. For this purpose, we describe a novel protocol to fabricate 1D micro-nanopatterned stripes by integrating the block copolymer micelle nanolithography (BCMNL) technique and maskless near UV lithography-based photopatterning. The photopatterned 10μm-wide stripes consist of a quasi-perfect hexagonal arrangement of gold nanoparticles, decorated with the RGD (arginine-glycine-aspartate) motif for single integrin heterodimer binding, and placed at a distance of 50, 80, and 100nm to regulate integrin clustering and focal adhesion dynamics. By employing time-lapse microscopy and immunostaining, we show that the displacement and speed of fibroblasts changes according to the nanoscale spacing of adhesion sites. We found that as the lateral spacing of adhesive peptides increased, fibroblast morphology was more elongated. This was accompanied by a decreased formation of mature focal adhesions and stress fibers, which increased cell displacement and speed. These results provide new insights into the migratory behavior of fibroblasts in 1D environments and our protocol offers a new platform to design and manufacture confined environments in 1D for integrin-mediated cell adhesion.

In preprints: improving and interrogating embryo models.

In preprints: improving and interrogating embryo models.

RanBP1 plays an essential role in directed migration of neural crest cells during development

Collective cell migration is essential for embryonic development, tissue regeneration and repair, and has been implicated in pathological conditions such as cancer metastasis. It is, in part, directed by external cues that promote front-to-rear polarity in individual cells. However, our understanding of the pathways that underpin the directional movement of cells in response to external cues remains incomplete. To examine this issue we made use of neural crest cells (NC), which migrate as a collective during development to generate vital structures including bones and cartilage. Using a candidate approach, we found an essential role for Ran-binding protein 1 (RanBP1), a key effector of the nucleocytoplasmic transport pathway, in enabling directed migration of these cells. Our results indicate that RanBP1 is required for establishing front-to-rear polarity, so that NCs are able to chemotax. Moreover, our work suggests that RanBP1 function in chemotaxis involves the polarity kinase LKB1/PAR4. We envisage that regulated nuclear export of LKB1 through Ran/RanBP1 is a key regulatory step required for establishing front-to-rear polarity and thus chemotaxis, during NC collective migration.

3D single cell migration driven by temporal correlation between oscillating force dipoles.

Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In some cells, symmetry breaking manifests itself in a directional flow of actin from the front to the rear of the cell. Many cells, especially in physiological 3D matrices, do not show such coherent actin dynamics and present seemingly competing protrusion/retraction dynamics at their front and back. How symmetry breaking manifests itself for such cells is therefore elusive. We take inspiration from the scallop theorem proposed by Purcell for micro-swimmers in Newtonian fluids: self-propelled objects undergoing persistent motion at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. We report similar observations for cells crawling in 3D. We quantified cell motion using a combination of 3D live cell imaging, visualization of the matrix displacement, and a minimal model with multipolar expansion. We show that our cells embedded in a 3D matrix form myosin-driven force dipoles at both sides of the nucleus, that locally and periodically pinch the matrix. The existence of a phase shift between the two dipoles is required for directed cell motion which manifests itself as cycles with finite area in the dipole-quadrupole diagram, a formal equivalence to the Purcell cycle. We confirm this mechanism by triggering local dipolar contractions with a laser. This leads to directed motion. Our study reveals that these cells control their motility by synchronizing dipolar forces distributed at front and back. This result opens new strategies to externally control cell motion as well as for the design of micro-crawlers.

Abstract 2534: Neural control of glioma infiltration

Abstract Adult glioblastoma multiforme (GBM) is the most common and deadly form of malignant brain cancer. A broad relationship between tumor progression and neuronal stimulation in the microenvironment is known in peripheral tumors, but only a few interactions between neuronal cues and GBM cells have been characterized. By combining in utero electroporation (IUE) with CRISPR/Cas9 and piggyBac transposase genetics, we have developed an endogenous, immunocompetent, in vivo model with which to study these interactions in a native tumor context. Combining this model with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) we interrogated tumor-neuron interactions. We assessed molecular and physiological changes in tumors during neural stimulation through tumor development with single cell sequencing and spatial transcriptomics. We examined expression data in populations present under stimulation that did not appear or appeared later in control tumors. These data showed axon guidance genes, a family of genes associated with directed cell motility, were upregulated in these populations. To functionally test these findings, we performed a barcoded screen of axon guidance genes in the IUE model followed by individual confirmation with gain and loss of function of individual genes. We found Sema4F affects infiltration of tumors as well as overall survival. Currently we wish to study the abrogation of this infiltration with Sema4F knockout in the context of DREADD stimulation. Completion of this study will provide new insights into neuronal interactions driving tumor progression in GBM. Citation Format: Emmet J. Huang-Hobbs, Yi-Ting Cheng, Yeungjun Ko, Junsung Woo, Akdes Harmanci, Ganesh Rao, Benjamin Deneen. Neural control of glioma infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2534.

A More Biomimetic Cell Migration Assay with High Reliability and Its Applications.

Cell migration refers to the directional movement of cells to the surrounding cell-free zone in response to chemical and mechanical stimuli. A cell migration assay is an essential device for studying pharmaceutical and medical problems. In this paper, we present a novel approach to a cell migration assay on a chip with two merits, namely (i) simultaneous creation of many cell samples on the same condition and (ii) cells migrating while being stressed in a fluidic environment. The first merit has addressed the problem of poor reproducibility in experimental studies for medical problems such as wound healing, and the second merit has made the cell migration device, which is an in vitro environment, more biomimetic. The two merits are attributed to a novel mechanical method to simultaneously create many cell-free zones and to the design of a microfluidic process to create shear stress in cells uniformly. Two applications were studied on our device to explore its effectiveness. The first application is regarding the combination chemotherapy of cisplatin and doxorubicin (Adriamycin) on cervical cancer cells (HeLa). The second application is regarding inhibiting the migration of endothelial cells (HUVEC) in the process of anti-angiogenesis.

ERAS Is Constitutively Expressed in the Tissues of Adult Horses and May Be a Key Player in Basal Autophagy.

ERas is a new gene of the Ras family found in murine embryonic stem (ES) cells. Its human ortholog is not expressed in human ES cells. So far ERas gene has only been found to be expressed in the tissues of adult cynomolgus monkeys and cattle; however, information about ERAS expression or its potential functions in equine tissues is lacking. This study was performed to investigate whether Eras is an equine functional gene and whether ERAS is expressed in the tissues of adult horses and determine its potential physiological role. Expression of the ERas gene was detected in all examined adult tissues, and the RT-PCR assay revealed ERAS transcripts. Protein expression was also detected by Western blot analysis. Quantitative real time RT-qPCR analysis revealed that different expression levels of ERAS transcripts were most highly expressed in the testis. Immunohistochemically, ERAS was found to be localized prevalently in the plasmatic membrane as well as cytoplasm of the cells. ERAS was a physical partner of activated PDGFβR leading to the AKT signaling. ERAS was found to interact with a network of proteins (BAG3, CHIP, Hsc70/Hsp70, HspB8, Synpo2, and p62) known to play a role in the chaperone-assisted selective autophagy (CASA), which is also known as BAG3-mediated selective macroautophagy, an adaptive mechanism to maintain cellular homeostasis. Furthermore, ERAS was found to interact with parkin. PINK1, BNIP3, laforin. All these proteins are known to play a role in parkin-dependent and -independent mitophagy. This is the first study demonstrating that Eras is a functional gene, and that ERAS is constitutively expressed in the tissues of adult horses. ERAS appears to play a physiological role in cellular proteostasis maintenance, thus mitigating the proteotoxicity of accumulated misfolded proteins and contributing to protection against disease. Finally, it is conceivable that activation of AKT pathway by PDGFRs promotes actin reorganization, directed cell movements, stimulation of cell growth.

Lights, Camera, PAKtion: Design and Engineering of a Subcellular Targeting Optogenetic PAK1 Kinase and Sensor

p21‐activated kinase 1 (PAK1) is a major ‘pathogenic kinase’ that directs cell motility, cytoskeletal remodeling and has been shown to function as a downstream regulator for various cancer signaling cascades. Such biological roles require a tight spatial and kinetic control of its localization and activity. Unfortunately, the spatiotemporal nature of these pathways renders the application of conventional tools inadequate for studying localized PAK1 induced cell behavior. Therefore, we have designed and engineered an optogenetic analog of PAK1 to address the difficulties in assessing PAK1 intracellular activity.Optogenetics is a powerful light‐triggered technology that, via control of protein biochemical activity, allows for the spatial and temporal control over a variety of biological processes. We developed an engineering strategy to construct a genetically expressed, light‐responsive optogenetic analog of PAK1 (optoPAK1) and demonstrated that, upon illumination, optoPAK1 migrates to specified intracellular sites. OptoPAK1 was designed to function independently of endogenous biochemical regulation in a constitutively active manner with minimal activity in the dark state. We employed the improved light‐induced dimer (iLid) system as a means to recruit and photoactivate the protein analog at discrete subcellular domains. Furthermore, we designed intracellular spatially segregated reporters of optoPAK1 activity and preliminary data displayed that optoPAK1 phosphorylates these reporters in a light‐dependent fashion. Taken together, optoPAK1 and its corresponding biosensor offer the opportunity to appropriately study the relationship between PAK1 compartmentalized biochemical activity and cell behavior.

1H, 13C and 15N chemical shift assignments of the ZnR and GYF cytoplasmic domains of the GltJ protein from Myxococcus xanthus.

Bacterial cell motility is essential for a range of physiological phenomena such as nutrient sensing, predation, biofilm formation and pathogenesis. One of the most intriguing motilities is bacterial gliding, which is defined as the ability of some bacteria to move across surfaces without an external appendage. In Myxococcus xanthus, gliding motility depends on the assembly of focal adhesion complexes (FAC) which include the Glt mutiprotein complex and allow directional movement of individual cells (A-motility). Within the Glt multiprotein complex, GltJ is one of the key proteins involved in FAC assembly. In this work we report complete backbone and side chain 1H, 13C and 15N chemical shifts of the two cytoplasmic domains of GltJ, GltJ-ZnR (BMRB No. 51104) and GltJ-GYF (BMRB No. 51096). These data provide the first step toward the first high resolution structures of protein domains from the Glt machinery and the atomic level characterization of GltJ cytoplasmic activity during FAC assembly.

Oscillating non-progressing flows induce directed cell motion

A novel deformation-dependent propulsion phenomenon for (blood) cells and soft capsules in oscillating microflows is presented. It enables the separation of cells with different deformabilities, such as healthy and malignant cells, without their labeling or obstacles in a microfluidic device. The propulsion phenomeon is based on a broken time reversal symmetry, which is achieved by a fast forward and slow backward movement of a fluid through microchannels that does not progress on average.

Design, simulation and application of multichannel microfluidic chip for cell migration

Cell migration is defined as the directional movement of cells toward a specific chemical concentration gradient, which plays a crucial role in embryo development, wound healing and tumor metastasis. However, current research methods showed low flux and are only suitable for single-factor assessment, and it was difficult to comprehensively consider the effects of other parameters such as different concentration gradients on cell migration behavior. In this paper, a four-channel microfluidic chip was designed. Its characteristics were as follows: it relied on laminar flow and diffusion mechanisms to establish and maintain a concentration gradient; it was suitable for observation of cell migration in different concentration gradient environment under a single microscope field; four cell isolation zones (20 μm width) were integrated into the microfluidic device to calibrate the initial cell position, which ensured the accuracy of the experimental results. In particular, we used COMSOL Multiphysics software to simulate the structure of the chip, which demonstrated the necessity of designing S-shaped microchannel and horizontal pressure balance channel to maintain concentration gradient. Finally, neutrophils were incubated with advanced glycation end products (AGEs, 0, 0.2, 0.5, 1.0 μmol·L -1), which were closely related to diabetes mellitus and its complications. The migration behavior of incubated neutrophils was studied in the 100 nmol·L -1 of chemokine (N-formylmethionyl-leucyl-phenyl-alanine) concentration gradient. The results prove the reliability and practicability of the microfluidic chip.

ALFY localizes to early endosomes and cellular protrusions to facilitate directional cell migration.

ABSTRACTCell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY; also known as WDFY3), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here, we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5- and EEA1-positive early endosomes in a PtdIns(3)P-dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility.

Multi-Cue Kinetic Model with Non-Local Sensing for Cell Migration on a Fiber Network with Chemotaxis

Cells perform directed motion in response to external stimuli that they detect by sensing the environment with their membrane protrusions. Precisely, several biochemical and biophysical cues give rise to tactic migration in the direction of their specific targets. Thus, this defines a multi-cue environment in which cells have to sort and combine different, and potentially competitive, stimuli. We propose a non-local kinetic model for cell migration in which cell polarization is influenced simultaneously by two external factors: contact guidance and chemotaxis. We propose two different sensing strategies, and we analyze the two resulting transport kinetic models by recovering the appropriate macroscopic limit in different regimes, in order to observe how the cell size, with respect to the variation of both external fields, influences the overall behavior. This analysis shows the importance of dealing with hyperbolic models, rather than drift-diffusion ones. Moreover, we numerically integrate the kinetic transport equations in a two-dimensional setting in order to investigate qualitatively various scenarios. Finally, we show how our setting is able to reproduce some experimental results concerning the influence of topographical and chemical cues in directing cell motility.

Enhanced chemotaxis through spatially regulated absolute concentration robustness

AbstractChemotaxis, the directional motility of cells in response to spatial gradients of chemical cues, is a fundamental process behind a wide range of biological events, including the innate immune response and cancer metastasis. Recent advances in cell biology have shown that the protrusions that enable amoeboid cells to move are driven by the stochastic threshold crossings of an underlying excitable system. As a cell encounters a chemoattractant gradient, the size of this threshold is regulated spatially so that the crossings are biased toward the front of the cell. For efficient directional migration, cells must limit undesirable lateral and rear‐directed protrusions. The inclusion of a control mechanism to suppress these unwanted firings would enhance chemotactic efficiency. It is known that absolute concentration robustness (ACR) exerts tight control over the mean and variance of species concentration. Here, we demonstrate how the coupling of the ACR mechanism to the cellular signaling machinery reduces the likelihood of threshold crossings in the excitable system. Moreover, we show that using the cell's innate gradient sensing apparatus to direct the action of ACR to the rear suppresses the lateral movement of the cells and that this results in improved chemotactic performance.

Deciphering vimentin-microtubule interactions in cell polarity

Vimentin intermediate filaments (IF) and the centrosome are a critical part of the cell cytoskeleton, a complex active network that directs many cellular functions. Recent studies have shown that centrosome positioning is critical for directional cell movement. On the other hand, loss of vimentin IF increases cell motility and directional persistence in microfluidic channels. These studies indicate both vimentin and centrosome are associated with directional cell migration but their interactions are widely unknown.

Puff up to decide: the role of regulatory volume changes in neutrophil polarity and chemotaxis

For directed cell movement, cells need to extend their plasma membrane towards sources of chemoattractant. Actin polymerization is known to play a critical role in this process, and we know many of the actin regulators that link attractant receptors with the actin cytoskeleton. Much less studied are the complementary regulators of membrane extension such as water influx, which is thought to collaborate with actin polymerization to inflate the leading edge for cell protrusion. We study this question in human neutrophils, cells of the innate immune system that migrate to sites of injury and infection to hunt and kill invading pathogens.

Regulation of directional cell motility in alveolar macrophages by a formin family protein Fhod1

Regulation of directional cell motility in alveolar macrophages by a formin family protein Fhod1

Computational modelling of nephron progenitor cell movement and aggregation during kidney organogenesis

During early kidney organogenesis, nephron progenitor (NP) cells move from the tip to the corner region of the ureteric bud (UB) branches in order to form the pretubular aggregate, the early structure giving rise to nephron formation. NP cells derive from metanephric mesenchymal cells and physically interact with them during the movement. Chemotaxis and cell–cell adhesion differences are believed to drive the cell patterning during this critical period of organogenesis. However, the effect of these forces to the cell patterns and their respective movements are known in limited details. We applied a Cellular Potts Model to explore how these forces and organizations contribute to directed cell movement and aggregation. Model parameters were estimated based on fitting to experimental data obtained in ex vivo kidney explant and dissociation–reaggregation organoid culture studies. Our simulations indicated that optimal enrichment and aggregation of NP cells in the UB corner niche requires chemoattractant secretion from both the UB epithelial cells and the NP cells themselves, as well as differences in cell–cell adhesion energies. Furthermore, NP cells were observed, both experimentally and by modelling, to move at higher speed in the UB corner as compared to the tip region where they originated. The existence of different cell speed domains along the UB was confirmed using self-organizing map analysis. In summary, we saw faster NP cell movements near aggregation. The applicability of Cellular Potts Model approach to simulate cell movement and patterning was found to be good during for this early nephrogenesis process. Further refinement of the model should allow us to recapitulate the effects of developmental changes of cell phenotypes and molecular crosstalk during further organ development.

Subcellular patterns of SPE-6 localization reveal unexpected complexities in Caenorhabditis elegans sperm activation and sperm function.

To acquire and maintain directed cell motility, Caenorhabditis elegans sperm must undergo extensive, regulated cellular remodeling, in the absence of new transcription or translation. To regulate sperm function, nematode sperm employ large numbers of protein kinases and phosphatases, including SPE-6, a member of C. elegans’ highly expanded casein kinase 1 superfamily. SPE-6 functions during multiple steps of spermatogenesis, including functioning as a “brake” to prevent premature sperm activation in the absence of normal extracellular signals. Here, we describe the subcellular localization patterns of SPE-6 during wild-type C. elegans sperm development and in various sperm activation mutants. While other members of the sperm activation pathway associate with the plasma membrane or localize to the sperm’s membranous organelles, SPE-6 surrounds the chromatin mass of unactivated sperm. During sperm activation by either of two semiautonomous signaling pathways, SPE-6 redistributes to the front, central region of the sperm’s pseudopod. When disrupted by reduction-of-function alleles, SPE-6 protein is either diminished in a temperature-sensitive manner (hc187) or is mislocalized in a stage-specific manner (hc163). During the multistep process of sperm activation, SPE-6 is released from its perinuclear location after the spike stage in a process that does not require the fusion of membranous organelles with the plasma membrane. After activation, spermatozoa exhibit variable proportions of perinuclear and pseudopod-localized SPE-6, depending on their location within the female reproductive tract. These findings provide new insights regarding SPE-6’s role in sperm activation and suggest that extracellular signals during sperm migration may further modulate SPE-6 localization and function.

Hindbrain neuropore tissue geometry determines asymmetric cell-mediated closure dynamics in mouse embryos

Gap closure is a common morphogenetic process. In mammals, failure to close the embryonic hindbrain neuropore (HNP) gap causes fatal anencephaly. We observed that surface ectoderm cells surrounding the mouse HNP assemble high-tension actomyosin purse strings at their leading edge and establish the initial contacts across the embryonic midline. Fibronectin and laminin are present, and tensin 1 accumulates in focal adhesion-like puncta at this leading edge. The HNP gap closes asymmetrically, faster from its rostral than caudal end, while maintaining an elongated aspect ratio. Cell-based physical modeling identifies two closure mechanisms sufficient to account for tissue-level HNP closure dynamics: purse-string contraction and directional cell motion implemented through active crawling. Combining both closure mechanisms hastens gap closure and produces a constant rate of gap shortening. Purse-string contraction reduces, whereas crawling increases gap aspect ratio, and their combination maintains it. Closure rate asymmetry can be explained by asymmetric embryo tissue geometry, namely a narrower rostral gap apex, whereas biomechanical tension inferred from laser ablation is equivalent at the gaps' rostral and caudal closure points. At the cellular level, the physical model predicts rearrangements of cells at the HNP rostral and caudal extremes as the gap shortens. These behaviors are reproducibly live imaged in mouse embryos. Thus, mammalian embryos coordinate cellular- and tissue-level mechanics to achieve this critical gap closure event.