Amoeboid Cell Migration Research Articles

Plasma Membrane Blebbing Is Controlled by Subcellular Distribution of Vimentin Intermediate Filaments.

The formation of specific cellular protrusions, plasma membrane blebs, underlies the amoeboid mode of cell motility, which is characteristic for free-living amoebae and leukocytes, and can also be adopted by stem and tumor cells to bypass unfavorable migration conditions and thus facilitate their long-distance migration. Not all cells are equally prone to bleb formation. We have previously shown that membrane blebbing can be experimentally induced in a subset of HT1080 fibrosarcoma cells, whereas other cells in the same culture under the same conditions retain non-blebbing mesenchymal morphology. Here we show that this heterogeneity is associated with the distribution of vimentin intermediate filaments (VIFs). Using different approaches to alter the VIF organization, we show that blebbing activity is biased toward cell edges lacking abundant VIFs, whereas the VIF-rich regions of the cell periphery exhibit low blebbing activity. This pattern is observed both in interphase fibroblasts, with and without experimentally induced blebbing, and during mitosis-associated blebbing. Moreover, the downregulation of vimentin expression or displacement of VIFs away from the cell periphery promotes blebbing even in cells resistant to bleb-inducing treatments. Thus, we reveal a new important function of VIFs in cell physiology that involves the regulation of non-apoptotic blebbing essential for amoeboid cell migration and mitosis.

Random walk and cell morphology dynamics in Naegleria gruberi.

Amoeboid cell movement and migration are wide-spread across various cell types and species. Microscopy-based analysis of the model systems Dictyostelium and neutrophils over the years have uncovered generality in their overall cell movement pattern. Under no directional cues, the centroid movement can be quantitatively characterized by their persistence to move in a straight line and the frequency of re-orientation. Mathematically, the cells essentially behave as a persistent random walker with memory of two characteristic time-scale. Such quantitative characterization is important from a cellular-level ethology point of view as it has direct connotation to their exploratory and foraging strategies. Interestingly, outside the amoebozoa and metazoa, there are largely uncharacterized species in the excavate taxon Heterolobosea including amoeboflagellate Naegleria. While classical works have shown that these cells indeed show typical amoeboid locomotion on an attached surface, their quantitative features are so far unexplored. Here, we analyzed the cell movement of Naegleria gruberi by employing long-time phase contrast imaging that automatically tracks individual cells. We show that the cells move as a persistent random walker with two time-scales that are close to those known in Dictyostelium and neutrophils. Similarities were also found in the shape dynamics which are characterized by the appearance, splitting and annihilation of the curvature waves along the cell edge. Our analysis based on the Fourier descriptor and a neural network classifier point to importance of morphology features unique to Naegleria including complex protrusions and the transient bipolar dumbbell morphologies.

Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. We perform a comprehensive, genome-wide assessment of the molecular factors critical to proliferation, differentiation, and cell migration in a neutrophil-like cell line. Through the development of multiple migration screen strategies, we specifically probe directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. We identify a role for mTORC1 signaling in cell differentiation, which influences neutrophil abundance, survival, and migratory behavior. Across our individual migration screens, we identify genes involved in adhesion-dependent and adhesion-independent cell migration, protein trafficking, and regulation of the actomyosin cytoskeleton. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

A computational model for early cell spreading, migration, and competing taxis

Cell motility represents one of the most fundamental functions in life such as organ development or tissue regeneration and diseases such as cancer. Here, we derived a computational model of cell motility that incorporates the most important mechanisms towards cell motility: cell protrusion, polarization, and retrograde flow. We first validate our model to explain the main phases of cell motility, i.e. symmetric cell spreading, cell polarization, and two important types of cell migration, confined and amoeboid cell migration. Then, we use our model to investigate the effect of chemical and mechanical signals in cell migration, that is chemotaxis and durotaxis. More importantly, we analyze the competition between durotaxis and chemotaxis. We show that chemotaxis rules over durotaxis in most situations although durotaxis can diminish chemotaxis. Moreover, we show that inhibiting the effect of GTPases in actin polymerization at the cell front may allow durotaxis to take control over chemotaxis in soft substrates. Understanding how the main forces in cell motility cooperate, and how a precise manipulation of external cues may control directed cell migration is not only key for a fundamental comprehension of cell biology but also plays a key role in the future of tissue and biomedical engineering. To this end, we provide a freely-available platform to predict all phases and modes of cell motility analyzed in this work.

Blebs-Formation, Regulation, Positioning, and Role in Amoeboid Cell Migration.

In the context of development, tissue homeostasis, immune surveillance, and pathological conditions such as cancer metastasis and inflammation, migrating amoeboid cells commonly form protrusions called blebs. For these spherical protrusions to inflate, the force for pushing the membrane forward depends on actomyosin contraction rather than active actin assembly. Accordingly, blebs exhibit distinct dynamics and regulation. In this review, we first examine the mechanisms that control the inflation of blebs and bias their formation in the direction of the cell’s leading edge and present current views concerning the role blebs play in promoting cell locomotion. While certain motile amoeboid cells exclusively form blebs, others form blebs as well as other protrusion types. We describe factors in the environment and cell-intrinsic activities that determine the proportion of the different forms of protrusions cells produce.

Piezo1 activation attenuates thrombin-induced blebbing in breast cancer cells.

ABSTRACTCancer cells exploit a variety of migration modes to leave primary tumors and establish metastases, including amoeboid cell migration, which is typically reliant on bleb formation. Here we demonstrate that thrombin induces dynamic blebbing in the MDA-MB-231 breast cancer cell line and confirm that protease-activated receptor 1 (PAR1) activation is sufficient to induce this effect. Cell confinement has been implicated as a driving force in bleb-based migration. Unexpectedly, we found that gentle contact compression, exerted using a custom built ‘cell press’ to mechanically stimulate cells, reduced thrombin-induced blebbing. Thrombin-induced blebbing was similarly attenuated using the small molecule Yoda1, an agonist of the mechanosensitive Ca2+ channel Piezo1, and this attenuation was impaired in Piezo1-depleted cells. Additionally, Piezo1 activation suppressed thrombin-induced phosphorylation of ezrin, radixin and moesin (ERM) proteins, which are implicated in the blebbing process. Our results provide mechanistic insights into Piezo1 activation as a suppressor of dynamic blebbing, specifically that which is induced by thrombin.

RhoA-ROCK competes with YAP to regulate amoeboid breast cancer cell migration in response to lymphatic-like flow.

Lymphatic drainage generates force that induces prostate cancer cell motility via activation of Yes‐associated protein (YAP), but whether this response to fluid force is conserved across cancer types is unclear. Here, we show that shear stress corresponding to fluid flow in the initial lymphatics modifies taxis in breast cancer, whereas some cell lines use rapid amoeboid migration behavior in response to fluid flow, a separate subset decrease movement. Positive responders displayed transcriptional profiles characteristic of an amoeboid cell state, which is typical of cells advancing at the edges of neoplastic tumors. Regulation of the HIPPO tumor suppressor pathway and YAP activity also differed between breast subsets and prostate cancer. Although subcellular localization of YAP to the nucleus positively correlated with overall velocity of locomotion, YAP gain‐ and loss‐of‐function demonstrates that YAP inhibits breast cancer motility but is outcompeted by other pro‐taxis mediators in the context of flow. Specifically, we show that RhoA dictates response to flow. GTPase activity of RhoA, but not Rac1 or Cdc42 Rho family GTPases, is elevated in cells that positively respond to flow and is unchanged in cells that decelerate under flow. Disruption of RhoA or the RhoA effector, Rho‐associated kinase (ROCK), blocked shear stress–induced motility. Collectively, these findings identify biomechanical force as a regulator amoeboid cell migration and demonstrate stratification of breast cancer subsets by flow‐sensing mechanotransduction pathways.

Cancer biology: Hypoxia-induced talin tail-docking sparks cancer metastasis

Hypoxia drives cancer metastasis and induces cancer cells to switch from collective to amoeboid migration. A new study identifies a molecular pathway in which hypoxia stimulates calpain-2-mediated cleavage of talin-1, resulting in a reduction of integrin β1 activity and the promotion of blebbing amoeboid cancer cell migration and metastasis.

Novel potential functions of amoeboid cells in thraustochytrids revealed by Aurantiochytrium limacinum BL10

The current study investigated the potential functions of amoeboid cell formation and migration in a thraustochytrid strain, Aurantiochytrium limacinum BL10. Our results showed that: (1) When the surface of an agar plate was inoculated with BL10, amoeboid cells mainly emerged on the periphery of isolated colonies. The amoeboid cells then migrated outwards to form small vegetative cell clusters, which favored rapid colony expansion. In addition, amoeboid cells were capable of self-recognition (i.e. they were able to distinguish BL10 from other thraustochytrid species), and could choose whether to evade (self colonies) or approach (non-self colonies). These observations indicated that amoeboid cells were employed by BL10 to help colonize empty territories and to outcompete other thraustochytrid species in previously colonized territories. (2) When the agar medium was soft, amoeboid cells were able to penetrate the surface and migrate throughout, thereby allowing BL10 to colonize the interior of the solid matrix. This finding suggested that amoeboid cell formation and migration may help Aurantiochytrium colonize the interior of solid matrices to obtain additional nutrients and spatial resources. The mechanisms underlying the regulation of amoeboid cell formation and migration as well as the extraordinary microbial social behaviors of BL10 are also discussed in this article.

Calpain-2 regulates hypoxia/HIF-induced plasticity toward amoeboid cancer cell migration and metastasis

Hypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression invarious cancer types. In epithelial cancer, hypoxia induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective intervention to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as a key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low extracellular matrix (ECM)-adhesive, predominantly bleb-based amoeboid movement, which was maintained by a low-oxidative and -glycolytic energy metabolism ("eco-mode"). Hypoxia induced calpain-2-mediated amoeboid conversion by deactivating β1 integrins through enzymatic cleavage of the focal adhesion adaptor protein talin-1. Consequently, targeted downregulation or pharmacological inhibition of calpain-2 restored talin-1 integrity and β1 integrin engagement and reverted amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced amoeboid conversion in the orthotopic mouse dermis and upregulated in invasive HN tumor xenografts invivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/β1 integrin axis as a druggable mechanosignaling program that conserves energy yet enables metastatic dissemination that can be reverted by interfering with calpain activity.

Three-dimensional migration of human amniotic fluid stem cells involves mesenchymal and amoeboid modes and is regulated by mTORC1.

Three‐dimensional (3D) cell migration is an integral part of many physiologic processes. Although being well studied in the context of adult tissue homeostasis and cancer development, remarkably little is known about the invasive behavior of human stem cells. Using two different kinds of invasion assays, this study aimed at investigating and characterizing the 3D migratory capacity of human amniotic fluid stem cells (hAFSCs), a well‐established fetal stem cell type. Eight hAFSC lines were found to harbor pronounced potential to penetrate basement membrane (BM)‐like matrices. Morphological examination and inhibitor approaches revealed that 3D migration of hAFSCs involves both the matrix metalloprotease‐dependent mesenchymal, elongated mode and the Rho‐associated protein kinase‐dependent amoeboid, round mode. Moreover, hAFSCs could be shown to harbor transendothelial migration capacity and to exhibit a motility‐associated marker expression pattern. Finally, the potential to cross extracellular matrix was found to be induced by mTORC1‐activating growth factors and reduced by blocking mTORC1 activity. Taken together, this report provides the first demonstration that human stem cells exhibit mTORC1‐dependent invasive capacity and can concurrently make use of mesenchymal and amoeboid 3D cell migration modes, which represents an important step toward the full biological characterization of fetal human stem cells with relevance to both developmental research and stem cell‐based therapy.

Cell scientist to watch – Verena Ruprecht

ABSTRACT Verena Ruprecht received her PhD in Biophysics from the Johannes Kepler University in 2010 for her work on developing single-molecule super-resolution imaging tools in the lab of Gerhard Schütz. Following a research visit in Didier Marguet's lab at the Centre d'Immunologie de Marseille-Luminy (CIML) in France, she moved to the Institute of Science and Technology (IST) in Austria for a postdoc, working jointly with Carl-Philipp Heisenberg and Michael Sixt. There, she discovered a unique amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Verena started her independent laboratory at the Centre for Genomic Regulation (CRG) in Barcelona, Spain, in September 2016. Her group combines genetic and biophysical methods with multi-scale imaging and mathematical modelling to study cellular dynamics in embryo development. In 2020, Verena was selected as an EMBO Young Investigator and in the same year awarded an HFSP Young Investigator Grant for a collaborative project to study the biophysics of zebrafish fertilization.

Three-dimensional stochastic simulation of chemoattractant-mediated excitability in cells.

During the last decade, a consensus has emerged that the stochastic triggering of an excitable system drives pseudopod formation and subsequent migration of amoeboid cells. The presence of chemoattractant stimuli alters the threshold for triggering this activity and can bias the direction of migration. Though noise plays an important role in these behaviors, mathematical models have typically ignored its origin and merely introduced it as an external signal into a series of reaction-diffusion equations. Here we consider a more realistic description based on a reaction-diffusion master equation formalism to implement these networks. In this scheme, noise arises naturally from a stochastic description of the various reaction and diffusion terms. Working on a three-dimensional geometry in which separate compartments are divided into a tetrahedral mesh, we implement a modular description of the system, consisting of G-protein coupled receptor signaling (GPCR), a local excitation-global inhibition mechanism (LEGI), and signal transduction excitable network (STEN). Our models implement detailed biochemical descriptions whenever this information is available, such as in the GPCR and G-protein interactions. In contrast, where the biochemical entities are less certain, such as the LEGI mechanism, we consider various possible schemes and highlight the differences between them. Our simulations show that even when the LEGI mechanism displays perfect adaptation in terms of the mean level of proteins, the variance shows a dose-dependence. This differs between the various models considered, suggesting a possible means for determining experimentally among the various potential networks. Overall, our simulations recreate temporal and spatial patterns observed experimentally in both wild-type and perturbed cells, providing further evidence for the excitable system paradigm. Moreover, because of the overall importance and ubiquity of the modules we consider, including GPCR signaling and adaptation, our results will be of interest beyond the field of directed migration.

Excitable actin dynamics and amoeboid cell migration.

Amoeboid cell migration is characterized by frequent changes of the direction of motion and resembles a persistent random walk on long time scales. Although it is well known that cell migration is typically driven by the actin cytoskeleton, the cause of this migratory behavior remains poorly understood. We analyze the spontaneous dynamics of actin assembly due to nucleation promoting factors, where actin filaments lead to an inactivation of these factors. We show that this system exhibits excitable dynamics and can spontaneously generate waves, which we analyze in detail. By using a phase-field approach, we show that these waves can generate cellular random walks. We explore how the characteristics of these persistent random walks depend on the parameters governing the actin-nucleator dynamics. In particular, we find that the effective diffusion constant and the persistence time depend strongly on the speed of filament assembly and the rate of nucleator inactivation. Our findings point to a deterministic origin of the random walk behavior and suggest that cells could adapt their migration pattern by modifying the pool of available actin.

Tailored nanotopography of photocurable composites for control of cell migration.

External mechanical stimuli represent elementary signals for living cells to adapt to their adjacent environment. These signals range from bulk material properties down to nanoscopic surface topography and trigger cell behaviour. Here, we present a novel approach to generate tailored surface roughnesses in the nanometer range to tune surface properties by particle size and volume ratio. Time-resolved local mean-squared displacement (LMSD) analysis of amoeboid cell migration reveals that nanorough surfaces alter effectively cell migration velocities and the active cell migration phases. Since the UV curable composite material is easy to fabricate and can be structured via different light based processes, it is possible to generate hierarchical 3D cell scaffolds for tissue engineering or lab-on-a-chip applications with adjustable surface roughness in the nanometre range.

Novel micropatterning technique reveals dependence of cell-substrate adhesion and migration of social amoebas on parental strain, development, and fluorescent markers.

Cell-substrate adhesion of the social amoeba Dictyostelium discoideum, a model organism often used for the study of chemotaxis, is non-specific and does not involve focal adhesion complexes. Therefore, micropatterned substrates where adherent Dictyostelium cells are constrained to designated microscopic regions are difficult to make. Here we present a micropatterning technique for Dictyostelium cells that relies on coating the substrate with an ∼1μm thick layer of polyethylene glycol (PEG) gel. We show that, when plated on a substrate with narrow parallel stripes of PEG-gel and glass, Dictyostelium cells nearly exclusive adhere to and migrate along the glass stripes, thus providing a model system to study one-dimensional migration of amoeboid cells. Surprisingly, we find substantial differences in the adhesion to PEG-gel and glass stripes between vegetative and developed cells and between two different axenic laboratory strains of Dictyostelium, AX2 and AX4. Even more surprisingly, we find that the distribution of Dictyostelium cells between PEG-gel and glass stripes is significantly affected by the expression of several fluorescent protein markers of the cytoskeleton. We carry out atomic force microscopy based single cell force spectroscopy measurements that confirm that the force of adhesion to PEG-gel substrate can be significantly different between vegetative and developed cells, AX2 and AX4 cells, and cells with and without fluorescent markers. Thus, the choice of parental background, the degree of development, and the expression of fluorescent protein markers can all have a profound effect on cell-substrate adhesion and should be considered when comparing migration of cells and when designing micropatterned substrates.

Microtubules control cellular shape and coherence in amoeboid migrating cells.

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.

A flexible network of vimentin intermediate filaments promotes migration of amoeboid cancer cells through confined environments

Tumor cells can spread to distant sites through their ability to switch between mesenchymal and amoeboid (bleb-based) migration. Because of this difference, inhibitors of metastasis must account for each migration mode. However, the role of vimentin in amoeboid migration has not been determined. Because amoeboid leader bleb-based migration (LBBM) occurs in confined spaces and vimentin is known to strongly influence cell-mechanical properties, we hypothesized that a flexible vimentin network is required for fast amoeboid migration. To this end, here we determined the precise role of the vimentin intermediate filament system in regulating the migration of amoeboid human cancer cells. Vimentin is a classic marker of epithelial-to-mesenchymal transition and is therefore an ideal target for a metastasis inhibitor. Using a previously developed polydimethylsiloxane slab-based approach to confine cells, RNAi-based vimentin silencing, vimentin overexpression, pharmacological treatments, and measurements of cell stiffness, we found that RNAi-mediated depletion of vimentin increases LBBM by ∼50% compared with control cells and that vimentin overexpression and simvastatin-induced vimentin bundling inhibit fast amoeboid migration and proliferation. Importantly, these effects were independent of changes in actomyosin contractility. Our results indicate that a flexible vimentin intermediate filament network promotes LBBM of amoeboid cancer cells in confined environments and that vimentin bundling perturbs cell-mechanical properties and inhibits the invasive properties of cancer cells.

Topographical Guidance of Highly Motile Amoeboid Cell Migration

Topographical Guidance of Highly Motile Amoeboid Cell Migration

Abstract B086: Cellular context-dependent regulation of cancer cell invasion and migration in pancreatic tumor spheroids co-cultured with pancreatic stellate cells

Abstract Purpose Invasion of cancer cells is the initial step of metastatic progression. Elucidation of the cellular and molecular mechanisms of invasion may provide novel strategies to modulate metastasis and survival. The plasticity of cancer cells and surrounding stroma induces cell migration of which three types have been reported: collective, mesenchymal, or amoeboid type of movement. Here we investigated differential invasion phenotype and underlying mechanisms using two pancreatic cancer cells, BxPC-3 (wild-type KRAS, epithelial type) and PANC-1 (mutated KRAS, intermediate type) under 3D culture conditions. Methods Multicellular tumor spheroids (TS) were cultured in 3D collagen gel matrix either as mono-culture or under pancreatic stellate cell (PSC) co-culture condition using micro-channel chips. TS formation, cell migration within and outside of collagen gel, ECM remodeling, and expression of proteins involved in epithelial-mesenchymal transition (EMT) as well as degradation of matrix proteins were evaluated using immunofluorescence confocal microscopy, real-time live cell imaging and subsequent image analysis. Results BxPC-3 cells formed highly compact TS with strong expression of E-cadherin compared to PANC-1 cells. BxPC-3 cells showed collective migration under mono-culture condition and showed 50% phenotypic conversion to amoeboid single cell migration under PSC co-culture condition. Lamellipodia formation with actin spikes were observed in both types of cell migration in BxPC-3 cells. PANC-1 cells showed mostly mesenchymal invasion with single cells migrating out of TS using invadopodia. PANC-1 cells showed significantly higher level of vimentin and TGF-β as compared to BxPC-3 cells. Extensive ECM remodeling with collagen densification and fiber alignment was shown with BxPC-3 cells, compared to that of PANC-1 cells, under both mono- and PSC co-culture conditions. Similar levels of collagen degradation and expression of MT1-MMP, integrin β1 and pFAK between the two cell lines strongly suggest additional mechanisms of mechanical deformation of ECM facilitated by traction force of BxPC-3 cells, which warrants further studies. When cancer cells and PSCs migrated out of collagen matrix channels to encounter, fibronectin-mediated cell-cell interaction leading to co-migration was observed in both cell lines. Conclusions We demonstrated cellular context-dependent regulation of cancer cell invasion and its association with cancer cell-ECM and cancer cell-PSC interaction in pancreatic TS co-cultured with PSC. Our 3D TS-PSC co-culture recapitulates the differential modes of cancer cell invasion; hence, it may serve as an efficient and in vivo mimic model to study cellular and molecular mechanisms of invasion. Citation Format: Seul-ki Kim, Min-Suk Oh, Hyo-Jeong Kuh. Cellular context-dependent regulation of cancer cell invasion and migration in pancreatic tumor spheroids co-cultured with pancreatic stellate cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B086. doi:10.1158/1535-7163.TARG-19-B086