Migration In 2D Research Articles

The Influence of Electric Field and Confinement on Cell Motility

The ability of cells to sense and respond to endogenous electric fields is important in processes such as wound healing, development, and nerve regeneration. In cell culture, many epithelial and endothelial cell types respond to an electric field of magnitude similar to endogenous electric fields by moving preferentially either parallel or antiparallel to the field vector, a process known as galvanotaxis. Here we report on the influence of dc electric field and confinement on the motility of fibroblast cells using a chip-based platform. From analysis of cell paths we show that the influence of electric field on motility is much more complex than simply imposing a directional bias towards the cathode or anode. The cell velocity, directedness, as well as the parallel and perpendicular components of the segments along the cell path are dependent on the magnitude of the electric field. Forces in the directions perpendicular and parallel to the electric field are in competition with one another in a voltage-dependent manner, which ultimately govern the trajectories of the cells in the presence of an electric field. To further investigate the effects of cell reorientation in the presence of a field, cells are confined within microchannels to physically prohibit the alignment seen in 2D environment. Interestingly, we found that confinement results in an increase in cell velocity both in the absence and presence of an electric field compared to migration in 2D.

Melanoma Cell Invasiveness Is Promoted at Least in Part by the Epidermal Growth Factor–Like Repeats of Tenascin-C

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like repeats (EGFL) as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased ROCK signaling concomitant with MLC2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNC EGFL play a role in melanoma invasion by modulating ROCK signaling and cell migration.

PKD Controls αvβ3 Integrin Recycling and Tumor Cell Invasive Migration through Its Substrate Rabaptin-5

Integrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvβ3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvβ3, which in turn inhibits α5β1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvβ3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvβ3-dependent invasion. Conversely, invasive migration that is dependent on α5β1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.

Abstract 485: Epidermal growth factor-like repeats of Tenascin C promote melanoma cell invasion, possibly by enabling mesenchymal to ameboid migration transition

Abstract In malignant melanoma, the most dangerous skin cancer, Tenascin-C (TNC) is overexpressed, especially at the invasive fronts of invasive melanomas. We found that the migration and invasiveness of a set of melanoma cell lines correlates with levels of TNC expression suggesting, surprisingly, that the melanoma cells themselves provide much of this matrix component. Adding TNC to matrices enhanced individual cell migration in cells that did not express much TNC, whereas it did not enhance the rapid motility of the cells expressing high levels. As TNC is a multidomain protein, the signaling pathways that promote this motogenic invasiveness are not obvious. Herein, we examined the potential role of the EGF-like repeats of TNC that have been shown to preferentially activate motogenic-signaling cascades. To analyze the effects of EGFL signaling on melanoma migration and invasion, we overexpressed TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). Interestingly, this led to impaired cell migration in in vitro wound healing assays and live cell tracking experiments. This was likely due to a shift in the adhesion-contractility balance during cell migration. TNCEGFL expressing cells presented a spindle-shaped morphology and formed cords in culture. Expression of TNCEGFL delayed melanoma cell attachment and spreading, as determined in cell adhesion and inverted centrifugation assays. This anti-adhesive phenotype was concomitant with increased MLC2 phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL expressing cells and improved migration in 2D. In distinction to the findings in 2D, TNCEGFL expressing cells had higher invasion potential in Matrigel invasion assays, with cells expressing high TNC having rounded “ameboid” morphology, underlying the importance of morphological shifts, and possibly ROCK signaling, in 3D movement through barrier matrices during invasion and dissemination. These results imply that TNCEGFL repeats are involved in melanoma invasion both by directly influencing cell migration and indirectly changing the tumor microenvironment composition, possibly allowing transition from mesenchymal to ameboid migrational mode. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 485. doi:1538-7445.AM2012-485

Paxillin Mediates Sensing of Physical Cues and Regulates Directional Cell Motility by Controlling Lamellipodia Positioning

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5–10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax−/− and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax−/− cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax−/− and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.

The Signaling Adaptor Eps8 Is an Essential Actin Capping Protein for Dendritic Cell Migration

Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D invitro and are delayed in reaching the draining lymph node (DLN) invivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.

The role of the cytoskeleton in cellular force generation in 2D and 3D environments

To adhere and migrate, cells generate forces through the cytoskeleton that are transmitted to the surrounding matrix. While cellular force generation has been studied on 2D substrates, less is known about cytoskeletal-mediated traction forces of cells embedded in more in vivo-like 3D matrices. Recent studies have revealed important differences between the cytoskeletal structure, adhesion, and migration of cells in 2D and 3D. Because the cytoskeleton mediates force, we sought to directly compare the role of the cytoskeleton in modulating cell force in 2D and 3D. MDA-MB-231 cells were treated with agents that perturbed actin, microtubules, or myosin, and analyzed for changes in cytoskeletal organization and force generation in both 2D and 3D. To quantify traction stresses in 2D, traction force microscopy was used; in 3D, force was assessed based on single cell-mediated collagen fibril reorganization imaged using confocal reflectance microscopy. Interestingly, even though previous studies have observed differences in cell behaviors like migration in 2D and 3D, our data indicate that forces generated on 2D substrates correlate with forces within 3D matrices. Disruption of actin, myosin or microtubules in either 2D or 3D microenvironments disrupts cell-generated force. These data suggest that despite differences in cytoskeletal organization in 2D and 3D, actin, microtubules and myosin contribute to contractility and matrix reorganization similarly in both microenvironments.

Following Actin Fibers in 3D During Cell Migration in Collagen Matrices

Actin polymerization is a major mechanism for the production of the force necessary for cell migration in 2D. The polymerization of actin and its retrograde motion at the leading edge of cell moving in 2D has been studied in great detail as well as the interaction of actin with focal adhesions. When cells grow in 3D collagen matrices, the extending lamellipodial protrusion is more difficult to visualize and it is likely not relavent for the movement of the cell over large distances. We use the modulation tracking 3D method to accurately image the cell protrusion. This method is capable of producing detailed images of 3D structures at the nanoscale and at the same time measure diffusion and aggregation of molecules in these structures. In 3D, cells produce very long protrusions that presumably grab on the surrounding collagen fibers to propel the rest of the cell body in a specific direction. What is unclear is if the cell is capable of forming stable adhesions with the collagen matrix and how the force is generated. Using the modulation tracking imaging method we can follow the changes in shape of the cellular protrusion and also image separately various proteins, including actin in the cytoplasm and in the membrane. In the thin long protrusion we observe both fast diffusing actin molecules and also relatively immobile species, presumably part of the actin cytoskeleton. We are developing a method to directly measure the movement of the entire actin bundle inside the very thin cells protrusions. The method is conceptually similar to speckle imaging; however, it works in 3D.

Genes Regulating the Progression of Human Ductal Carcinoma In Situ to Invasive Breast Cancer.

Abstract Rationale: The progression of human ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC) represents a major potentially life-threatening event. Our understanding of this process is rudimentary. Improved understanding could lead to better ability to predict and new therapeutic strategies to prevent progression, which would be major contributions to health care. Objectives: To identify genes involved in regulating the progression of human DCIS to IBC, and to confirm their involvement utilizing relevant in vitro and in vivo model systems. Methods: Gene expression profiles were evaluated in five complimentary cohorts of human DCIS and IBC (total n=59 in each category) using cDNA microarray technology. Supervised statistical comparisons were used to identify differentially expressed genes in each cohort. The genes were distilled to top priority candidates by the following vetting strategy: (1) expression changed in same direction in 2 or more cohorts; and (2) they possessed known biological functions which might reasonably be involved in regulating tumor invasion; and (3) they had not been studied in the context of breast cancer invasion. Initial studies have focused on candidate invasion-suppressor genes (down-regulated in IBC vs. DCIS). A human DCIS-like cell line (DCIS.COM) was stably transduced with shRNAi against the genes (3-5 hairpins each) and evaluated in vitro and in vivo for changes in invasion-related behaviors vs. controls (growth and migration in 2D and 3D cell cultures, invasion in 3D culture, and a human-in-mouse [HIM} DCIS xenograft model). The latter is a novel method we developed where cells are injected into the nipple duct of intact mammary glands of immune-compromised mice. They initially grow as DCIS which progress to IBC at varying rates depending on the cells. Results: Two novel invasion-suppressor genes (CSTA and FAT1) were identified based on demonstrating significantly increased growth, migration, and invasion in the assays described. In particular, shCSTA (n=22) and shFAT1 (n=22) HIM DCIS xenografts progressed to large IBCs by 10 weeks or less, significantly (P<.0001) greater than observed in controls (n=26) (67%, 64%, vs. 26%, respectively). Conclusions: We have identified two genes (CSTA and FAT1) whose normal functions appear to suppress the progression of human DCIS to IBC. These results could lead to new therapeutic strategies to prevent or suppress progression. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6141.

Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells

BackgroundMany studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.MethodsThe effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.ResultsWe observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.ConclusionTaken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.

Delayed-shot 3D depth migration

For 3D seismic imaging in structurally complex areas, the use of migration by wavefield extrapolation has become widespread. By its very nature, this family of migration methods operates on data sets that satisfy a wave equation in the context of a single, physically realizable field experiment, such as a common-shot record. However, common-shot migration of data recorded over dipping structures requires a migration aperture much larger than the recording aperture, resulting in extra computations. A different type of wave-equation record, the response to a linear or planar source, can be synthesized from all the common-shot records. Synthesizing these records from common-shot records involves slant-stack processing, or applying delays to the various shots; we call these records delayed-shot records. Delayed-shot records don't suffer from the aperture problems of common-shot records since their recording aperture is the length of the seismic survey. Consequently, delayed-shot records hold potential for efficient, accurate imaging by wavefield extrapolation. We present a formulation of delayed-shot migration in 2D and 3D (linear sources) and its application to 3D marine streamer data. This formulation includes a discussion of sampling theory issues associated with the formation of delayed-shot records. For typical marine data, 2D and 3D delayed-shot migration can be significantly more efficient than common-shot migration. Synthetic and real data examples show that delayed-shot migration produces images comparable to those from common-shot migration.

Monte-Carlo simulation of order–disorder kinetics in 2D and fcc binary alloys

The thermodynamic parameters that drive the atomic migration in 2D and 3D binary alloys are studied using Monte-Carlo simulations. The model is based on a vacancy–atom jump mechanism between nearest neighbour sites, with a constant vacancy concentration, so that only the migration counterpart (EM) of the total activation energy (EA=EM+EF, where EF is the vacancy formation energy) is determined. The ordering kinetics are well described by exponential like behaviours with one (respectively two) relaxation time in the 2D (respectively 3D) cases. The temperature dependence of the long relaxation time is an Arrhenius law yielding effective migration energies. Slowing down of the relaxation is observed at the Order–Disorder transitions. In both cases, we observe a linear variation of the effective migration energy with the Order–Disorder critical temperature.

Background velocity estimation using non‐linear optimization for reflection tomography and migration misfit

We show that it is possible to estimate the background velocity for prestack depth migration in 2D laterally varying media using a non‐linear optimization technique called very fast simulated annealing (VFSA). We use cubic splines in the velocity model parametrization and make use of either successive pairs of shot gathers or several constant‐offset sections as input data for the inversion. A Kirchhoff summation scheme based on first‐arrival traveltimes is used to migrate/model the input data during the velocity analysis. We evaluate and compare two different measures of error. The first is defined in the recorded data or (x,t) domain and is based on a reflection‐tomography criterion. The second is defined in the migrated data or (x,z) domain and is based on a migration‐misfit criterion. Depth relaxation is used to improve the convergence and quality of the velocity analysis while simultaneously reducing the computational cost. Further, we show that by coarse sampling in the offset domain the method is still robust.Our non‐linear optimization approach to migration velocity analysis is evaluated for both synthetic and real seismic data. For the velocity‐analysis method based on the reflection‐tomography criterion, traveltimes do not have to be picked. Similarly, the migration‐misfit criterion does not require that depth images be manually compared. Interpreter intervention is required only to restrict the search space used in the velocity‐analysis problem. Extension of the proposed schemes to 3D models is straightforward but practical only for the fastest available computers.