Random Cell Motility Research Articles

ActivinA: a new leukemia-promoting factor conferring migratory advantage to B-cell precursor-acute lymphoblastic leukemic cells.

B-cell precursor-acute lymphoblastic leukemia modulates the bone marrow (BM) niche to become leukemia-supporting and chemo-protective by reprogramming the stromal microenvironment. New therapies targeting the interplay between leukemia and stroma can help improve disease outcome. We identified ActivinA, a TGF-β family member with a well-described role in promoting several solid malignancies, as a factor favoring leukemia that could represent a new potential target for therapy. ActivinA resulted over-expressed in the leukemic BM and its production was strongly induced in mesenchymal stromal cells after culture with leukemic cells. Moreover, MSCs isolated from BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA compared to their normal counterparts. The pro-inflammatory leukemic BM microenvironment synergized with leukemic cells to induce stromal-derived ActivinA. Gene expression analysis of ActivinA-treated leukemic cells showed that this protein was able to significantly influence motility-associated pathways. Interestingly, ActivinA promoted random motility and CXCL12-driven migration of leukemic cells, even at suboptimal chemokine concentrations, characterizing the leukemic niche. Conversely, ActivinA severely impaired CXCL12-induced migration of healthy CD34+ cells. This opposite effect can be explained by the ability of ActivinA to increase intracellular calcium only in leukemic cells, boosting cytoskeleton dynamics through a higher rate of actin polymerization. Moreover, by stimulating the invasiveness of the leukemic cells, ActivinA was found to be a leukemia-promoting factor. Importantly, the ability of ActivinA to enhance BM engraftment and the metastatic potential of leukemic cells was confirmed in a xenograft mouse model of the disease. Overall, ActivinA was seen to be a key factor in conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche.

AMF siRNA treatment of keloid through inhibition signaling pathway of RhoA/ROCK1

A keloid (KD) is a benign dermal fibrotic tumor. Treatment of KDs is challenging and the recurrence rate is high; thus, there is an unmet need to explore new target sites and new treatment methods. As a tumor-associated cytokine, autocrine motility factor (AMF) can effectively stimulate the random and directional movement of cells. We first found that AMF was overexpressed in keloid fibroblasts (KFs) and the proliferation and migration of KFs were promoted by AMF stimulation. After treatment with Y-27632, RhoA kinase inhibitor, the proliferation and migration capacity of KFs declined significantly, and type I collagen protein, active RhoA and ROCK1 also were downregulated. In addition, a KD transplantation model was established under the skin of nude mice, with KD intramural injection AMF siRNA, we found that the weight of the KD was smaller than in the control group (P < 0.05), KD tissue sections stained by HE and Masson showed that fibers became loose and the blood vessels were visibly reduced. In conclusion, AMF siRNA is expected to be a novel strategy to treat KD by inhibiting signaling pathway of RhoA/ROCK1.

Retraction of "Motility-Based Cell Sorting by Planar Cell Chromatography".

RETURN TO ISSUEPREVRetractionNEXTORIGINAL ARTICLEThis notice is a retraction.Retraction of “Motility-Based Cell Sorting by Planar Cell Chromatography”Carlos C. CoCarlos C. CoMore by Carlos C. Co, Chia-Chi Ho*Chia-Chi HoMore by Chia-Chi Ho, and Girish KumarGirish KumarMore by Girish KumarCite this: Anal. Chem. 2018, 90, 9, 5972Publication Date (Web):April 18, 2018Publication History Published online18 April 2018Published inissue 1 May 2018https://doi.org/10.1021/acs.analchem.8b01500Copyright © 2018 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views1165Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (156 KB) Get e-Alerts Get e-Alerts

Statistical modelling of cell movement

Collective cell movement affects vital biological processes in the human organism such as wound healing, immune response, and cancer metastasis. A better understanding of the mechanisms driving cell movement is then essential for the advancement of medical treatments. In this paper, we demonstrate how the unscented Kalman filter, a technique used extensively in engineering in the context of filtering, can be applied to estimate random or directed cell movement. Our proposed model, formulated using stochastic differential equations, is fitted on data describing the movement of Dictyostelium cells, an amoeba routinely used as a proxy for eukaryotic cell movement.

Investigation of the Heat Loss Effect on Cellular Flames via Proper Orthogonal Decomposition

ABSTRACTFor better control of a lean premixed combustion system, it is necessary to understand the mechanism of unsteady cellular flame. The objective of the present study is to investigate how heat loss influences the cellular instability of lean premixed flame. The lean premixed CH4/H2/air cellular flames generated on a McKenna burner were studied, and heat loss was responsible for the manifestation of unsteady cellular structure. A method of applying proper orthogonal decomposition in scale analysis is presented, which has proven helpful in characterizing the cellular flame. The multiple scale nature of unstable cellular flame was identified, and the small scales were found to act in the random movement of cells. The contribution of each scale is obtained. The energy distribution on modes indicates that the energy on higher-order modes, equivalently the contribution of smaller scales, is decreased with decreasing heat loss. Through analyzing the principal modes and temporal coefficients, heat loss is found to influence a flame’s unstable behavior by promoting the oscillation of large scales, and the wrinkling factor is estimated. It is found that there is a cut-off length scale, below which the wrinkles make little contribution to the enhancement of combustion intensity.

Determining whether observed eukaryotic cell migration indicates chemotactic responsiveness or random chemokinetic motion

Chemotaxis, the motion of cells directed by a gradient of chemoattractant molecules, guides cells in immune response, development, wound healing, and cancer. Unfortunately, this process is difficult to distinguish from chemokinesis, i.e., stimulated random cell motion. Chemotaxis is frequently inferred by determining how many cells cross a boundary in a chemotaxis assay, for example how many cells crawl into a chemoattractant-infused filter, or how many cells enter a defined region in an under-agarose assay or agarose spot assay. To mitigate possible ambiguity in whether motion observed in these assays is directed by the chemoattractant gradient or by chemokinesis, we developed a mathematical model to determine when such methods indeed indicate directed motion of cells. In contrast to previous analyses of chemotaxis assays, we report not just the gradients that arise in the assays but also resulting cell motion. We applied the model to data obtained from rigorous measurements and show, as examples, that MDA-MB-231 breast-cancer cells are at least 20 times less sensitive to gradients of EGF or CXCL12 than neutrophils are to formyl peptides; we then used this information to determine the extent to which gradient sensing increases the rate of boundary crossing relative to a random-motility control. Results show, for example, that in the filter assay, 2–4 times as many neutrophils pass through the filter when exposed to a gradient as when the gradient is absent. However, in the other combinations of cells and assays we considered, only 10–20% more cells are counted as having migrated in a directed, rather than random, motility condition. We also discuss the design of appropriate controls for these assays, which is difficult for the under-agarose and agarose spot assays. Moreover, although straightforward to perform with the filter assay, reliable controls are often not done. Consequently, we infer that chemotaxis is frequently over-reported, especially for cells like MDA-MB-231 cells, which move slowly and are relatively insensitive to gradients. Such results provide insights into the use of chemotaxis assays, particularly if one wants to acquire and analyze quantitative data.

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

Human pluripotent stem cells, after directed differentiation in vitro, can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation.

Measurement of Cell Motility Using Microgrooved Substrates.

Cells migrate to perform specific functions such as tissue/organ formation during development or to repair injured tissue. Therefore, the study of cell migration is important not only for fundamental cell biology research, but also to understand tissue morphogenesis and wound healing. In this chapter, we describe a method developed in our laboratory to measure cell migration in a uniaxial direction. In this approach, linear microgrooves, fabricated on a transparent poly(dimethylsiloxane) (PDMS) substrate, allow cells to move in a uniaxial direction, in contrast to the random movement of cells in all directions on a smooth substrate surface. This chapter describes in detail the materials and methods needed to measure cell movement using microgrooved substrates, as well as applications of this method.

Abstract A42: A mechanically coupled reaction-diffusion model for predicting in vivo C6 glioma growth in rats

Abstract Introduction: Accurate biophysical models of tumor growth that can be used to provide predictions of future tumor growth and therapy response on an individual basis would be a powerful tool for clinicians to select and guide patient therapy. The reaction-diffusion equation is one such model that has been extensively used to model in vivo glioma growth. This model describes the change in tumor cell number over time due to the net proliferation of tumor cells (reaction) and the random movement of tumor cells (diffusion). One shortcoming of this model is that tumor growth is essentially spatially unrestricted resulting in inaccurate tumor growth predictions. It is well-known, however, that tumor expansion is restricted in response to increased mechanical stresses, and we have incorporated these effects into a more realistic mechanically coupled reaction-diffusion model. Using in vivo MRI measurements of glioma growth, model parameters for both the mechanically and non-mechanically coupled models can be estimated and used to generate individualized tumor growth predictions. Thus, the goal of this study is to evaluate the accuracy of tumor growth predictions made with the mechanically and non-mechanically coupled reaction-diffusion models to in vivo measurements of glioma growth. Methods: The mechanical reaction-diffusion model alters the standard model by changing the tumor cell diffusion coefficient spatially and temporally as a function of the local von Mises stress. The von Mises stress is determined by calculating the tissue displacement caused by the growing tumor. As the tumor grows and causes increased tissue displacement, the von Mises stress also increases, resulting in a decrease in the tumor cell diffusion coefficient. To evaluate this model, rats (N = 2) with C6 gliomas were imaged over ten days with diffusion-weighted MRI (DW-MRI) and contrast enhanced MRI (CE-MRI). Signal enhancement observed in the CE-MRI datasets were used to identify tumor regions. Tumor cellularity was then estimated within these enhancing regions using DW-MRI data. Model parameters (i.e., tumor cell proliferation rate and tumor cell diffusion coefficient) from the standard and mechanical models were then estimated from day 10, day 12, and day 14's cellularity measurements. For the mechanical model, several weights of mechanical coupling were investigated to determine the optimal value for a given tumor. These estimated parameters were then used to evaluate the model from days 14 through 20 to predict future tumor growth. Predicted tumor growth obtained from the standard and mechanical models were compared quantitatively to the in vivo measurement of tumor growth at the local (concordance correlation coefficient (CCC)) and at the global (percent error in total volume and Dice value) levels. Results: The standard model predictions ranged from 23.3% to 91.6% error in tumor volume estimates on days 15, 16, 18, and 20. Mechanical model predictions ranged from in 3.6% to 15.5% error on days 15, 16, 18, and 20. The standard model resulted in Dice values ranging from 0.60 to 0.75, while the mechanical model had values ranging from 0.77 to 0.83. However, a poor level of agreement was observed for voxel predictions of tumor cellularity resulting in low CCCs (less than 0.03) for both the standard and mechanical models. Conclusions: Incorporating the inhibiting effects of mechanical stress on tumor expansion resulted in improved global level predictions (i.e., decreased percent error in tumor volume, and increased Dice values) compared to the standard model. Both models, however, poorly predicted the observed intratumor heterogeneity in cell number. Therefore, the mechanical model is a more accurate description of in vivo C6 glioma growth at the global level, but further improvements are needed to provide a more complete description intratumor cellularity changes. Citation Format: David A. Hormuth, II, Jared A. Weis, Michael I. Miga, Thomas E. Yankeelov. A mechanically coupled reaction-diffusion model for predicting in vivo C6 glioma growth in rats. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A42.

PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion

Molecular therapies are hallmarks of "personalized" medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, "spatiotemporal" mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.

Specific dephosphorylation at tyr-554 of git1 by ptprz promotes its association with paxillin and hic-5.

G protein-coupled receptor kinase-interactor 1 (Git1) is involved in cell motility control by serving as an adaptor that links signaling proteins such as Pix and PAK to focal adhesion proteins. We previously demonstrated that Git1 was a multiply tyrosine-phosphorylated protein, its primary phosphorylation site was Tyr-554 in the vicinity of the focal adhesion targeting-homology (FAH) domain, and this site was selectively dephosphorylated by protein tyrosine phosphatase receptor type Z (Ptprz). In the present study, we showed that Tyr-554 phosphorylation reduced the association of Git1 with the FAH-domain-binding proteins, paxillin and Hic-5, based on immunoprecipitation experiments using the Tyr-554 mutants of Git1. The Tyr-554 phosphorylation of Git1 was higher, and its binding to paxillin was consistently lower in the brains of Ptprz-deficient mice than in those of wild-type mice. We then investigated the role of Tyr-554 phosphorylation in cell motility control using three different methods: random cell motility, wound healing, and Boyden chamber assays. The shRNA-mediated knockdown of endogenous Git1 impaired cell motility in A7r5 smooth muscle cells. The motility defect was rescued by the exogenous expression of wild-type Git1 and a Git1 mutant, which only retained Tyr-554 among the multiple potential tyrosine phosphorylation sites, but not by the Tyr-554 phosphorylation-defective or phosphorylation-state mimic Git1 mutant. Our results suggested that cyclic phosphorylation-dephosphorylation at Tyr-554 of Git1 was crucial for dynamic interactions between Git1 and paxillin/Hic-5 in order to ensure coordinated cell motility.

The amoeboidal Dictyostelium aquaporin AqpB is gated via Tyr216 and aqpB gene deletion affects random cell motility.

Regulation of aquaporin (AQPs) water channels mostly occurs on the transcriptional level or by intracellular trafficking. Direct AQP gating is comparatively rare and is described mainly for fungi and plants. Earlier, we identified a gated water-specific AQP in Dictyostelium discoideum, AqpB, which is opened by truncation of an extended intracellular loop D (AqpB Δ208-219). We show that Tyr216 of loop D is a key residue in the gating mechanism possibly involving phosphorylation. Mutation of Tyr216 to aspartate or glutamate initiated water permeability (Pf =69μm/s) to the same extent as AqpB Δ208-219, whereas neither replacement of Tyr216 by a positive arginine (Y216R) nor introduction of a negative charge in a neighbouring position (T217D) opened the channel. We overexpressed AqpB wildtype and AqpB Δ208-219 as GFP fusion constructs in Dictyostelium amoebae and found that the truncated, permanently open AqpB yielded cells with reduced capability to cope with hypotonic stress. Genetic deletion of aqpB yielded a strain with significantly reduced speed of random motility (4.5μm/min vs. 6.3μm/min of wildtype cells). Yet, chemotaxis towards folate and cAMP was unaffected. In this context, we identified a second, so-far uncharacterised amoeboidal AQP, AqpD, whose role in directed cell locomotion needs to be established. Our data add to the completion of the Dictyostelium model for cell motility and show that knowledge on the expression, permeability properties and localisation of amoeboidal AQPs are crucial for further development of the system.

γ-Actin plays a key role in endothelial cell motility and neovessel maintenance.

BackgroundAngiogenesis plays a crucial role in development, wound healing as well as tumour growth and metastasis. Although the general implication of the cytoskeleton in angiogenesis has been partially unravelled, little is known about the specific role of actin isoforms in this process. Herein, we aimed at deciphering the function of γ-actin in angiogenesis.MethodsLocalization of β- and γ-actin in vascular endothelial cells was investigated by co-immunofluorescence staining using monoclonal antibodies, followed by the functional analysis of γ-actin using siRNA. The impact of γ-actin knockdown on the random motility and morphological differentiation of endothelial cells into vascular networks was investigated by timelapse videomicroscopy while the effect on chemotaxis was assessed using modified Boyden chambers. The implication of VE-cadherin, VEGFR-2 and ROCK signalling was then examined by Western blotting and using pharmacological inhibitors.ResultsThe two main cytoplasmic isoforms of actin strongly co-localized in vascular endothelial cells, albeit with some degree of spatial preference. While β-actin knockdown was not achievable without major cytotoxicity, γ-actin knockdown did not alter the viability of endothelial cells. Timelapse videomicroscopy experiments revealed that γ-actin knockdown cells were able to initiate morphological differentiation into capillary-like tubes but were unable to maintain these structures, which rapidly regressed. This vascular regression was associated with altered regulation of VE-cadherin expression. Interestingly, knocking down γ-actin expression had no effect on endothelial cell adhesion to various substrates but significantly decreased their motility and migration. This anti-migratory effect was associated with an accumulation of thick actin stress fibres, large focal adhesions and increased phosphorylation of myosin regulatory light chain, suggesting activation of the ROCK signalling pathway. Incubation with ROCK inhibitors, H-1152 and Y-27632, completely rescued the motility phenotype induced by γ-actin knockdown but only partially restored the angiogenic potential of endothelial cells.ConclusionsOur study thus demonstrates for the first time that β-actin is essential for endothelial cell survival and γ-actin plays a crucial role in angiogenesis, through both ROCK-dependent and -independent mechanisms. This provides new insights into the role of the actin cytoskeleton in angiogenesis and may open new therapeutic avenues for the treatment of angiogenesis-related disorders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13221-014-0027-2) contains supplementary material, which is available to authorized users.

Abstract LB-70: BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression

Abstract Functional loss of BRCA1 has been implicated in genomic instability and cancer progression. A recent study demonstrated that BRCA1 plays a role in cancer cell migration. Using a random cell motility assay, we showed that BRCA1 promotes ovarian cancer cell migration. A proteomics study of BRCA1 +/- cell lines and tissues identified Profilin1 (Pfn1) as one of the differentially expressed proteins. This was further confirmed by Western blot and IHC analyses with tissue samples and cell lines. Pfn1 is an actin cytoskeleton-associated protein which promotes breast cancer cell motility/invasion and dissemination when downregulated. To further study whether BRCA1 impacts cell migration through Pfn1, Pfn1 was silenced which revealed enhanced motility in multiple BRCA1-WT ovarian cancer cell lines. Furthermore, overexpression of Pfn1 in BRCA1 cell lines reduced random motility which further implicates the importance of Pfn1 in ovarian cancer motility. This is the first evaluation of both motility and the functional role of Pfn1 in ovarian cancer cells. While BRCA1 has been extensively studied in the context of DNA repair, there is emerging evidence that BRCA1 proteins may play a role in cell migration. The increased motility seen in wild-type cells compared to BRCA1-mutated cells may portend increased metastatic potential in wild-type cancers. Our findings demonstrate that 1) BRCA1 positively influences ovarian cancer cell motility, and 2) BRCA1 status determines Pfn1's role in ovarian cancer cell motility suggesting a possible novel mechanism association between Pfn1 and BRCA1 and ovarian cancer cell motility. Future studies should explore whether Pfn1 downregulation enhances the metastatic potential of ovarian cancer cells with intact BRCA1 function and whether Pfn1 may serve as a possible therapeutic target. Citation Format: David M. Gau, Jamie Lesnock, Thomas Krivak, Robert Edwards, Partha Roy. BRCA1 impacts ovarian cancer cell migration through modulating Pfn1 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-70. doi:10.1158/1538-7445.AM2014-LB-70

Naringenin inhibits the growth of Dictyostelium and MDCK-derived cysts in a TRPP2 (polycystin-2)-dependent manner.

Background and PurposeIdentifying and characterizing potential new therapeutic agents to target cell proliferation may provide improved treatments for neoplastic disorders such as cancer and polycystic diseases.Experimental ApproachWe used the simple, tractable biomedical model Dictyostelium to investigate the molecular mechanism of naringenin, a dietary flavonoid with antiproliferative and chemopreventive actions in vitro and in animal models of carcinogenesis. We then translated these results to a mammalian kidney model, Madin-Darby canine kidney (MDCK) tubule cells, grown in culture and as cysts in a collagen matrix.Key ResultsNaringenin inhibited Dictyostelium growth, but not development. Screening of a library of random gene knockout mutants identified a mutant lacking TRPP2 (polycystin-2) that was resistant to the effect of naringenin on growth and random cell movement. TRPP2 is a divalent transient receptor potential cation channel, where mutations in the protein give rise to type 2 autosomal dominant polycystic kidney disease (ADPKD). Naringenin inhibited MDCK cell growth and inhibited cyst growth. Knockdown of TRPP2 levels by siRNA in this model conferred partial resistance to naringenin such that cysts treated with 3 and 10 μM naringenin were larger following TRPP2 knockdown compared with controls. Naringenin did not affect chloride secretion.Conclusions and ImplicationsThe action of naringenin on cell growth in the phylogenetically diverse systems of Dictyostelium and mammalian kidney cells, suggests a conserved effect mediated by TRPP2 (polycystin-2). Further studies will investigate naringenin as a potential new therapeutic agent in ADPKD.Linked ArticlesThis article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10

Distinguishing between mechanisms of cell aggregation using pair-correlation functions

Many cell types form clumps or aggregates when cultured in vitro through a variety of mechanisms including rapid cell proliferation, chemotaxis, or direct cell-to-cell contact. In this paper we develop an agent-based model to explore the formation of aggregates in cultures where cells are initially distributed uniformly, at random, on a two-dimensional substrate. Our model includes unbiased random cell motion, together with two mechanisms which can produce cell aggregates: (i) rapid cell proliferation and (ii) a biased cell motility mechanism where cells can sense other cells within a finite range, and will tend to move towards areas with higher numbers of cells. We then introduce a pair-correlation function which allows us to quantify aspects of the spatial patterns produced by our agent-based model. In particular, these pair-correlation functions are able to detect differences between domains populated uniformly at random (i.e. at the exclusion complete spatial randomness (ECSR) state) and those where the proliferation and biased motion rules have been employed – even when such differences are not obvious to the naked eye. The pair-correlation function can also detect the emergence of a characteristic inter-aggregate distance which occurs when the biased motion mechanism is dominant, and is not observed when cell proliferation is the main mechanism of aggregate formation. This suggests that applying the pair-correlation function to experimental images of cell aggregates may provide information about the mechanism associated with observed aggregates. As a proof of concept, we perform such analysis for images of cancer cell aggregates, which are known to be associated with rapid proliferation. The results of our analysis are consistent with the predictions of the proliferation-based simulations, which supports the potential usefulness of pair correlation functions for providing insight into the mechanisms of aggregate formation.

Matricellular Signal Transduction Involving Calmodulin in the Social Amoebozoan Dictyostelium

The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM) sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL) repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa) releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa) in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes.

Motility-based cell sorting by planar cell chromatography.

We report here a new methodology for sorting mammalian cells based on their intrinsic motility on planar substrates, independent of chemoattractants and external fields. This biological analogue of thin layer chromatography consists of arrays of asymmetric adhesive islands on tissue culture dishes that rectify the random movement of cells and direct their migration in a specific direction. We demonstrated the use of planar cell chromatography in the separation of mixtures of 3T3 fibroblasts that express constitutively active Rac1 or RhoA and mixtures of 3T3 fibroblasts and SH-SY5Y neuroblastoma cells.

Human Axonal Survival of Motor Neuron (a-SMN) Protein Stimulates Axon Growth, Cell Motility, C-C Motif Ligand 2 (CCL2), and Insulin-like Growth Factor-1 (IGF1) Production

Spinal muscular atrophy is a fatal genetic disease of motoneurons due to loss of full-length survival of motor neuron protein, the main product of the disease gene SMN1. Axonal SMN (a-SMN) is an alternatively spliced isoform of SMN1, generated by retention of intron 3. To study a-SMN function, we generated cellular clones for the expression of the protein in mouse motoneuron-like NSC34 cells. The model was instrumental in providing evidence that a-SMN decreases cell growth and plays an important role in the processes of axon growth and cellular motility. In our conditions, low levels of a-SMN expression were sufficient to trigger the observed biological effects, which were not modified by further increasing the amounts of the expressed protein. Differential transcriptome analysis led to the identification of novel a-SMN-regulated factors, i.e. the transcripts coding for the two chemokines, C-C motif ligands 2 and 7 (CCL2 and CCL7), as well as the neuronal and myotrophic factor, insulin-like growth factor-1 (IGF1). a-SMN-dependent induction of CCL2 and IGF1 mRNAs resulted in increased intracellular levels and secretion of the respective protein products. Induction of CCL2 contributes to the a-SMN effects, mediating part of the action on axon growth and random cell motility, as indicated by chemokine knockdown and re-addition studies. Our results shed new light on a-SMN function and the underlying molecular mechanisms. The data provide a rational framework to understand the role of a-SMN deficiency in the etiopathogenesis of spinal muscular atrophy.

EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins

DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.