Automated time-lapse microscopy and high-resolution tracking of cell migration

Fotos, Joseph S.; Patel, Vivek P.; Karin, Norman J.; Temburni, Murali K.; Koh, John T.; Galileo, Deni S.

doi:10.1007/s10616-006-9006-7

Cited by 29 publications

(31 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…GBM21/shL1 cells also showed reduction of L1 expression compared to controls. Quantitative Super Scratch assays [7, 39, 40] were performed using time-lapse microscopy, and T98G/shL1 cells migrated at a lower average velocity throughout the experiment (Fig. 4b) and had significantly reduced overall average velocity compared to T98G/control cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…By using a highly quantitative Super Scratch time-lapse motility assay [39, 40], which allowed tracking of individual cell velocities throughout the time course of the experiment, it was clear that L1 attenuation caused a highly significant decrease of cell motility in T98G/shL1 cells. Using L1 ectodomain-conditioned medium and the time-lapse motility assay, we not only demonstrated the recovery of cell motility in L1-attenuated T98G/shL1 cells, but also showed the increase of cell motility in U-118 MG cells, which do not express endogenous L1 protein.…”

Section: Discussionmentioning

confidence: 99%

“…Cell motility of U-118 MG, T98G/shL1, and T98G/control cells was measured by using the time-lapse microscopy Super Scratch assay as previously described [7, 39, 40]. Cells were grown to confluence on plastic tissue culture dishes and then “wounded” with a sterile plastic 1 ml pipettor tip in serum-free media.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

L1 stimulation of human glioma cell motility correlates with FAK activation

Yang

Chilukuri

et al. 2011

J Neurooncol

Self Cite

View full text Add to dashboard Cite

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass upregulate ADAM10 to proteolyze surface L1 and the resultant ectodomain increases human glioma cell migration and invasion by binding to integrin receptors, activating FAK, and increasing turnover of focal complexes.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

L1 stimulation of human glioma cell motility correlates with FAK activation

Yang

Chilukuri

et al. 2011

J Neurooncol

Self Cite

View full text Add to dashboard Cite

show abstract

“…It is likely that bone cells in the vicinity of skeletal damage are exposed to high levels of LPA released from hematomas. Pre-osteoblast migration is essential for proper fracture healing [19], and LPA has robust chemotactic effects on osteoblastic cells [1, 20, 21]. LPA induced membrane blebbing in primary cultured calvarial osteoblasts and stimulated the formation of membrane extensions in MC3T3-E1 pre-osteoblastic cells and MLO-Y4 osteocytic cells [21–23].…”

Section: Introductionmentioning

confidence: 99%

Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth

Waters

Jacobs

Gritsenko

et al. 2011

Bone

Self Cite

View full text Add to dashboard Cite

Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24 hours in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6 hours and 844 proteins at 24 hours. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6 hours and 24 hours of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among the proteins with elevated abundances in dendrites were molecules that regulate cytoskeletal function, cell motility and membrane adhesion. Our combined transcriptomic/proteomic analysis of the response of MLO-Y4 osteocytic cells to LPA indicates that dendritogenesis is a membrane- and cytoskeleton-driven process with actin dynamics playing a particularly critical role.

show abstract

“…Experimentally, tumor cells from lines like U-87 human glioma can move at a rate of about [8], [4]. Many bacteria can swim as fast as 50 ηm/s [14], and the maximum velocity of wild-type Salmonella can reach 68 ηm/s [13].…”

Section: Implementation and Parametersmentioning

confidence: 99%

Computer simulation of Salmonella typhimurium accumulation within tumors

Gao

Tabirca

Tangney

2011

Proceedings of the 9th International Conference on Computational Methods in Systems Biology

View full text Add to dashboard Cite

Salmonella typhimurium exhibit the ability to specifically chemotax and proliferate in heterogeneous tumor tissue in vitro. Based on this observation, we present a computer simulation to describe how S. typhimurium strains respond to chemotaxis signals that are secreted by different type of tumor cells. To test the effect of each chemoattractant on the chemotaxis of S. typhimurium toward different tumor regions, a range of simulations have been carried out by quantifying the accumulation of chemotaxis machinery knockouts, including generalized strains lacking individual cell surface chemoreceptors. To measure the bacterial accumulation affected by tumor size and structures, we compared the average bacterial density in small tumors (diameter < 500 ηm without quiescent region) with large tumors (diameter > 500 ηm contains quiescent and necrotic regions). Our work demonstrates the value of combining computer simulation with experiments to uncover potential mechanisms that are involved in bacterial chemotaxis tumor targeting and accumulation. In addition, it also demonstrates the utility of the Glazier-Graner-Hogeweg methodology (GGH) for modeling the links between cellular dynamics and environment signaling as well as predicting unanticipated results.

show abstract

Automated time-lapse microscopy and high-resolution tracking of cell migration

Cited by 29 publications

References 33 publications

L1 stimulation of human glioma cell motility correlates with FAK activation

L1 stimulation of human glioma cell motility correlates with FAK activation

Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth

Computer simulation of Salmonella typhimurium accumulation within tumors

Contact Info

Product

Resources

About