Movement Of Single Cells Research Articles

Single File or En Masse

TGF-β signaling causes metastasizing breast cancer cells to change from collective to single cell movement.

A Novel Peptide Mediates Aggregation and Migration of Hemocytes from an Insect

Insect blood cells (hemocytes) comprise an essential arm of the immune system [1-7]. Several factors mediating recognition and phagocytosis of foreign intruders by hemocytes have been identified, but the mechanisms regulating hemocyte movement remain fragmentary. Embryonic hemocytes from Drosophila migrate along stereotypical routes in response to chemotactic signals from PVF ligands, members of the platelet-derived growth factor family [8-12]. Embryonic and larval hemocytes also accumulate at external wounds [11-13], but PVFs are not required for this response, suggesting involvement by other, unknown factors. Here we report the identification of hemocyte chemotactic peptide (HCP) from the moth Pseudaletia separata and present evidence that it stimulates aggregation and directed movement of phagocytic hemocytes. Spatiotemporal studies revealed that HCP is expressed in both epidermal cells and hemocytes, whereas structure-function studies identified post-translational modifications important for activity. HCP also shares similarities with another group of cytokines from moths called ENF peptides [14-17]. Taken together, our results identify HCP as a chemotactic cytokine that enhances clotting at wound sites in larvae.

Differential RhoA Dynamics in Migratory and Stationary Cells Measured by FRET and Automated Image Analysis

Genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to study the spatiotemporal regulation of molecular activity in live cells with high resolution. The efficient and accurate quantification of the large amount of imaging data from these single-cell FRET measurements demands robust and automated data analysis. However, the nonlinear movement of live cells presents tremendous challenge for this task. Based on image registration of the single-cell movement, we have developed automated image analysis methods to track and quantify the FRET signals within user-defined subcellular regions. In addition, the subcellular pixels were classified according to their associated FRET signals and the dynamics of the clusters analyzed. The results revealed that the EGF-induced reduction of RhoA activity in migratory HeLa cells is significantly less than that in stationary cells. Furthermore, the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. In contrast, there is a lack of consistent preference in RhoA polarity among stationary cells. Therefore, our image analysis methods can provide powerful tools for high-throughput and systematic investigation of the spatiotemporal molecular activities in regulating functions of live cells with their shapes and positions continuously changing in time.

Modeling single and collective cell movements in 3D matrices

Modeling single and collective cell movements in 3D matrices

Human Fibroblast Growth Factor Receptor 1-IIIb Is a Functional Fibroblast Growth Factor Receptor Expressed in the Pancreas and Involved in Proliferation and Movement of Pancreatic Ductal Cells

The possible functions of the human IIIb-messenger RNA splice variant of fibroblast growth factor (FGF) receptor 1 (FGFR-1 IIIb) are yet to be delineated. In this study, the expression and functionality of the human FGFR-1 IIIb were characterized in the pancreas. In situ hybridization with a specific FGFR-1 IIIb probe in human pancreatic tissues demonstrated that FGFR-1 IIIb localized in normal pancreatic acinar and in ductal-like pancreatic cancer cells. To further assess the potential role of this receptor, a full-length human FGFR-1 IIIb was stably expressed in TAKA-1 pancreatic ductal cells not expressing endogenous FGFR-1. The FGFR-1 IIIb-expressing TAKA-1 cells synthesized a glycosylated 110-kd protein capable of inducing proliferation on incubation with exogenous FGF-1, -2, and -4. These effects were paralleled by tyrosine phosphorylation of FGFR substrate 2 and association of FGFR substrate 2 with FGFR-1 IIIb. The FGF-1, -2, and -10 induced the activation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, and c-Jun N-terminal kinase. Pharmacological inhibition revealed that FGF-induced proliferation was dependent on the concomitant activation of p44/42 MAPK and c-Jun N-terminal kinase. The FGFR-1 IIIb expression enhanced single-cell movement and plating efficacy. Our results demonstrate that the human FGFR-1 IIIb variant is a functional FGFR expressed in the pancreas that can alter pancreatic functions that regulate proliferation, adhesion, and movement.

Identification of a Fibroblast Growth Factor Receptor 1 Splice Variant That Inhibits Pancreatic Cancer Cell Growth

Fibroblast growth factor receptors (FGFR) play important roles in many biological processes. Nothing is presently known about possible roles of the human FGFR1-IIIb mRNA splice variant. In this study, we characterized for the first time the effects of FGFR1-IIIb expression on the transformed phenotype of human pancreatic cancer cells. The full-length FGFR1-IIIb cDNA was generated and stably expressed in PANC-1 and MIA PaCa-2 pancreatic cancer and TAKA-1 pancreatic ductal cells. FGFR1-IIIb-expressing cells synthesized a glycosylated 110-kDa protein enhancing tyrosine phosphorylation of FGFR substrate-2 on FGF-1 stimulation. The basal anchorage-dependent and anchorage-independent cell growth was significantly inhibited. These effects were associated with a marked reduction of p44/42 mitogen-activated protein kinase (MAPK) phosphorylation in combination with enhanced activity of p38 MAPK and c-Jun NH(2)-terminal kinase. FGFR1-IIIb expression inhibited single-cell movement and in vitro invasion as determined by time-lapse microscopy and Boyden chamber assay as well as in vivo tumor formation and growth in nude mice. Microscopic analysis of the xenograft tumors revealed a reduced Ki-67 labeling and a lower amount of tumor necrosis in FGFR1-IIIb-expressing tumors. Our results show that FGFR1-IIIb is a functional FGFR that inhibits the transformed phenotype of human pancreatic cancer cells.

EXPRESSION OF FGFR1-IIIB INHIBITS PANCREATIC CANCER CELL GROWTH IN VITRO AND IN VIVO

Fibroblast growth factors (FGFs) and their high affinity receptors (FGFRs) play important roles in many malignancies being involved in processes like angiogenesis and mitogenesis. Ligand binding specificities are determined predominantly by alternative mRNA splicing of the second half of Ig-like domain III of the FGFRs. Cells of epithelial origin mostly express IIIb and cells of mesenchymal origin more often the IIIc variants. We recently demonstrated that FGFR1-IIIc dramatically enhanced the transforming potential of TAKA-1 pancreatic ductal cells and that human pancreatic carcinoma tissue and cultured pancreatic cancer cells predominantly express FGFR1-IIIc. Presently only little is known about the recently cloned human FGFR1-IIIb. The aim of this study was to characterize possible functions and the transforming potential of human FGFR1-IIIb in pancreatic ductal cells. In-situ-hybridization demonstrated that FGFR1-IIIb localized in normal pancreatic acinar and ductal pancreatic cancer cells. Full-length human FGFR1-IIIb was stably expressed in TAKA-1 pancreatic ductal cells and PANC-1 pancreatic cancer cells next. FGFR1-IIIb was expressed as a glycosylated 110 kDa protein. Basal anchorage-dependent and-independent growth, determined by MTT and soft agar assays, respectively, was significantly inhibited by at least 35% and 75%, respectively. These effects were paralleled by a reduced phosphorylation of p44/42 MAPK and increased p38 MAPK and c-Jun-N-terminal kinase activity. Additionally, expression of FGFR1-IIIb in PANC-1 cells reduced single cell movement determined by time-lapse microscopy and invasion in the Boyden chamber assay. Xenograft formation was inhibited. Thus, only thirty of 40 sites injected with FGFR1-IIIb expressing PANC-1 cells developed tumors compared to 38 of 40 sites injected with control-transfected cells. The growth of the developing xenograft tumors and their KI-67 labeling index were further significantly reduced. Establishment of clones in MIA PaCa-2 cells showed inhibitory functions by FGFR1-IIIb expression and enhancement of proliferation by IIIc. In this study, for the first time, functions of human FGFR1-IIIb are described in the pancreas demonstrating inhibition of transformation in contrast to FGFR1-IIIc in vitro and in vivo. Our results suggest that exon switching of FGFR1 from IIIc to IIIb may significantly influence the transforming potential of pancreatic cancer cells.

Behavior of cellulose production of Acetobacter xylinum in 13C-enriched cultivation media including movements on nematic ordered cellulose templates

Acetobacter xylinum NQ-5 (ATCC 53582) and AY-201 (ATCC 23769) cultivated in the Hestrin–Schramm medium containing d-glucose with a natural 13C-abundance of 1.1% were investigated regarding their cell division rates and the bacterial movements. Comparative studies were carried out in the presence of d-glucose-U- 13C 6 with a uniform 13C-labeling of 99% as the sole carbon source. The bacterial growth rates in numbers were found to increase in the 13C-enriched media by about 13% for NQ-5 and 26% for AY-201, respectively. The movements of single cells caused by the inverse force of the secretion and deposition of cellulose nanofibers on nematic ordered cellulose (NOC) templates were investigated by real-time video analyzes using light microscopy. As a result, d-glucose-U- 13C 6 reduced the speed of motion for both strains, which was an opposite trend to the above growth rates in the cell division. The deposited cellulose fibers were proved to be in a cellulose crystalline form by CP/MAS 13C NMR and NIR FT Raman spectroscopic measurements, although the biosynthesized and thereafter-deposited cellulose has a strong interaction with the surface of NOC.

Autonomous Modes of Behavior in Primordial Germ Cell Migration

Zebrafish primordial germ cells (PGCs) are guided toward their targets by the chemokine SDF-1a. PGCs were followed during three phases of their migration: when migrating as individual cells, while remaining in a clustered configuration, and when moving as a cell cluster within the embryo. We found that individually migrating PGCs alternate between migratory and pausing modes. Pausing intervals are characterized by loss of cell polarity and correlate with subsequent changes in the direction of migration. These properties constitute an intrinsic behavior of PGCs, enabling erasure of prior polarity and re-sampling of the environment. Following migration arrest at a site of high SDF-1a levels, PGCs resume migration as a cluster. The seemingly coordinated cluster migration is a result of single-cell movement in response to local variations in SDF-1a distribution. Together, these behavioral modes allow the cells to arrive at specific destinations with high fidelity and remain at their target site.

Simultaneous Measurement of Growth and Movement of Cells Exploiting On-Chip Single-Cell Cultivation Assay

We have developed an on-chip single-cell microcultivation assay as a means of simultaneously observing the growth and movement of single bacterial cells during long-term cultivation. This assay enables the direct observation of single cells captured in microchambers fabricated on thin glass slides and having semipermeable membrane lids, in which the cells can swim within the space without escape for the long periods. Using this system, the relationship between the cell cycle and the tendency of movement was observed and it was found that the mean free path length did not change during the cell cycle, and that the growth and the swimming were not synchronized. The result indicates that the ability of movement of the cells was independent of the cell cycle.

Rhythm 'n' biology: what happens if cell biology and techno music meet?

Beauty is in the eye of the beholder. Peering down a microscope, what a layperson may regard as strange fuzzy structures could be an exciting discovery for the trained scientist. On the other hand, while the scientist might only see useful information, the layperson may marvel at the hidden beauty of the microscopic world. To watch bacteria swim towards a food source or an amoeba slowly crawl toward and digest an unfortunate bacterium does hold a certain fascination of watching life taking place at the smallest level. By granting access into these minute worlds, the scanning electron microscope, for instance, has not only allowed scientists to study the surfaces of many cells and organisms at high magnifications, but has also produced many of the images that have graced the covers of or have been featured as full‐page illustrations in non‐scientific magazines. > The colourful patterns from immunofluorescence or confocal laser scanning microscopy also have a striking beauty that goes beyond the scientific content Similarly, fluorescently tagged antibodies and the green fluorescent protein (GFP) have revolutionised light microscopy, and many biological publications today contain familiar images of brightly labelled intracellular structures. And it does not stop at simple pictures; conferences on microbiology or cell biology feature an increasing number of presentations that rely on movies of cellular events. But the colourful patterns produced by immunofluorescence or confocal laser scanning microscopy also have a striking beauty that goes beyond the scientific content they represent. This awareness led some scientists to develop a new concept of depicting their work: a form of multimedia presentation that relates a biological story through text, animation, sound, video and even music such that the speaker is redundant. This approach was demonstrated to the auditorium of the Cinema of the Cell session at this year's ELSO conference, organised by …

Bmp Activity Gradient Regulates Convergent Extension during Zebrafish Gastrulation

During vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. Here, by measuring movements of cell populations in vivo, we reveal the presence of three domains of convergent extension movements in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, we demonstrate that these domains are specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicated that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses revealed that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. We show that high Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension.

3I0930 マイクロ加工と画像処理を用いた大腸菌 : 細胞の成長過程における運動パターン変化解析技術の開発(26.計測,一般講演,日本生物物理学会第40回年会)

3I0930 マイクロ加工と画像処理を用いた大腸菌 : 細胞の成長過程における運動パターン変化解析技術の開発(26.計測,一般講演,日本生物物理学会第40回年会)

Interactive image processing system for assessment of cell movement.

The study of cancer cell motility is considered to be important in understanding cancer metastasis. The movement behaviour of cells within clustered cell colonies is of particular interest. Changes in cell movement, area and velocity can be an indicator of cell spreading. The aim of the study is to develop and apply a computerised interactive image processing system to quantify the movement of cells within cell clusters. A semi-automatic boundary description method based on two-dimensional rendering is devised. The system is later combined with image-processing methods that facilitate the relocation of the cell boundary over time; this forms a new approach to assessing cell movement. These methods are incorporated into a software system, enabling an interactive procedure to define and monitor the movement of single cells in cell clusters from digitised microscope images. Validation of the method shows a maximum error of 10% in defining the area through a cubic spline interpolation. The system is applied to analyse the movement and area of HT115 human colon cancer cells. The system provides tools for the analysis of movement, area and velocity of single cells in cancer cell colonies and may thus be of value in further understanding cancer cell motility.

A model for hepatocarcinogenesis treating phenotypical changes in focal hepatocellular lesions as epigenetic events.

The major paradigm for mathematically describing the carcinogenic process has been through the use of multistage models. Multistage models are made up of numerous compartments representing cells in various stages on the way to malignancy and where movement from one cell class to another is assumed to have exponential waiting time. Once a cell is in a particular class, clonal expansion through a linear birth-death process increases the size of the compartment. These models are characterized by movement of single cells from one compartment to another rather than clonal colonies of cells. However, there is some evidence to suggest that, in certain organs for certain types of agents, preneoplastic lesions with different phenotypes arise directly from an entire clonal colony rather than from a single cell within that colony. This manuscript describes a simple mathematical model of carcinogenesis using both persistent changes of single or several cells (to start the process) and shifting of colonies to describe the stages of the model. Likelihoods for the use of the model with data on colonies of preneoplastic lesions are described and applied to real data.

PDGF-BB increases endothelial migration and cord movements during angiogenesis in vitro

To explore direct effects of platelet-derived growth factor (PDGF) on endothelial cells during angiogenesis in vitro, we have used cloned bovine aortic endothelial cells that spontaneously form cord structures. Recently we have shown that cells forming these endothelial cords express PDGF beta-receptors and that PDGF-BB can contribute to cellular proliferation and cord formation. In this study we investigated whether PDGF-induced cellular migration might also contribute to endothelial repair and angiogenesis in vitro. Ten individual endothelial cells in cords were tracked at an early stage of cord formation by video-timelapse microscopy. PDGF-BB (100 ng/ml) induced an increase in endothelial cell movement of 67 +/- 15% as compared with diluent control. Interestingly, PDGF-BB also increased movements of entire cord structures, followed at branching points, by 53 +/- 12% over diluent control. Taken together, these video-timelapse experiments suggested that the apparent movements of single endothelial cord cells might also be due to the motion of entire underlying cord structures in response to PDGF. To analyze the response of single endothelial cord cells we therefore examined whether PDGF-induced migration contributes to endothelial repair. Abrasions were applied with a razor blade to confluent monolayers of endothelial cells at an intermediate stage of cord formation. PDGF-BB concentration-dependently increased the distance to which cord-forming endothelial cells migrated into the abrasion. An increased number of elongated, i.e., probably migrating, endothelial cells was found in the abrasion in response to PDGF-BB. However, there was no effect of PDGF-BB on the total number of endothelial cells found in the abrasion. PDGF-AA affected neither the distance to which the cells migrated nor the number of elongated cells. Actin and tubulin stainings revealed that these cytoskeletal structures were not appreciably altered by PDGF-BB. Furthermore, urokinase-type plasminogen activator transcripts were not modulated in response to PDGF-BB. We conclude that in this model of angiogenesis in vitro PDGF-BB can elicit the movement of entire cord structures, possibly via u-PA-independent mechanisms. PDGF-BB also controls the migration of single cord-forming endothelial cells. Thus, PDGF-BB possibly contributes to endothelial repair and angiogenesis by direct effects on proliferation and composite movements of PDGF beta-receptor-expressing endothelial cells and cords.

Morphometric Description of the Wandering Behavior in Drosophila Larvae: Aberrant Locomotion in Na+ and K+ Channel Mutants Revealed by Computer-Assisted Motion Analysis

Wandering is a simple behavior in Drosophila larvae prior to metamorphosis. Using the Dynamic Image Analysis System (DIAS) initially developed for analyzing amoeboic movements of single cells, we have analyzed videotaped behaviors of Drosophila larvae at the wandering stage. Previous studies show that mutations in the Na+ channel gene paralytic (para) cause paralysis at 29°C, and mutations in the K+ channel β subunit gene Hyperkinetic (Hk) lead to leg-shaking under ether anesthesia. The application of DIAS revealed quantifiable abnormalities in the larval locomotion of both ion channel mutants even under “permissive” conditions. Analysis of centroid movement indicates that, compared to wild type, both Hk and para larvae crawled at a slower average speed, but at a similar peak instantaneous speed during a contraction cycle. Nevertheless, contraction in the body length was greater in mutants, implying a lower efficiency in conversion of muscular contraction to distance translocation. In addition, each mutant produced a characteristic crawling pattern distinct from the wild-type control. The larval crawling pattern was determined by periods of linear locomotion interposed by non-locomotive, “searching and decision-making” episodes, after which the crawling was resumed in a new direction. Our results demonstrate that mutations in single ion channel subunits resulted in stereotypic modifications in locomotion control and crawling patterns, and that DIAS is a powerful tool in revealing subtle differences in animal behavior and quantifying mutational effects on the interplay of discrete behavioral components.

A new laser photometric technique for the measurement of erythrocyte aggregation and sedimentation kinetics.

The aggregation kinetics of erythrocytes is studied by using laser light scattering. Laser light is passed through a well mixed sample of blood and the forward-scattered light intensity is recorded continuously. During the process of aggregation and sedimentation under a gravitational field, erythrocytes are subjected to different orientations. The events of a single cell's movement, formation of aggregates and their sedimentation produce a variation in scattered-light intensity. The mean intensity variation that bears this information is effectively employed to arrive at the kinetics of single cells, one-dimensional aggregates and three-dimensional aggregates during sedimentation. In addition to this, the time intervals of different phases of aggregation and sedimentation are also measured. All these parameters are obtained for erythrocytes suspended in native plasma. Then, the results are compared with erythrocytes suspended in dextran (Dx) 70 and Dx 500.

Magnetophoresis: I. detection of magnetically labeled cells

A new method for the detection of magnetically labeled cells in suspension using an optical microscope is presented. The movement of individual cells in a nonuniform magnetic field (magnetophoresis) can be studied and the direction and velocity of their movement can be determined. Cell-associated magnetic particles (e.g., attached magnetic immunomicrospheres) influence these parameters. We developed a magnetophoresis chamber for measuring the movement of single cells in a high-gradient magnetic field. Paramagnetic erythrocytes were used to monitor the level of magnetic force in the chamber. Bw6 antigen-positive REH cells were used as a biological model. Magnetic microspheres coupled with anti-Bw6 antibody were used as magnetic labels. The movement of magnetically labeled REH cells was investigated versus the intensity of magnetic field. The magnetization of microspheres was experimentally determined by the Faraday balance method. Experimental and theoretical results substantiate the feasibility of detecting a single cell associated with a single magnetic microsphere. Thus, magnetophoresis provides a sensitive biometrical system for magnetically labeled entities.

Towards the molecular physiology of cell movements

The above passage, taken from a letter written by Anthony van Leeuwenhoek to a Mr Oldenberg in 1674, is probably the first recorded observation of the movement of single cells. The specimen was water, freshly collected from a lake a short distance from Leeuwenhoek's house in Delft, and it seems probable that some of the little creatures cfescribed were ciliated protozoa. Certainly in later observations Leeuwenhoek described in unmistakable detail, and in some cases sketched, ciliated protozoa such as Euglena, Stylonichia and the stalked Vorticello ('the lowliest animal I ever saw'); he was even able to see bacteria through his simple lenses. Movement was one of the few characteristics of life that could be identified readily using the rudimentary techniques then available, and a source of amazement to early microscopists. Leeuwenhoek, himself a cautious empiricist, recorded what he saw in enthusiastic but painstaking and relatively unadorned detail, but other early microscopists had more florid interpretations. Thus Henry Baker, in his Employment for the Microscope, published in 17.53, described: