Initiating and guiding migration: lessons from border cells

Cell migration occurs in many different contexts. Amoebae and other isolated cells migrate in culture. In animals, 'professional' migratory cells of the immune system constantly survey the body for intruders, whereas other cell types perform specific developmentally regulated migrations. One simple model for the latter type of event is migration of border cells during Drosophila oogenesis. Recent findings have shed light on how border cell fate is induced and on how the migration is guided. This article discusses the implications of these studies and compares (invasive) migration through a tissue with what is known about cells crawling on a flat substratum.     

The role of root border cells in plant defense

The survival of a plant depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Perhaps because of the root tip's vital role in plant health, it is ensheathed by large populations of detached somatic cells - root 'border' cells - which have the ability to engineer the chemical and physical properties of the external environment. Of particular significance, is the production by border cells of specific chemicals that can dramatically alter the behavior of populations of soilborne microflora. Molecular approaches are being used to identify and manipulate the expression of plant genes that control the production and the specialized properties of border cells in transgenic plants. Such plants can be used to test the hypothesis that these unusual cells act as a phalanx of biological 'goalies', which neutralize dangers to newly generated root tissue as the root tip makes its way through soil.     

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte.     

Cell migration in invertebrates: clues from border and distal tip cells.

Recent studies in two invertebrate systems, border cells in Drosophila melanogaster and distal tip cells in Caenorhabditis elegans, have provided important insight into the mechanisms of directed cell migration. These migrating cells are guided by extracellular signals, such as EGF, TGF-beta and netrin. In addition, metalloproteases alter the extracellular matrix of the tissue through which these cells migrate. Along the migratory path, migrating cells respond to changes in guidance signals by altering the expression of receptor signaling pathways. Finally, Dock180, CrkII and the GTPase Rac link the extracellular signals to the cellular machinery that controls cell motility.     

Formation and separation of root border cells

Plant roots release a large number of border cells into the rhizosphere, which are believed to play a key role in root development and health. The formation and loss of these cells from the root cap region is a developmentally regulated process that is also controlled by phytohormones and environmental factors. The separation of border cells involves the complete dissociation of individual cells from each other and from root tissue. This process requires the activity of cell wall-degrading enzymes that solubilize the cell wall connections between cells. We present and discuss the solubilization process with an emphasis on pectin-degrading enzymes as well as the recently discovered root border-like cells of Arabidopsis thaliana.     

Epigenomic profiling of cancer cells

DNA methylation, post-translational modifications of histones and high order organization of chromatin in cell nuclei are the components of the epigenome. Epigenetic regulation of gene expression is specific for each cell type, within different tissues, according to stages of development and (in the adult organism) of differentiation. Almost invariably, this regulation is altered in disease states, including cancer. The complete understanding of the identity of the epigenome of cancer has been so far hampered, due to the technical limitations and costs of the genome-wide analyses required. The recent development of next generation sequencing (NGS) technologies, however, holds the promise of fast, reliable and cost-effective analyses. Here we review the main approaches employed thus far to identify altered epigenetic patterns in cancer cells, and analyse how they are predicted to evolve in the scenario of the ultra high-throughput (UHT) screenings.     

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.     

Root border cells take up and release glucose-C

BACKGROUND AND AIMS: Border cells are released from the root tips of many plant species, and can remain viable in the rhizosphere for 1 week. Whether border cells are capable of controlled glucose exchange with their environment was investigated. METHODS: Border cells were removed from Zea mays L. root tips, and immersed in (14)C-labelled D-glucose. In one experiment, the hexose transport inhibitor, phlorizin, was used to investigate active glucose uptake from a range of glucose concentrations. In another experiment, glucose efflux from border cells was monitored over time. KEY RESULTS: Glucose uptake by the border cells increased with increasing glucose concentration from 0.2 to 20 mm. At 0.2 mm glucose, uptake was mainly active, as evidenced by the approx. 60 % inhibition with phlorizin. At 2 and 20 mm glucose, however, uptake was mainly via diffusion, as phlorizin inhibition was negligible. Glucose efflux increased with time for live border cells in both 2 and 20 mm glucose. There was no clear efflux/time pattern for heat-killed border cells. CONCLUSIONS: Border cells actively take up glucose, and also release it. Under our experimental conditions, glucose uptake and efflux were of similar order of magnitude. In the rhizosphere net glucose exchange will almost certainly depend on local soil conditions.     

Migration of fibroblastoid stromal cells in murine blood

This paper describes the kinetics of fibroblastic colony forming units (CFU-f) in murine blood after phenylhydrazine-induced haemolytic anaemia and their subsequent migration into haemopoietic organs. Murine blood contained 5.3 +/- 0.8 CFU-f per 10(6) nucleated cells. Absence of particle ingestion and factor VIII-related antigen in addition to the enzyme pattern in CFU-f-derived cells confirmed that these cells did not have a macrophage-like or endothelial nature. Phenylhydrazine treatment of mice resulted in a 3-fold increase in blood CFU-f numbers which was accompanied by increases in blood cellularity and granulocyte-macrophage progenitor numbers. When both partners of CBA/N and CBA/T6T6 mice in parabiosis had been treated with phenylhydrazine, spleens and femoral bone marrow of both mice were shown to contain partner-derived CFU-f. These data suggest that circulating CFU-f represent a stromal cell population which can migrate into haemopoietic organs.     

Migration and keratinization of cells in wool follicles

Migration of cells in wool follicles of an adult Merino sheep was studied autoradiographically in skin samples taken at intervals after an intravenous injection of [3H]thymidine. Fibre and inner root sheath cells incorporated [3H]thymidine in a cone-shape region of the follicle bulb. Labelled inner sheath cells migrated out of the bulb ahead of contemporaneous cells in the fibre and remained in advance, although to a progressively lesser extent, until the inner sheath cells sloughed into the follicle lumen. Outer root sheath cells incorporated [3H]thymidine along the length of the follicle. Cells in the proximal half of the outer sheath migrated inwards and distally and sloughed into the follicle lumen before contemporaneous inner sheath cells. Other cells in the distal half of the outer sheath migrated past the level where cells from the proximal population were shed and also sloughed into the lumen. In the most distal part of the outer sheath, which formed the epidermis-like lining of the follicle canal, little migration of cells was observed during 8 days of observation. The specific activity of tritium in fibres plucked from the same sheep at intervals after the intravenous injection of [3H]thymidine was determined by scintillation counting and assessed in terms of cell migration and hardening of the fibres. The time which the specific activity of solvent-degreased fibres reached a maximum was found to give an estimate of the time for cells in the fibre to migrate to the upper limit of the keratogenous zone. When the plucked fibres were extracted with 8 M urea the times of the maximum specific activities of the urea-dispersible and urea-insoluble material provided respectively estimates of the times at which hardening of the fibres began and ended. The effects of different planes of nutrition were examined in two other Merino sheep by radioassay of fibres plucked after intravenous injections of [3H]thymidine given after equilibration period of at least 2 months on each level of feeding. A high plane of nutrition the rate of cell migration and hastened the onset of hardening of the fibres, but prolonged the hardening process. The prolongation of the hardening process was confirmed by the specific activities of fibres plucked after intravenous injections of [35S]cystine.     

Migration of multipotent interstitial stem cells in Hydra

Stem cells in Hydra represent one of the phylogenetically most ancient stem cell systems and, therefore, provide information for reconstructing the early history of stem cell control mechanisms. Hydra's interstitial stem cells are multipotent and differentiate into both somatic cell types and germ line cells. Although it is well accepted that cells of the interstitial cell lineage are migratory, the in vivo migratory potential of multipotent interstitial stem cells has never been explored. Combining in vivo tracing of genetically labeled interstitial stem cells and tissue transplantation, we show that in contrast to precursor cells, multipotent interstitial stem cells are stationary. Only when exposed to tissue depleted of the interstitial cell lineage, interstitial stem cells start to migrate and to repopulate emptied stem cell niches. We conclude that multipotent interstitial stem cells in Hydra are static and that microenvironmental cues including signals derived from the interstitial cell lineage or from niche cells can trigger a shift in collective stem cell behavior to start migration.     

禽类原始生殖细胞的迁移能力

Blood samples were collected from chicken embryos at stage 11-15,and labeled with fluorescent dye PKH26.Primordial germ cells (PGCs)were then isolated from blood samples by nycodenz density gradient centrifugation.After PGCs were labeled and isolated,about 200 PGCs in one microliter were injected into the subgerminal cavity of quail blastoderm at stageX.After 48 hours incubation,chicken PGCs were identified by fluorescent microscopy.Red fluorescence emitted from PKH26 labeled chicken PGCs was observed in the head, the heart and the developing gonadal anlage of quail embryos.The result suggests that chicken PGCs still keep migration ability after 56 hours[Acta Zoologica Sinica 49(6):868-872,2003].     

Metabolite profiling of CHO cells: Molecular reflections of bioprocessing effectiveness

Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC‐MS) analytics to define the molecular loci by which two yield‐enhancing feeds improve recombinant antibody yields from a model GS‐CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing.     

Operomics: Integrated genomic and proteomic profiling of cells and tissues

In the post-genome era, technologies are becoming availablethat allow the profiling of tissues and cell populations atmultiple levels including genomic (DNA and RNA), proteomic (proteinsand peptides) and post-proteomic (eg metabolomic). Operomicsrefers to the molecular analysis of tissues and cells at thethree levels that are connected through the coding process —namely, DNA, RNA and protein. The premise is that no one levelor type of analysis fully captures gene expression and thatfunctional changes at the proteome level cannot be simply predictedfrom analyses at the DNA or RNA levels. An important determinantthat weakens a direct link between RNA and protein levels istranslational control that differentially regulates mRNA translation.In this paper, the approaches for genomic and proteomic profilingand the contribution of translational control are reviewed.      

Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo

As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, β-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.