To clarify the role of Ret signaling components in enteric nervous system (ENS) development, we evaluated ENS anatomy and intestinal contractility in mice heterozygous for Ret, GFRα1 and Ret ligands. These analyses demonstrate that glial cell line-derived neurotrophic factor (GDNF) and neurturin are important for different aspects of ENS development. Neurturin is essential for maintaining the size of mature enteric neurons and the extent of neuronal projections, but does not influence enteric neuron number. GDNF availability determines enteric neuron number by controlling ENS precursor proliferation. However, we were unable to find evidence of programmed cell death in the wild type ENS by immunohistochemistry for activated caspase 3. In addition, enteric neuron number is normal in Bax -/-and Bid -/-mice, suggesting that, in contrast to most of the rest of the nervous system, programmed cell death is not important for determining enteric neuron numbers. Only mild reductions in neuron size and neuronal fiber counts occur in Ret +/-and Gfra1 +/-mice. All of these heterozygous mice, however, have striking problems with intestinal contractility and neurotransmitter release, demonstrating that Ret signaling is critical for both ENS structure and function.
The role of cell adhesion molecules in mediating interactions with neighboring cells and the extracellular matrix has long been appreciated. More recently, these molecules have been shown to modulate intracellular signal transduction cascades critical for cell growth and proliferation. Expression of adhesion molecule on glia (AMOG) is downregulated in human and mouse gliomas, suggesting that AMOG may be important for growth regulation in the brain. In this report, we examined the role of AMOG expression on cell growth and intracellular signal transduction. We show that AMOG does not negatively regulate cell growth in vitro or in vivo. Instead, expression of AMOG in AMOG-deficient cells results in a dramatic increase in cell size associated with protein kinase B/Akt hyperactivation, which occurs independent of phosphatidylinositol 3-kinase activation. AMOG-mediated Akt phosphorylation specifically activates the mTOR/p70 S6 kinase pathway previously implicated in cell size regulation, but it does not depend on tuberous sclerosis complex/Ras homolog enriched in brain (Rheb) signaling. These data support a novel role for a glial adhesion molecule in cell size regulation through selective activation of the Akt/mTOR/S6K signal transduction pathway.Cues received from the extracellular environment by membrane receptors influence diverse intracellular signaling pathways that regulate cell survival, differentiation, and growth. Cell adhesion molecules have been primarily implicated in maintaining cell-cell and cell-matrix interactions important for maintaining tissue integrity. However, recent evidence indicates that these adhesion molecules, like other membrane-localized receptors, can influence intracellular signal transduction (34, 61). Numerous adhesion molecules, including cadherins, integrins, and immunoglobulin-like adhesion molecules, modulate these signaling pathways' effects on cell growth and proliferation.In the central nervous system (CNS), altered expression of a number of cellular adhesion molecules has been associated with brain tumor formation, including neural cell adhesion molecule (NCAM), the L1 adhesion molecule, and multiple members of the cadherin family. Increased expression of NCAM, a member of the immunoglobulin superfamily, has been implicated in invasion of glioma cells (47). Upon clustering of the 140-kDa NCAM protein by homophilic binding or interactions with heparan sulfate proteoglycans, the NCAM cytoplasmic tail activates the Ras/mitogen-activated protein (MAP) kinase (MAPK) signaling cascade (56), which likely contributes to increased tumor proliferation. In addition, overexpression of the L1 adhesion molecule in high-grade gliomas promotes cell-matrix and intercellular interactions and facilitates glioma cell migration (33, 59). Similarly, numerous members of the cadherin family have been implicated in brain tumor formation. N-cadherin promotes oligodendrocyte migration and adhesion to astrocytes (57), and E-cadherin expression in WC5 rat astrocyte-like cells results in increased cell ad...
The LEAP (Laser‐Enabled Analysis and Processing) platform combines in situ imaging with laser manipulation to efficiently identify, purify, and monitor expansion of high secreting clones. It also allows for rapid analysis of cell population heterogeneity. This unit describes the LEAP instrumentation as well as basic and alternate protocols of the major applications in recombinant human or humanized IgG expression. The protocols include fluorescent cell counting, secreted recombinant IgG capture and detection, and IgG‐secreting clone selection by laser processing. Curr. Protocol. Cytom. 43:2.14.1‐2.14.27. © 2008 by John Wiley & Sons, Inc.
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