Localization of intracellular proteins at acetylcholine receptor clusters induced by electric fields in Xenopus muscle cells

Electric fields cause acetylcholine receptor (AChR) patches to form on the cathodal sides of cultured muscle cells. These patches are stable for several hours following cessation of an electric field treat-ment, indicating that the receptors are anchored to the cluster sites. Furthermore, at the ultrastructural level, AChR patches induced by electric fields are marked by an accumulation of extracellular matrix material and a sarcolemmal density. Thus, these AChR patches are similar to those induced by other stimuli, including nerve, polycation-coated beads, and the tissue culture substratum. Proteins that may be involved in anchoring AChRs have been colocalized with AChR patches induced by the latter three stimuli, but not at AChR patches induced by electric fields. In this study, we demon-strate that three putative anchoring proteins, 43K (K=IQ3MT) protein, 58K protein and talin, are associated with field-induced AChR patches. We also show that these proteins persist at field-induced AChR patches following removal of the field, indicating that they are stabilized at the AChR patch. Our data are consistent with the possibility that these proteins contribute to the stabilization of AChRs at patches induced by the electric field. Since 43K, 58K and talin are intracellular proteins, and therefore could not undergo field-induced lat-eral electrophoresis, our observations support the notion that the electric field triggers the formation of an AChR-stabilizing specialization. Key words: electric field, acetylcholine receptor, 43K protein, 58K protein, talin

Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and trigeminal axons

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in vitro sensitivity of trigeminal axons to neurotrophins and target-derived diffusible factors and now report on these properties for geniculate axons. GDNF, BDNF, and NT-4, but not NT-3 or NGF, stimulate geniculate axon outgrowth during the ages investigated, embryonic days 12-14. Sensitivity to effective neurotrophins is developmentally regulated and different from that of the trigeminal ganglion. In vitro coculture studies revealed that geniculate axons were repelled by branchial arch explants that were previously shown to be repellent to trigeminal axons (Rochlin and Farbman [1998] J Neurosci 18:6840-6852). In addition, some branchial arch explants and untransfected COS7 cells repelled geniculate but not trigeminal axons. Sema3A, a ligand for neuropilin-1, is effective in repelling geniculate and trigeminal axons, and antineuropilin-1, but not antineuropilin-2, completely blocks the repulsion by arch explants that repel axon outgrowth from both ganglia. Sema3A mRNA is concentrated in branchial arch epithelium at the appropriate time to mediate the repulsion. In Sema3A knockout mice, geniculate and trigeminal afferents explore medial regions of the immature tongue and surrounding territories not explored in heterozygotes, supporting our previous hypothesis that Sema3A-based repulsion mediates the early restriction of sensory afferents away from midline structures

Supplementary Material for: Neurotrophin-4 Is More Potent than Brain-Derived Neurotrophic Factor in Promoting, Attracting and Suppressing Geniculate Ganglion Neurite Outgrowth

The geniculate ganglion, which provides innervation to taste buds in the anterior tongue and palate, is unique among sensory ganglia in that its neurons depend on both neurotrophin-4 (NT4) and brain-derived neurotrophic factor (BDNF) for survival. Whereas BDNF is additionally implicated in taste axon guidance at targeting stages, much less is known about the guidance role of NT4 during targeting, or about either neurotrophin during initial pathfinding. NT4 and BDNF have distinct expression patterns in vivo, raising the possibility of distinct roles. We characterized the influence of NT4 and BDNF on geniculate neurites in collagen I gels at early embryonic through postnatal stages. During early pathfinding to the tongue (embryonic days 12–13; E12–13), NT4 and BDNF promote significantly longer outgrowth than during intralingual targeting (E15–18). NT4 is more potent than BDNF at stimulating neurite outgrowth and both factors exhibit concentration optima, i.e. intermediate concentrations (0.25 ng/ml NT4 or 25 ng/ml BDNF) promote maximal neurite extension and high concentrations (10 ng/ml NT4 or 200 ng/ml BDNF) suppress it. Only partial suppression was seen at E12 (when axons first emerge from the ganglion in vivo) and postnatally, but nearly complete suppression occurred from E13 to E18. We show that cell death is not responsible for suppression. Although blocking the p75 receptor reduces outgrowth at the optimum concentrations of NT4 and BDNF, it did not reduce suppression of outgrowth. We also report that NT4, like BDNF, can act as a chemoattractant for geniculate neurites, and that the tropic influence is strongest during intralingual targeting (E15–18). NT4 does not appear to act as an attractant in vivo, but it may prevent premature invasion of the epithelium by suppressing axon growth

Molecular Regulation of Contractile Smooth Muscle Cell Phenotype: Implications for Vascular Tissue Engineering

The molecular regulation of smooth muscle cell (SMC) behavior is reviewed, with particular emphasis on stimuli that promote the contractile phenotype. SMCs can shift reversibly along a continuum from a quiescent, contractile phenotype to a synthetic phenotype, which is characterized by proliferation and extracellular matrix (ECM) synthesis. This phenotypic plasticity can be harnessed for tissue engineering. Cultured synthetic SMCs have been used to engineer smooth muscle tissues with organized ECM and cell populations. However, returning SMCs to a contractile phenotype remains a key challenge. This review will integrate recent work on how soluble signaling factors, ECM, mechanical stimulation, and other cells contribute to the regulation of contractile SMC phenotype. The signal transduction pathways and mechanisms of gene expression induced by these stimuli are beginning to be elucidated and provide useful information for the quantitative analysis of SMC phenotype in engineered tissues. Progress in the development of tissue-engineered scaffold systems that implement biochemical, mechanical, or novel polymer fabrication approaches to promote contractile phenotype will also be reviewed. The application of an improved molecular understanding of SMC biology will facilitate the design of more potent cell-instructive scaffold systems to regulate SMC behavior

Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5(NM1)), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5(NM1) was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function