Endothelial Jarid2/Jumonji is required for normal cardiac development and proper Notch1 expression

Jarid2/Jumonji critically regulates developmental processes including cardiovascular development. Jarid2 knock-out mice exhibit cardiac defects including hypertrabeculation with noncompaction of the ventricular wall. However, molecular mechanisms underlying Jarid2-mediated cardiac development remain unknown. To determine the cardiac lineage-specific roles of Jarid2, we generated myocardial, epicardial, cardiac neural crest, or endothelial conditional Jarid2 knock-out mice using Cre-loxP technology. Only mice with an endothelial deletion of Jarid2 recapitulate phenotypic defects observed in whole body mutants including hypertrabeculation and noncompaction of the ventricle. To identify potential targets of Jarid2, combinatorial approaches using microarray and candidate gene analyses were employed on Jarid2 knock-out embryonic hearts. Whole body or endothelial deletion of Jarid2 leads to increased endocardial Notch1 expression in the developing ventricle, resulting in increased Notch1-dependent signaling to the adjacent myocardium. Using quantitative chromatin immunoprecipitation analysis, Jarid2 was found to occupy a specific region on the endogenous Notch1 locus. We propose that failure to properly regulate Notch signaling in Jarid2 mutants likely leads to the defects in the developing ventricular chamber. The identification of Jarid2 as a potential regulator of Notch1 signaling has broad implications for many cellular processes including development, stem cell maintenance, and tumor formation.     

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.     

Immuno-histochemical expression of alpha1, alpha2 and alpha3 integrin subunits during angiogenesis in vitro

Aortic explants were obtained from mouse fetuses and cultured in collagen gels. Immuno-fluorescence microscopy, antibodies (anti alpha1, alpha2 and alpha3 integrin subunits) were used. Fibroblastic cells migrated from the aortic explant after one day of cultivation. The migrating cells located in the peripheral part of the aortic explant were positive for alpha1 and alpha2 integrin subunit antibodies. Immuno-fluorescence-positive staining for the alpha3 integrin subunit antibody was clearly seen in the migrating cells located near the aortic explant and surrounding tube-like structures. In an immuno-electron microscope study performed by pre-embedding immuno labeling, gold particles associated with the alpha3 integrin subunit were found to reside on the membranes of the cells surrounding the capillary-like tubes. Two synthetic peptides, GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) and KDGEA (Lys-Asp-Gly-Glu-Ala), were added to the growth medium to study their effects on cell migration. KDGEA, a compound containing the recognition sequence for alpha2beta1 integrin, decreased cell migration, while GRGDSP exhibited no effect. The migration of fibroblastic cells is an important phenomenon for tube formation. The present study suggested that the alpha1 and alpha2 integrin subunits are both involved in the cell migration, and more specifically, that the alpha2 integrin subunit participates in cell migration through the KDGEA sequence. The alpha3 integrin subunit played a role in tube formation.     

Integrin alpha2beta1 is the required receptor for endorepellin angiostatic activity

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the alpha2beta1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the alpha2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of alpha2beta1. Microvascular endothelial cells from alpha2beta1(-/-) mice, but not those isolated from either wild-type or alpha1beta1(-/-) mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in alpha2beta1(-/-) mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin alpha2beta1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the alpha2beta1 integrin receptor.     

Localization of the alpha 1 and alpha 2 subunits of the dihydropyridine receptor and ankyrin in skeletal muscle triads

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the dihydropyridine (DHP) receptor and ankyrin in rat skeletal muscle with immunofluorescence and immunogold labeling techniques. All three proteins were concentrated in the triad junction formed between the T-tubules and sarcoplasmic reticulum. The alpha 1 and alpha 2 subunits of the DHP receptor were colocalized in the junctional T-tubule membrane, supporting their proposed association in a functional complex and the possible participation of the alpha 2 subunit in excitation-contraction coupling. Ankyrin label in the triad showed a distribution different from that of the DHP receptor subunits. In addition, ankyrin was found in longitudinally oriented structures outside the triad. Thus, ankyrin might be involved in organizing the triad and in immobilizing integral membrane proteins in T-tubules and the sarcoplasmic reticulum.     

beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2

Recently, a requirement for beta-arrestin-mediated endocytosis in the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by several G protein-coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Galphaq-coupled proteinase-activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, beta-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2deltaST363/6A), which is unable to interact with beta-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(deltaST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, beta-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.     

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.     

Pofut1 is required for the proper localization of the Notch receptor during mouse development

Protein O-fucosyltransferase 1 (Pofut1), which catalyzes the addition of O-linked fucose to the EGF domains of the Notch receptor, is indispensable for Notch signaling activation. However, the mechanism of action of Pofut1 in mice is still unclear. Mouse embryos lacking Pofut1 shows defects in valve formation and trabeculation in the cardiovascular system, which are almost identical abnormalities to those of the RBP-Jk mutants. In our current study, we have examined the epistatic relationship between the functions of Pofut1 and activated-Notch1 (NICD1) by taking advantage of the fact that forced expression of NICD1 results in myocardial defects. These defects were still evident in NICD1-expressing embryos irrespective of the presence or absence of Pofut1, which indicates that Pofut1 is required for Notch signaling upstream of NICD1. We further found that Pofut1-null cells do not possess normally localized Notch1 receptors, which may results in their lack of interaction with the Dll1 ligand in the presomitic mesoderm where Notch signaling plays a pivotal role. We propose that altered trafficking pathways may account for the abnormal accumulation of the Notch1 receptor in the endoplasmic reticulum in Pofut1-null mouse embryos.     

Interaction between GRIP and liprin-alpha/SYD2 is required for AMPA receptor targeting

Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors, but the underlying mechanism is unknown. We show that GRIP binds to the liprin-alpha/SYD2 family of proteins that interact with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) and that are implicated in presynaptic development. In neurons, liprin-alpha and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-alpha is important for postsynaptic as well as presynaptic maturation.     

The FgfrL1 receptor is required for development of slow muscle fibers

FgfrL1, which interacts with Fgf ligands and heparin, is a member of the fibroblast growth factor receptor (Fgfr) family. FgfrL1-deficient mice show two significant alterations when compared to wildtype mice: They die at birth due to a malformed diaphragm and they lack metanephric kidneys. Utilizing gene arrays, qPCR and in situ hybridization we show here that the diaphragm of FgfrL1 knockout animals lacks any slow muscle fibers at E18.5 as indicated by the absence of slow fiber markers Myh7, Myl2 and Myl3. Similar lesions are also found in other skeletal muscles that contain a high proportion of slow fibers at birth, such as the extraocular muscles. In contrast to the slow fibers, fast fibers do not appear to be affected as shown by expression of fast fiber markers Myh3, Myh8, Myl1 and MylPF. At early developmental stages (E10.5, E15.5), FgfrL1-deficient animals express slow fiber genes at normal levels. The loss of slow fibers cannot be attributed to the lack of kidneys, since Wnt4 knockout mice, which also lack metanephric kidneys, show normal expression of Myh7, Myl2 and Myl3. Thus, FgfrL1 is specifically required for embryonic development of slow muscle fibers.     

The TSC1-TSC2 complex is required for proper activation of mTOR complex 2

The mammalian target of rapamycin (mTOR) is a protein kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. Both complexes phosphorylate a hydrophobic motif on downstream protein kinases, which contributes to the activation of these kinases. mTOR complex 1 (mTORC1) phosphorylates S6K1, while mTORC2 phosphorylates Akt. The TSC1-TSC2 complex is a critical negative regulator of mTORC1. However, how mTORC2 is regulated and whether the TSC1-TSC2 complex is involved are unknown. We find that mTORC2 isolated from a variety of cells lacking a functional TSC1-TSC2 complex is impaired in its kinase activity toward Akt. Importantly, the defect in mTORC2 activity in these cells can be separated from effects on mTORC1 signaling and known feedback mechanisms affecting insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Our data also suggest that the TSC1-TSC2 complex positively regulates mTORC2 in a manner independent of its GTPase-activating protein activity toward Rheb. Finally, we find that the TSC1-TSC2 complex can physically associate with mTORC2 but not mTORC1. These data demonstrate that the TSC1-TSC2 complex inhibits mTORC1 and activates mTORC2, which through different mechanisms promotes Akt activation.     

ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion

Skeletal muscle satellite cells, which are found between the muscle fiber and the basal lamina, remain quiescent and undifferentiated unless stimulated to remodel skeletal muscle or repair injured skeletal muscle tissue. Quiescent satellite cells express c-met and fibroblast growth factor receptors (FGFR) 1 and 4, suggesting these receptors are involved in maintaining the undifferentiated quiescent state or involved in satellite cell activation. Although the signaling pathways involved are poorly understood, the mitogen activated protein kinase (MAPK) cascade has been implicated in the regulation of skeletal muscle growth and differentiation by FGFs. In this study, we investigated if activation of the Raf-MKK1/2-ERK1/2 signaling cascade plays a role in FGF-dependent repression of differentiation and proliferation of MM14 cells, a skeletal muscle satellite cell line. Inactivation ofthe Raf-MKK1/2-ERK1/2 pathway in myoblasts through the overexpression of dominant negative mutants of Raf-1 blocks ERK1/2 activity and prevents myoblast proliferation. Additionally, inhibition of MKK1/2 by treatment with pharmacological inhibitors also blocks FGF-mediated stimulation of ERK1/2 and blocks the G1 to S phase transition of myoblasts. Unexpectedly, we found that inactivation of the Raf-ERK pathway does not activate a muscle reporter, nor does inactivation of this pathway promote myogenic differentiation. We conclude that FGF-stimulated ERK1/2 signaling is required during the G1 phase of the cell cycle for commitment of myoblasts to DNA synthesis but is not required for mitosis once cells have entered the S-phase. Moreover, ERK1/2 signaling is not required either to repress differentiation, to promote skeletal muscle gene expression, or to promote myoblast fusion.     

PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation

Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.     

Developmental changes in the expression of GABAA receptor subunits alpha1, alpha2, and alpha3 in the rat pre-Botzinger complex.

Previously, we reported that the pre-B?tzinger complex (PBC) exhibited a dramatic reduction in cytochrome oxidase activity at postnatal day (P) 12. This coincided in time with decreases in glutamate and NMDA receptor subunit 1 and increases in GABA, GABAB, glycine receptor, and glutamate receptor GluR2. To test our hypothesis that various alpha-subunits of GABAA receptors also undergo changes in their expression during postnatal development, as they do in other brain regions, we undertook an in-depth immunohistochemical study of GABAA receptor subunits alpha1, alpha2, and alpha3 in the PBC of P0 to P21 rats. We found that 1) GABAA alpha3-subunit was expressed at relatively high levels at P0, which then declined with age; 2) GABAA alpha1-subunit was expressed at relatively low levels at P0 but increased with age; 3) the developmental trends of subunits alpha1 and alpha3 intersected at P12; and 4) GABAA alpha2-subunit expression was moderate to light at P0 and remained quite constant during development, being lowest at P21. These findings suggest that the apparent switch in relative expressions of subunits alpha3 and alpha1 during development and the intersection of slopes around P12 may be associated with possible changes in GABAA receptor subtypes that would mediate different functional properties of GABA transmission, such as primarily a less efficient inhibitory transmission before P12 and a more mature inhibitory effect at P12 and thereafter, as suggested by the kinetics of distinct postsynaptic potentials. This mechanism may contribute partially to the dramatic reduction in cytochrome oxidase activity within the PBC at P12, as shown previously.     

Importin KPNA2 is required for proper nuclear localization and multiple functions of NBS1

Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1, is a component of the MRE11-RAD50-NBS1 (MRN) complex, a central player associated with double strand break (DSB) repair. In response to radiation, NBS1 is phosphorylated by ATM, and the MRN complex relocalizes to form punctate nuclear foci for DNA repair. NBS1 controls both the nuclear localization of the MRN complexes and radiation-induced focus formation. We report here that the KPNA2 (importin alpha1) is important for the normal nuclear localization of the MRN complex and its proper formation of the nuclear foci. KPNA2 is the only member of the importin alpha family that physically interacts with NBS1, and the KPNA2-mediated nucleus localization sequence (NLS) is mapped to amino acid residues 461-467 of NBS1 that is sufficient for both the interaction with KPNA2 and the proper nuclear localization. Inhibition of KPNA2 or blockage of the KPNA2 interaction with NBS1 results in a reduction of radiation-induced nuclear focus accumulation, DSB repair, and cell cycle checkpoint signaling of NBS1. Collectively, our results strongly suggest that an interaction with KPNA2 contributes to nuclear localization and multiple tumor suppression functions of the NBS1 complex.     

Heterologous expression of BI Ca2+ channels in dysgenic skeletal muscle

We have examined the ability of BI (class A) Ca2+ channels, cloned from rabbit brain, to mediate excitation-contraction (E-C) coupling in skeletal muscle. Expression plasmids carrying cDNA encoding BI channels were microinjected into the nuclei of dysgenic mouse myotubes grown in primary culture. Ionic currents and intramembrane charge movements produced by the BI channels were recorded using the whole-cell patch- clamp technique. Injected myotubes expressed high densities of ionic BI Ca2+ channel current (average 31 pA/pF) but did not display spontaneous contractions, and only very rarely displayed evoked contractions. The expressed ionic current was pharmacologically distinguished from the endogenous L-type current of dysgenic skeletal muscle (Idys) by its insensitivity to the dihydropyridine antagonist (+)-PN 200-110. Peak BI Ca2+ currents activated with a time constant (tau a) of approximately 2 ms and inactivated with a time constant (tau h) of approximately 260 ms (20-23 degrees C). The time constant of inactivation (tau h) was not increased by substituting Ba2+ for Ca2+ as charge carrier, demonstrating that BI channels expressed in dysgenic myotubes do not undergo Ca(2+)-dependent inactivation. The average maximal Ca2+ conductance (Gmax) produced by the BI channels was quite large (approximately 534 S/F). In contrast, the average maximal charge movement (Qmax) produced in the same myotubes (approximately 2.7 nC/microF) was quite small, being barely larger than Qmax in control dysgenic myotubes (approximately 2.3 nC/microF). Thus, the ratio Gmax/Qmax for the BI channels was considerably higher than previously found for cardiac or skeletal muscle L-type Ca2+ channels expressed in the same system, indicating that neuronal BI Ca2+ channels exhibit a much higher open probability than these L-type Ca2+ channels.     

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes.     

Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Background

BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/Principal Findings

Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/Significance

BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.     

Electrical properties of normal and dysgenic mouse skeletal muscle in culture

Electrical properties of normal and dysgenic mouse skeletal muscle were studied by intracellular recording from embryonic cells developing in vitro. Passive membrane constants were determined from records of transmembrane potential responses to hyperpolarizing pulses of current using two types of analyses, assuming the tubes to be finite cylinders: the off transient and steady state analyses. The following properties of normal and dysgenic fibers were also studied. (a) membrane potentials (b) acetylcholine sensitivity (c) α-Bungarotoxin binding and (d) maximum rate of rise, overshoot and one-half fall time of the action potential. Rare electrotonic coupling between fibroblasts and myotubes was noted. An anomalous type of rectification Was observed in some fibers in which the transmembrane potential responses possessed under and overshoots. These responses may have affected the values of membrane constants as derived by the off transient analysis. In all parameters studied, including membrane constants derived by the steady state analysis, the cultured mouse cells resembled adult denervated mammalian muscle rather than innervated muscle. There were no differences between normal and dysgenic fibers with respect to any of the parameters studied. Dysgenic fibers did not contract although they displayed passive and active membrane properties like those in normal, non-dysgenic fibers.