Skeletal muscle fibers of newborn rats are coupled by gap junctions

In the soleus muscle and in the short muscles of the hindfoot of the newborn rat many muscle fibers are arranged in clusters comprising two to four fibers enclosed by a common basal lamina. The fibers of a cluster are connected by gap junctions which were seen in thin sections and in freeze-fractures. One week after birth, almost all fibers have detached; no gap junctions were found.     

Direct activation of phospholipase C-gamma by fibroblast growth factor receptor is not required for mesoderm induction in Xenopus animal caps.

Members of the fibroblast growth factor (FGF) family induce mesoderm formation in explants of Xenopus embryonic ectoderm (animal caps). Recent studies have been directed at determining signaling pathways downstream of the FGF receptor that are important in mesoderm induction. We have recently shown that a point mutation in the FGF receptor changing tyrosine 766 to phenylalanine (Y/F mutation) abolishes phospholipase C-gamma (PLC-gamma) activation in mammalian cells. To explore the role of PLC-gamma activation in FGF-stimulated mesoderm induction, we constructed two chimeric receptors, each consisting of the extracellular portion of the platelet-derived growth factor beta receptor, with one having the transmembrane and intracellular portions of the wild-type FGF receptor 1 (PR-FR wt) and the other having the corresponding region of the Y/F766 mutant FGF receptor 1 (PR-FR Y/F766). When expressed in Xenopus oocytes, only PR-FR wt was able to mediate PLC gamma phosphorylation, inositol-1,4,5-trisphosphate accumulation, and calcium efflux in response to platelet-derived growth factor stimulation. However, both receptors mediated mesoderm induction in Xenopus animal caps as measured by cap elongation, muscle-specific actin mRNA induction, and skeletal muscle formation. These results demonstrate that PLC gamma activation by the FGF receptor is not required for FGF-stimulated mesoderm induction.     

Internalized gap junctions are degraded by autophagy

Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously-and effectively after treatment with natural inflammatory mediators-internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.     

Internalized gap junctions are degraded by autophagy

Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously—and effectively after treatment with natural inflammatory mediators—internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.     

Embryonic induction and muscle gene activation

Embryonic induction, a process in which the differentiation of a cell is determined by its proximity to other kinds of cells, is of major importance in animal development. We review here what is known of the steps by which a muscle-specific actin gene is first activated by embryonic induction in early amphibian embryos.     

Mast cell activation is not required for induction of airway hyperresponsiveness by ozone in mice

Exposure to ambient ozone(O₃) is associated withincreased exacerbations of asthma. We sought to determine whether mastcell degranulation is induced by in vivo exposure toO₃ in mice and whether mast cellsplay an essential role in the development of pulmonarypathophysiological alterations induced byO₃. For this we exposed mastcell-deficientWBB6F₁-kit^W/kit^W-v(kit^W/kit^W-v)mice and the congenic normalWBB6F₁ (+/+) mice to air or to 1 or 3 parts/million O₃ for 4 h andstudied them at different intervals from 4 to 72 h later. We foundevidence of O₃-induced cutaneous,as well as bronchial, mast cell degranulation. Polymorphonuclear cellinflux into the pulmonary parenchyma was observed after exposure to 1 part/milllion O₃ only in mice thatpossessed mast cells. Airway hyperresponsiveness to intravenousmethacholine measured in vivo under pentobarbital anesthesia wasobserved in bothkit^W/kit^W-vand +/+ mice after exposure to O₃.Thus, although mast cells are activated in vivo byO₃ and participate inO₃-induced polymorphonuclear cellinfiltration into the pulmonary parenchyma, they do not participate detectably in the development ofO₃-induced airwayhyperresponsiveness in mice.

     

Formation of gap junctions by expression of connexins in Xenopus oocyte pairs

RNAs coding for connexins 32, 43, and the putative lens gap junction protein MP26 were tested for their ability to induce cell-cell coupling in Xenopus oocyte pairs. Large, voltage-insensitive conductances developed when connexin32 and 43 RNA-injected oocytes were paired both with themselves and with each other. Oocyte pairs injected with water manifested small conductances, which were symmetrically voltage-dependent. MP26 RNA-injected pairs displayed no conductances above control values. Unexpectedly, connexin43/water oocyte pairs developed high, asymmetrically voltage-dependent conductances, a property not displayed by the connexin32/water pairs. In single oocytes, these proteins remained intracellular until pairing, at which time the connexins, but not MP26, concentrated at the appositional areas.     

Rectangular arrays of particles on freeze-cleaved plasma membranes are not gap junctions

Freeze-cleave replicas of adult rat diaphragm have revealed the presence of numerous small rectangular arrays of 60 Å particles (respectively pits) on the fracture faces of the sarcolemmas of the myofibers. Since these fibers are separated by thick basal laminae and are not electrically coupled we conclude that the rectangular arrays are not morphological equivalents of gap junctions as suggested by Staehelin [14]. The term “type III gap junctions” for these arrays therefore should be discontinued.     

Cooperative binding is not required for activation of muscle phosphorylase.

Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oocyte-granulosa cell heterologous gap junctions are required for the coordination of nuclear and cytoplasmic meiotic competence

Homologous gap junctions are generally recognized as a means of coordinating cellular behavior under developmental and homeostatic conditions. In the mammalian ovary, heterologous gap junctions between the oocyte and the granulosa cells have been widely implicated in the regulation of meiotic maturation late in oogenesis. However, the role of oocyte-granulosa cell gap junctions at earlier stages of oogenesis is poorly understood. Stage-specific defects in both oocyte and follicle development have been identified in juvenile mice deficient in heterologous oocyte-granulosa cell gap junctions due to targeted deletion of Gja4, the gene encoding connexin-37. Follicle development arrests at the type 4 preantral stage and although oocytes commence growth, oocyte growth ceases at a diameter of 52 microm (74.3% of control size). Analysis of cell cycle and cytoskeletal markers indicates that oocytes arrest in a G(2) state based on uniform decondensed GV chromatin, interphase microtubule arrays, and nonphosphorylated cytoplasmic centrosomes. Functional assays of meiotic competence confirm that oocytes from connexin-37-deficient mice are unable to enter M phase (initiate meiotic maturation) unless treated with the phosphatase inhibitor okadaic acid (OA). Unlike growing oocytes from heterozygous control animals, OA-treated oocytes from connexin-37-deficient mice respond acutely and progress rapidly to the circular bivalent stage of meiosis I and upon removal from OA rapidly revert to an interphase state. In contrast, OA-treated control incompetent oocytes are slow to respond, exhibit a lower proportion of chromosomal bivalent stage oocytes, but remain in and progress into meiotic M phase upon removal from OA. This study demonstrates that heterologous gap-junctional communication is required for the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence.     

Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression.

Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. We show that the expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. As we demonstrate that the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, we find that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. Finally, we find that by removing Otx2 function, development of rhombomere 3 is rescued in Gbx2(-/-) embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. Our results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes.     

Mast cells are not required for anaphylatoxin-induced ileal smooth muscle contraction

The complement-derived anaphylatoxin peptides, C3a and C5a, have long been considered to manifest their spasmogenic activities primarily through stimulation of mast cells. Although mast cells represent the major non-circulating repository for histamine, these cells also elaborate a number of additional, highly potent spasmogenic mediators derived from arachidonic acid. The same lipid mediators can be released by many other cell types. As a result, evaluation of the role of mast cells in anaphylatoxin-dependent responses cannot be based exclusively upon an analysis of the mediators released. We evaluated the role of mast cells in anaphylatoxin-induced ileal smooth muscle contractions by testing isolated segments of ileal tissues derived from genetically mast cell-deficient mice and their congenic normal (+/+) littermates. Isolated tissues from either congenic normal (+/+) or mast cell-deficient Sl/Sld mice responded similarly to acetylcholine, histamine, serotonin, prostaglandin E2, and the thromboxane A2 analog, U-46619. At 1 microgram/ml, histamine induced contractions of greater magnitude in tissues from mast cell-deficient animals; however, this mediator also desensitized the tissues to repeat challenge with histamine at the same concentration. C5a at 1 nM resulted in contractions equivalent to approximately 50% of the maximal KCl response; normal and mast cell-deficient tissues responded in a similar manner. C5a also released histamine from the normal mouse ileum, in addition to causing contraction of the tissues. C3a at 200 nM also produced similar contractile responses in both +/+ and S1/S1d tissues. These studies show that the anaphylatoxin peptides C3a and C5a are capable of contracting smooth muscle-containing tissues by a mechanism completely independent of mast cells. In addition, we also demonstrated that mast cell degranulation does not necessarily provoke ileal contraction. Thus compound 48/80, a mast cell degranulating agent unrelated to the anaphylatoxins, did not induce contractions in ileal tissues, even when used at concentrations as high as 100 micrograms/ml. Compound 48/80 did release histamine from the +/+ ileum, however, indicating that the agent was able to cause degranulation of ileal mast cells. Taken together, these data indicate that spasmogenic responses to anaphylatoxins (and possibly other agents) that are associated with mast cell degranulation need not necessarily require mast cell mediator release for their expression.     

Two functional heads are required for full activation of smooth muscle myosin

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.