Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells.
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Animals↗
Biomedical subjects
A Füchtbauer
Publications and source records attributed to A Füchtbauer.
Repression of muscle-specific gene activation by the murine Twist protein.
Inhibition of myogenic differentiation can be achieved by various mechanisms. The murine bHLH protein Twist has been shown to inhibit muscle differentiation in mammalian cells. Here we demonstrate that this inhibition is cell autonomous and does not alter cell proliferation. By overexpression of E12, we can distinguish the inhibitory mechanisms of Twist and the dominant negative HLH factor Id. A difference is seen both for the native muscle-specific enhancers of myogenin and myosin light chain 1/3 and for an enhancer consisting only of four E-boxes. Mutagenesis experiments revealed that both the basic region and an evolutionarily conserved carboxy-terminal domain are required for the Twist-specific type of inhibition. Loss of either of these regions renders Twist less efficient and more similar to Id. Gel mobility shift assays demonstrate that Twist can bind to the muscle creatine kinase E-box and inhibit DNA binding of heterodimers of E12 with myogenic bHLH transcription factors like MyoD. However, a fourfold excess of Twist compared to MyoD is required for both effects. Our results suggest that Twist inhibits muscle-specific gene activation by formation of actively inhibitory complexes rather than by sequestering E-proteins.
Animals↗
Maid: a maternally transcribed novel gene encoding a potential negative regulator of bHLH proteins in the mouse egg and zygote.
We isolated an abundant novel cDNA SSEC-8 from a subtraction cDNA library enriched for maternal transcripts that are still present in the mouse 2 cell stage embryo. This gene is evolutionarily conserved and maps to the distal region of mouse chromosome 2. The deduced polypeptide sequence of the encoded protein contains a conserved helix-loop-helix (HLH) motif without a basic DNA binding domain, suggesting that it functions as a negative regulator of basic (b) HLH transcription factors. Gel mobility shift assays show that in vitro translated protein prevents the E12/MyoD bHLH dimer from binding to DNA. Also, transient overexpression of this protein in C2C12 cells reduced the transcription of a CAT-reporter regulated by an E12/MyoD driven enhancer. The 3'-UTR contains consensus sequences of cytoplasmic polyadenylation elements (CPE's), and the length of its poly (A) tail changes during oocyte maturation, indicating that its expression is controlled by timely activation of translation. This new gene, Maid, models the translational and transcriptional regulation of gene expression during the transition from gamete to embryo.
Amino Acid Sequence↗
Antibodies against vertebrate microfilament proteins in the analysis of cellular motility and adhesion.
Microinjection of specific antibodies can be an alternative and a supplement to genetic engineering in dissecting the function of individual cytoskeletal components. In this report, we describe some of the requirements for using this technique, its potential application in conjunction with morphological and biochemical analyses, and its limitations. Examples are given for the injection of antibodies to alpha-actinin, vinculin and myosin, and the effects of such treatment on adhesion, motility and cytokinesis of the recipient cells.
Animals↗
Differential effects of gelsolins on tissue culture cells.
Gelsolins, prepared from a number of different sources, showed similar severing activity on F-actin in vitro or on stress fibers of detergent-extracted cells but differed in their effects on actin in stress fibers of microinjected cells. When human gelsolin isolated from plasma was injected into cells in a Ca(++)-containing buffer, stress fibers were degraded, the cellular morphology was changed, and numerous actin patches appeared. These effects were particularly striking when the Ca(++)-insensitive N-terminal proteolytic fragment of this gelsolin was injected. By contrast, Ca(++)-sensitive gelsolins isolated from human platelets, pig stomach smooth muscle and pig plasma showed no comparable activity. Furthermore, the Ca(++)-independent N-terminal proteolytic fragments prepared from these gelsolins also had no effect despite their in vitro actin severing activity. Most striking was the finding that human plasma gelsolin expressed in E. coli did not degrade stress fibers, in contrast to the same protein isolated from plasma; nor was there any stress fiber disruption observed with the N-terminal half of human gelsolin expressed in Escherichia coli. The different behavior of these gelsolins in cells cannot be explained by sequence diversity between plasma and cytoplasmic forms, nor by variability in the Ca++ sensitivity of the preparations. It suggests the presence of factors, as yet unidentified, that may regulate gelsolin activity in the cytoplasm of living cells and discriminate between gelsolins of different origin. Such discrimination could be achieved as a result of post-translational modification of the gelsolin; only in this way can differences between apparently identical proteins isolated from human plasma and expressed in E. coli be reconciled.
Actin Cytoskeleton↗
Long-term expression of isomyosins and myoendocrine functions in ectopic grafts of atrial tissue.
Tissue fragments of newborn rat atria were transplanted under the dorsal skin or into the bed of the anterior tibial muscle of nude mice. After 5-11 weeks, the grafts, which had reorganized into beating atrium-like structures, were analyzed and compared to ventricular tissue transplanted the same way. As revealed by monoclonal antibodies against alpha- and beta-type myosin heavy chains, atrial grafts retained a typical pattern of myosin expression distinct from that of ventricular grafts. The majority of ectopic atrial myocytes contained specific atrial granules in which cardiodilatin-immunoreactive material has been localized. Specific granules and cardiodilatin immunoreactivity were not found in myocytes of ventricular grafts. We conclude that the long-term maintenance of isomyosin expression and of the myoendocrine function of atrial tissue is largely independent of the anatomical environment.
Animals↗
Actin-severing activity copurifies with phosphofructokinase.
Microinjection of muscle 6-phosphofructokinase (PFK; EC 2.7.1.11) into tissue culture cells led to a reversible disintegration of microfilament bundles (stress fibers). The mode of disruption as well as of recovery of stress fibers was very similar to that found previously in experiments performed with the actin-severing protein brevin, an extracellular variant of gelsolin. PFK, like brevin, was also capable of disrupting stress fibers in detergent-extracted cells and in ethanol-fixed cells, in a Ca2+-dependent manner. When compared with heart muscle gelsolin, PFK comigrated with the 85- to 90-kDa band. Antibodies against PFK crossreacted with gelsolin from the same species. These results point to a tight association between polypeptides with similar biochemical and immunological parameters present in both preparations. They suggest hitherto unexpected cellular control mechanisms for both microfilament functions and glycolysis.
Actin Cytoskeleton↗
Disruption of microtubules in living cells and cell models by high affinity antibodies to beta-tubulin.
Polyclonal antibodies with high affinity for beta-tubulin were found to disrupt cytoplasmic microtubules efficiently after microinjection into tissue culture cells. The degree of microtubular fragmentation was directly proportional to the amount of the injected antibody. At molar ratios of 1 antibody per 100 tubulin dimers, most microtubules were disrupted within 90 min after injection. In contrast, the time course of disintegration was relatively independent of the antibody concentration. Within the range of 1 antibody per 10(2)-10(4) tubulin dimers, the maximal values for microtubular disintegration were reached approximately 1-1.5 h after injection. Mitotic microtubules were found to be resistant to all antibody concentrations used. In living cells, microtubules recovered within a few hours after antibody-induced decay. The time course of recovery, like the extent of disintegration, was a function of the antibody concentration. The antibody acted also on microtubules in detergent-extracted cell models and on microtubules polymerised in vitro. When added to microtubular protein, the bivalent antibody as well as its Fab fragments prevented polymerisation. The data suggest that these antibodies disrupt microtubules because their affinity to tubulin is at least 100 times higher than the affinities found for tubulin:tubulin interaction. Fragmented microtubules are probably unstable and decompose into smaller units.
Animals↗
Organization and function of structural elements in focal contacts of tissue culture cells.
The role of structural elements in the organization and maintenance of focal contacts was studied by microinjecting into tissue culture cells specific probes which interfere with filamentous actin or with vinculin: actin interaction. Injection of actin capping proteins from Physarum and brain resulted in breakdown of microfilament bundles starting at their distal ends and in loss of focal contacts. This process was fully reversible. Injection of a high affinity antibody against chicken gizzard vinculin led to partial breakdown of microfilament bundles con-concomitant with disruption of focal contacts with vinculin remaining at the plasma membrane. This process was irreversible.
Actinin↗
Effects of aldehydes and organic solvents on concanavalin A binding sites in cerebellar tissue sections.
Fresh frozen cerebellar sections of adult mice treated with aldehydes and organic solvents reveal differences in detectability of Concanavalin A (Con A) binding sites. While fluorescein coupled Con A shows intense labeling of synaptic glomeruli and granule cell bodies under all conditions, the molecular layer labels intensely after treatment with paraformaldehyde, glutaraldehyde, acetone, ethanol and butanol. Complete loss of staining in molecular and granular layers and substantial increase in white matter labeling occurs after chloroform-methanol treatment. Except for glutaraldehyde treated sections, all labeling is specifically inhibited by methyl-alpha-D-mannoside, but not by galactose.
Aldehydes↗
Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells.
Capping proteins are F-actin binding proteins which interfere with the in vitro growth of an actin filament by blocking one of its ends (for recent reviews see refs 1-3). The majority of such proteins described so far "cap' the fast-growing (positive) end of the polar filament, thus reducing the velocity of filament growth while increasing the number of filaments being formed de novo from a monomer pool. We have studied the effects of capping proteins on the organization of actin filaments in living tissue culture cells by microinjection in conjunction with fluorescence, reflection contrast and electron microscopy. Our results, reported here, indicate that capping proteins from different sources disrupt microfilament bundles in a variety of cell types causing their disintegration from the distal end towards the centre of the cell.
Animals↗