scholarly journals A marginal band-associated protein has properties of both microtubule- and microfilament-associated proteins.

1989 ◽  
Vol 109 (4) ◽  
pp. 1609-1620 ◽  
Author(s):  
E Birgbauer ◽  
F Solomon
Keyword(s):  
Cultured Cells ◽  
Significant Proportion ◽  
Growth Cones ◽  
Dual Nature ◽  
Spatial Control ◽  
Marginal Band ◽  
Single Protein ◽  
Associated Proteins ◽  
Nonionic Detergent ◽  
Chicken Erythrocytes

The marginal band of nucleated erythrocytes is a microtubule organelle under rigorous quantitative and spatial control, with properties quite different from those of the microtubule organelles of cultured cells. Previous results suggest that proteins other than tubulin may participate in organizing the marginal band, and may interact with elements of the erythrocyte cytoskeleton in addition to microtubules. To identify such species, we raised mAbs against the proteins that assemble from chicken brain homogenates with tubulin. One such antibody binds to a single protein in chicken erythrocytes, and produces an immunofluorescence pattern colocalizing with marginal band microtubules. Several properties of this protein are identical to those of ezrin, a protein isolated from brush border and localized to motile elements of cultured cells. A significant proportion of the antigen is not soluble in erythrocytes, as determined by extraction with nonionic detergent. This cytoskeleton-associated fraction is unaffected by treatments that solubilize the marginal band microtubules. The protein has properties of both microtubule- and microfilament-associated proteins. In the accompanying manuscript (Goslin, K., E. Birgbauer, G. Banker, and F. Solomon. 1989. J. Cell Biol. 109:1621-1631), we show that the same antibody recognizes a component of growth cones with a similar dual nature. In early embryonic red blood cells, the antigen is dispersed throughout the cell and does not colocalize with assembled tubulin. Its confinement to the marginal band during development follows rather than precedes that of microtubules. These results, along with previous work, suggest models for the formation of the marginal band.

scholarly journals The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization.

1989 ◽  
Vol 109 (4) ◽  
pp. 1621-1631 ◽  
Author(s):  
K Goslin ◽  
E Birgbauer ◽  
G Banker ◽  
F Solomon
Keyword(s):  
Hippocampal Neurons ◽  
Growth Cones ◽  
Microtubule Depolymerization ◽  
Dual Nature ◽  
Phalloidin Staining ◽  
The Reformation ◽  
Associated Proteins ◽  
Chicken Erythrocytes ◽  
The Relationship

We are interested in the relationship between the cytoskeleton and the organization of polarized cell morphology. We show here that the growth cones of hippocampal neurons in culture are specifically stained by a monoclonal antibody called 13H9. In other systems, the antigen recognized by 13H9 is associated with marginal bands of chicken erythrocytes and shows properties of both microtubule-and microfilament-associated proteins (Birgbauer, E., and F. Solomon. 1989 J. Cell Biol. 109:1609-1620). This dual nature is manifest in hippocampal neurons as well. At early stages after plating, the antibody stains the circumferential lamellipodia that mediate initial cell spreading. As processes emerge, 13H9 staining is heavily concentrated in the distal regions of growth cones, particularly in lamellipodial fans. In these cells, the 13H9 staining is complementary to the localization of assembled microtubules. It colocalizes partially, but not entirely, with phalloidin staining of assembled actin. Incubation with nocodazole rapidly induces microtubule depolymerization, which proceeds in the distal-to-proximal direction in the processes. At the same time, a rapid and dramatic redistribution of the 13H9 staining occurs; it delocalizes along the axon shaft, becoming clearly distinct from the phalloidin staining and always remaining distal to the receding front of assembled microtubules. After longer times without assembled microtubules, no staining of 13H9 can be detected. Removal of the nocodazole allows the microtubules to reform, in an ordered proximal-to-distal fashion. The 13H9 immunoreactivity also reappears, but only in the growth cones, not in any intermediate positions along the axon, and only after the reformation of microtubules is complete. The results indicate that the antigen recognized by 13H9 is highly concentrated in growth cones, closely associated with polymerized actin, and that its proper localization depends upon intact microtubules.

scholarly journals Identification of a novel Ca(2+)-regulated protein that is associated with the marginal band and centrosomes of chicken erythrocytes

1995 ◽  
Vol 108 (2) ◽  
pp. 685-698 ◽  
Author(s):  
J. Zhu ◽  
S.E. Bloom ◽  
E. Lazarides ◽  
C. Woods
Keyword(s):  
Sequence Analysis ◽  
Nuclear Membrane ◽  
Binding Sites ◽  
Cultured Cells ◽  
Marginal Band ◽  
Avian Erythrocyte ◽  
Chicken Erythrocytes ◽  
Definitive Erythropoiesis ◽  
Developmental Analysis

We have identified a novel Ca(2+)-regulated protein, p23, that is expressed specifically in avian erythrocyte and thrombocyte lineages. Sequence analysis of this 23 kDa protein reveals that it bears no homology to any known sequence. In mature definitive erythrocytes p23 exists in equilibrium between a soluble and a cytoskeletal bound pool. The cytoskeletal fraction is associated with the marginal band of microtubules, centrosomes and nuclear membrane under conditions of low free [Ca2+]. An increase in free [Ca2+] to 10(−6) M is sufficient to induce dissociation of > 95% of bound p23 from its target cytoskeletal binding sites, yet this [Ca2+] has little effect on calmodulin-mediated MB depolymerization. Analysis of p23 expression and localization during erythropoiesis together with results from heterologous p23 expression in tissue cultured cells demonstrated that this protein does not behave as a bone fide microtubule-associated protein. In addition, the developmental analysis revealed that although p23 is expressed early in definitive erythropoeisis, its association with the MB, centrosome and nuclear membrane occurs only in the final stages of differentiation. This cytoskeletal association correlates with marked p23 stabilization and accumulation at a time p23 expression is being markedly downregulated. We hypothesize that the mechanism of p23 association to the MB and centrosomes may be induced in part by a decrease in intracellular [Ca2+] during the terminal stages of definitive erythropoiesis.

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Development ◽  
1989 ◽  
Vol 105 (3) ◽  
pp. 505-519 ◽  
Author(s):  
P.C. Letourneau ◽  
T.A. Shattuck
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Growth Cones ◽  
Tissue Cell ◽  
Axonal Pathfinding ◽  
Cell Behaviour ◽  
Nerve Growth ◽  
Associated Proteins ◽  
Nerve Growth Cones

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.

scholarly journals Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 260 ◽  
Author(s):  
Yumiko Saijo-Hamano ◽  
Hideyuki Matsunami ◽  
Keiichi Namba ◽  
Katsumi Imada
Keyword(s):  
Mechanical Properties ◽  
Structural Model ◽  
Sequence Similarity ◽  
Significant Sequence Similarity ◽  
Protein Subunits ◽  
Bacterial Flagellum ◽  
Curved Tube ◽  
Single Protein ◽  
Associated Proteins ◽  
Density Map

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components—the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook.

scholarly journals A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration

2019 ◽  
Vol 295 (8) ◽  
pp. 2175-2183 ◽  
Author(s):  
Yuichi Sekine ◽  
Jane A. Lindborg ◽  
Stephen M. Strittmatter
Keyword(s):  
Spinal Cord ◽  
Axonal Regeneration ◽  
Axon Regeneration ◽  
Cultured Cells ◽  
Enzyme Inhibitor ◽  
Recombinant Protein Expression ◽  
Cord Injury ◽  
Central Nervous System Trauma ◽  
Associated Proteins ◽  
Sirna Knockdown

Glial signals are known to inhibit axonal regeneration and functional recovery after mammalian central nervous system trauma, including spinal cord injury. Such signals include membrane-associated proteins of the oligodendrocyte plasma membrane and astrocyte-derived, matrix-associated proteins. Here, using cell lines and primary cortical neuron cultures, recombinant protein expression, immunoprecipitation and immunoblot assays, transmission EM of exosomes, and axon regeneration assays, we explored the secretion and activity of the myelin-associated neurite outgrowth inhibitor Nogo-A and observed exosomal release of a 24-kDa C-terminal Nogo-A fragment from cultured cells. We found that the cleavage site in this 1192-amino-acid-long fragment is located between amino acids 961–971. We also detected a Nogo-66 receptor (NgR1)–interacting Nogo-66 domain on the exosome surface. Enzyme inhibitor treatment and siRNA knockdown revealed that β-secretase 1 (BACE1) is the protease responsible for Nogo-A cleavage. Functionally, exosomes with the Nogo-66 domain on their surface potently inhibited axonal regeneration of mechanically injured cerebral cortex neurons from mice. Production of this fragment was observed in the exosomal fraction from neuronal tissue lysates after spinal cord crush injury of mice. We also noted that, relative to the exosomal marker Alix, a Nogo-immunoreactive, 24-kDa protein is enriched in exosomes 2-fold after injury. We conclude that membrane-associated Nogo-A produced in oligodendrocytes is processed proteolytically by BACE1, is released via exosomes, and is a potent diffusible inhibitor of regenerative growth in NgR1-expressing axons.

scholarly journals Dynamics of microtubules from erythrocyte marginal bands.

1993 ◽  
Vol 4 (3) ◽  
pp. 323-335 ◽  
Author(s):  
B Trinczek ◽  
A Marx ◽  
E M Mandelkow ◽  
D B Murphy ◽  
E Mandelkow
Keyword(s):  
Phosphate Buffer ◽  
Dynamic Instability ◽  
Critical Concentration ◽  
Mammalian Brain ◽  
Tubulin Isoforms ◽  
Marginal Band ◽  
Associated Proteins ◽  
Free Microtubules ◽  
The Difference ◽  
Brain Microtubules

Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.

scholarly journals Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins.

1985 ◽  
Vol 101 (4) ◽  
pp. 1379-1385 ◽  
Author(s):  
C L Howe ◽  
L M Sacramone ◽  
M S Mooseker ◽  
J S Morrow
Keyword(s):  
Brush Border ◽  
Human Erythrocyte ◽  
Binding Capacity ◽  
Cytoskeletal Regulation ◽  
Associated Proteins ◽  
Chicken Erythrocytes ◽  
Related Proteins ◽  
Inside Out

The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.

A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome

1994 ◽  
Vol 107 (2) ◽  
pp. 601-611 ◽  
Author(s):  
J.E. Dominguez ◽  
B. Buendia ◽  
C. Lopez-Otin ◽  
C. Antony ◽  
E. Karsenti ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Polyclonal Antibodies ◽  
Microtubule Organizing Center ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Pericentriolar Material ◽  
Peptide Maps

The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.

Distribution of Na+ and K+ Currents in Soma, Axons and Growth Cones of Leech Retzius Neurones in Culture

1990 ◽  
Vol 150 (1) ◽  
pp. 1-17 ◽  
Author(s):  
U. GARCÍA ◽  
S. GRUMBACHER-REINERT ◽  
R. BOOKMAN ◽  
H. REUTER
Keyword(s):  
Cultured Cells ◽  
Growth Cones ◽  
Proximal Segment ◽  
Mapping Procedure ◽  
Na Currents ◽  
K Current ◽  
Current Densities ◽  
A New Technique ◽  
Retzius Cells ◽  
K Currents

1. Leech Retzius neurones were isolated by a new technique which allowed investigation of macroscopic currents over the surface of the cell body and the axons using loose patch-clamp. The distribution of ion current densities was measured for neurones that had just been removed from the CNS, and for cultured cells in which neurite outgrowth had begun. To standardize the mapping procedure, the same patch electrode was used at various sites along the neurone. 2. Immediately after isolation of the cell, rapidly activating and inactivating Na+ currents were recorded from distal segments of the axons, but not from the soma or the proximal segment. Na+ currents were isolated by using patch electrodes containing tetraethylammonium (TEA+) and 4-aminopyridine (4-AP) to block K+ channels and Cd2+ to block calcium channels. Na+ currents in all regions of the neurone where they could be recorded were similar in their voltage dependence and kinetics. The Na+ current density was highest at the broken tips of the axon stumps. 3. Neurites began to extend from the broken axon tips approximately 30min after isolation. Newly grown processes showed a high density of Na+ currents at their growth cones. After 2 days in culture the current densities became more uniformly distributed and Na+ currents could then be recorded in the soma and proximal axon segments. 4. In agreement with earlier studies made with conventional two-electrode voltage-clamp, three principal K+ currents were detected in Retzius cells: a rapidly activating and inactivatingA-type current blocked by 4-AP (IA); a more slowly activating and inactivating delayed K+ current blocked by TEA+ (IK1); and a Ca2+-activated K+ current (IC). Immediately after isolation of the Retzius cell, both rapid A-type and slow delayed K+ currents were distributed more uniformly than Na+ currents over the soma and axons. In their voltage sensitivities and kinetics, these two K+ currents were markedly different from each other; their characteristics were, however, constant in different regions of the cell. 5. Ca2+ currents were too small to be measured directly during depolarizing pulses. However, tail currents were large enough to demonstrate the presence of Ca2+ channels in the proximal segment of the axon and in the soma; the currents were not sufficiently large to resolve their spatial distribution. 6. It is concluded that ion channels are present in newly grown membranes and that the density of Na+ channels is highest in the tips of distal axon stumps from which outgrowth begins. By contrast, K+ currents are distributed more uniformly along the neurone.

scholarly journals Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo.

1987 ◽  
Vol 104 (1) ◽  
pp. 51-59 ◽  
Author(s):  
S Kim ◽  
M Magendantz ◽  
W Katz ◽  
F Solomon
Keyword(s):  
Drug Sensitivity ◽  
Cultured Cells ◽  
Animal Cell ◽  
Single Plane ◽  
Differentiated Cells ◽  
Marginal Band ◽  
Immature Cells ◽  
Postmitotic Cell ◽  
Microtubule Structure

The microtubules of mature nucleated erythrocytes are organized into a marginal band that is confined to a single plane at the periphery and that contains essentially the same number of microtubule profiles in each individual cell. Developing erythrocytes can be isolated in homogeneous and synchronously developing populations from chicken embryos. For these reasons, these cells offer a particularly accessible system for study of the pathway leading to a specific microtubule structure in a normal, terminally differentiated animal cell. Along this developmental course, striking changes occur in the properties of the microtubules. Between the postmitotic cell and the formation of the band, a novel arrangement is found: bundles of laterally associated microtubules in each cell, coursing through the cytoplasm but not confined to the periphery. The microtubule organizing centers evident at early stages disappear by the time the band forms. The microtubules in early cells are readily depolymerized by drugs, but that drug sensitivity is lost in the mature cells. The microtubule arrangement of mature cells is faithfully recapitulated after reversible depolymerization, while that of the immature cells is not. Finally, as the band forms, the microtubules and microfilaments increasingly become coaligned. In sum, the microtubules of immature cells have many properties in common with those of cultured cells, but during maturation those properties change. The results suggest that lateral interactions become increasingly important in stabilizing and organizing the microtubules. The properties of marginal band microtubules, and comparable properties of axonal microtubules, may reflect differences between the requirements for cytoskeletal structures of cycling cells and terminally differentiated cells.

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