scholarly journals Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

1995 ◽  
Vol 6 (8) ◽  
pp. 981-996 ◽  
Author(s):  
R Takemura ◽  
S Okabe ◽  
T Umeyama ◽  
N Hirokawa
Keyword(s):  
Electron Microscope ◽  
Model System ◽  
Sf9 Cells ◽  
Assembly Process ◽  
Microtubule Associated Proteins ◽  
Cos Cells ◽  
Microtubule Polarity ◽  
Neuronal Processes ◽  
Associated Proteins ◽  
Peripheral Cytoplasm

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.

scholarly journals MAP6 is an intraluminal protein that induces neuronal microtubules to coil

2020 ◽  
Vol 6 (14) ◽  
pp. eaaz4344 ◽  
Author(s):  
Camille Cuveillier ◽  
Julie Delaroche ◽  
Maxime Seggio ◽  
Sylvie Gory-Fauré ◽  
Christophe Bosc ◽  
...  
Keyword(s):  
Nervous System ◽  
Mechanical Stress ◽  
Microtubule Associated Proteins ◽  
Left Handed ◽  
Neuronal Processes ◽  
Associated Proteins ◽  
The Face ◽  
Cold Stability

Neuronal activities depend heavily on microtubules, which shape neuronal processes and transport myriad molecules within them. Although constantly remodeled through growth and shrinkage events, neuronal microtubules must be sufficiently stable to maintain nervous system wiring. This stability is somehow maintained by various microtubule-associated proteins (MAPs), but little is known about how these proteins work. Here, we show that MAP6, previously known to confer cold stability to microtubules, promotes growth. More unexpectedly, MAP6 localizes in the lumen of microtubules, induces the microtubules to coil into a left-handed helix, and forms apertures in the lattice, likely to relieve mechanical stress. These features have not been seen in microtubules before and could play roles in maintaining axonal width or providing flexibility in the face of compressive forces during development.

scholarly journals Domains of Neuronal Microtubule-associated Proteins and Flexural Rigidity of Microtubules

1997 ◽  
Vol 138 (5) ◽  
pp. 1067-1075 ◽  
Author(s):  
Harald Felgner ◽  
Rainer Frank ◽  
Jacek Biernat ◽  
Eva-Maria Mandelkow ◽  
Eckhard Mandelkow ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Flexural Rigidity ◽  
Microtubule Binding ◽  
Fourfold Increase ◽  
Microtubule Associated Proteins ◽  
Relative Contribution ◽  
Low Concentrations ◽  
Neuronal Processes ◽  
Associated Proteins ◽  
And Function

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at ∼20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.

scholarly journals The Development of Cell Processes Induced by tau Protein Requires Phosphorylation of Serine 262 and 356 in the Repeat Domain and Is Inhibited by Phosphorylation in the Proline-rich Domains

1999 ◽  
Vol 10 (3) ◽  
pp. 727-740 ◽  
Author(s):  
Jacek Biernat ◽  
Eva-Maria Mandelkow
Keyword(s):  
Tau Protein ◽  
Approximate Model ◽  
Necessary Condition ◽  
Sf9 Cells ◽  
Microtubule Associated Proteins ◽  
Repeat Domain ◽  
Phosphorylation Pattern ◽  
Associated Proteins ◽  
Cell Processes ◽  
A Minor

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer’s disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.

scholarly journals Overexpression of tau in a nonneuronal cell induces long cellular processes.

1991 ◽  
Vol 114 (4) ◽  
pp. 725-733 ◽  
Author(s):  
J Knops ◽  
K S Kosik ◽  
G Lee ◽  
J D Pardee ◽  
L Cohen-Gould ◽  
...  
Keyword(s):  
Cell Shape ◽  
Single Gene ◽  
Sf9 Cells ◽  
Cdna Insert ◽  
Microscopic Appearance ◽  
Microtubule Associated Proteins ◽  
Microtubule Stabilization ◽  
Cellular Processes ◽  
Associated Proteins ◽  
Axonal Compartment

The ways in which the various microtubule-associated proteins (MAPs) contribute to cellular function are unknown beyond the ability of these proteins to modify microtubule dynamics. One member of the MAP family, tau protein, is restricted in its distribution to the axonal compartment of neurons, and has therefore prompted studies that attempt to relate tau function to the generation or maintenance of this structure. Sf9 cells from a moth ovary, when infected with a baculovirus containing a tau cDNA insert, elaborate very long processes. This single gene product expressed in a foreign host cell grossly alters the normal rounded morphology of these cells. The slender, relatively nonbranched appearance of these processes as well as their uniform caliber resembles the light-microscopic appearance of axons observed in several neuronal culture systems. Immunolabeling of the tau-expressing Sf9 cells demonstrated tau reactivity in the induced processes, and EM that microtubule bundles were present in the processes. Microtubule stabilization alone was insufficient to generate processes, since taxol treatment did not alter the overall cell shape, despite the induction of microtubule bundling within the cell body.

scholarly journals Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta'-iminodipropionitrile-intoxicated axon as a model system.

1985 ◽  
Vol 101 (1) ◽  
pp. 227-239 ◽  
Author(s):  
N Hirokawa ◽  
G S Bloom ◽  
R B Vallee
Keyword(s):  
Electron Microscopy ◽  
Axonal Transport ◽  
Immunofluorescence Microscopy ◽  
Model System ◽  
Cross Link ◽  
Fixed Tissue ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Rapid Transport ◽  
Neurotoxic Agent

Axons from rats treated with the neurotoxic agent beta,beta'-iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue. To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN-treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy using colloidal gold-labeled second antibodies revealed that both anti-MAP 1A and anti-MAP 1B stained fuzzy filamentous structures between microtubules. In view of earlier work indicating that rapid transport is associated with the microtubule domain in the IDPN-treated axon, it now appears that MAP 1A and MAP 1B may play a role in this process. We believe that MAP 1A and MAP 1B are major components of the microtubule-associated fibrillar matrix in the axon.

Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau

1992 ◽  
Vol 103 (4) ◽  
pp. 953-964 ◽  
Author(s):  
R. Takemura ◽  
S. Okabe ◽  
T. Umeyama ◽  
Y. Kanai ◽  
N.J. Cowan ◽  
...  
Keyword(s):  
Post Translational Modification ◽  
3T3 Cells ◽  
Microtubule Organization ◽  
Alpha Tubulin ◽  
Transfected Cells ◽  
Microtubule Associated Proteins ◽  
Cos Cells ◽  
Tubulin Acetylation ◽  
Microtubule Stability ◽  
Associated Proteins

We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin.

Towards an understanding of microtubule function and cell organization: an overview

1992 ◽  
Vol 70 (10-11) ◽  
pp. 835-841 ◽  
Author(s):  
Thomas H. MacRae
Keyword(s):  
Dynamic Instability ◽  
Functional Integration ◽  
Structural Protein ◽  
Microtubule Associated Proteins ◽  
Microtubule Stability ◽  
Microtubule Polarity ◽  
Tubulin Isoforms ◽  
Associated Proteins ◽  
Cell Organization ◽  
Cellular Components

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin–GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed α-tubulin and β-tubulin. Most eukaryotic cells possess several isoforms of the α- and β-tubulins, as well as γ-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural–functional integration that characterizes eukaryotic cells.Key words: tubulin, microtubules, microtubule-associated proteins.

Characterization of microtubules by dark-field STEM and replication

1991 ◽  
Vol 49 ◽  
pp. 152-153
Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese
Keyword(s):  
Protein Interactions ◽  
Low Dose ◽  
Unit Length ◽  
Dark Field ◽  
Carbon Films ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Freeze Dried ◽  
Protein Motors ◽  
Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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