Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins

Schliwa, Manfred; Euteneuer, Ursula; Bulinski, J. Chloë; Izant, Jonathan G.

doi:10.1073/pnas.78.2.1037

Cited by 220 publications

(109 citation statements)

References 21 publications

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“…Studies in detergentextracted cells showed that microtubules polymerize at low calcium concentrations, whereas increasing calcium concentrations to the micromolar range directly induced microtubule disassembly (Fuller and Brinkley, 1976;Schliwa, 1976). These effects have been hypothesized to be mediated by calcium-dependent regulators of microtubule assembly, such as the abundant neuronal protein calmodulin (Marcum et al, 1978;Schliwa et al, 1981;Lee and Wolff, 1982;Deery et al, 1984). A recent report furthermore suggests that posttranslational modifications that are associated with microtubule stability, such as glutamylation, are regulated by synaptic activity (Maas et al, 2009).…”

Section: Nmda Receptor Activation Suppresses Microtubule Growth In Dementioning

confidence: 99%

NMDA Receptor Activation Suppresses Microtubule Growth and Spine Entry

Kapitein

Yau²,

Gouveia³

et al. 2011

J. Neurosci.

108

105

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Dynamic microtubules are important to maintain neuronal morphology and function, but whether neuronal activity affects the organization of dynamic microtubules is unknown. Here, we show that a protocol to induce NMDA-dependent long-term depression (LTD) rapidly attenuates microtubule dynamics in primary rat hippocampal neurons, removing the microtubule-binding protein EB3 from the growing microtubule plus-ends in dendrites. This effect requires the entry of calcium and is mediated by activation of NR2B-containing NMDA-type glutamate receptor. The rapid NMDA effect is followed by a second, more prolonged response, during which EB3 accumulates along MAP2-positive microtubule bundles in the dendritic shaft. MAP2 is both required and sufficient for this activity-dependent redistribution of EB3. Importantly, NMDA receptor activation suppresses microtubule entry in dendritic spines, whereas overexpression of EB3-GFP prevents NMDA-induced spine shrinkage. These results suggest that short-lasting and long-lasting changes in dendritic microtubule dynamics are important determinants for NMDA-induced LTD.

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Section: Nmda Receptor Activation Suppresses Microtubule Growth In Dementioning

confidence: 99%

NMDA Receptor Activation Suppresses Microtubule Growth and Spine Entry

Kapitein

Yau²,

Gouveia³

et al. 2011

J. Neurosci.

108

105

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“…All of the serum-deprived cells within a cell-free zone were microinjected with either the polyclonal or the monoclonal TTL antibodies and incubated for 1-72 h. At that time the cells were rinsed once in warm PHEM buffer (60 mM Pipes, pH 6.95, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCIz) (Schliwa et al, 1981), lysed for I rain with PHEM containing 0.1% (vol/vol) Triton X-100, and then fixed for 20 min in PHEM containing 5 mM EGS. Cells were immunostained with Glu IgG and Tyr IgM antibodies and fluorescein and Texas Red-conjugated secondary antibodies specific for mouse IgG and lgM, respectively.…”

Section: Conversion Kinetics and Ac Tubulin Lmmunostainingmentioning

confidence: 99%

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

Webster

Wehland

Weber

et al. 1990

The Journal of Cell Biology

141

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Abstract.The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence ~1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of ,'~ 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.

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“…Following electrophoresis the gels were fixed for 2 h in 20 % methanol supplemented with 10 % fetal calf serum and 1 mM Bt,cAMP for four days at 37 "C. The coverslips were washed three times with Pipes/Hepes/MgCI,/EGTA buffer (60 mM Pipes, 25 mM Hepes, 2 mM MgCI,, 10 mM EGTA, pH 6.9) and the cells were lysed in this buffer containing 0.2 Brij 58 for 5 min at room temperature [36]. The supernatant was removed and the extracted cells were then fixed with 0.5 glutaraldehyde in Pipes/Hepes/MgCI,/EGTA buffer for 15 min.…”

Section: Antibody Staining Of Proteins Resolved By Electrophoresis Onmentioning

confidence: 99%

Mapping of Distinct Structural Domains on Microtubule‐Associated Protein 2 by Monoclonal Antibodies

Scherson

Geiger

Eshhar

et al. 1982

European Journal of Biochemistry

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Monoclonal antibodes against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by a-chyrnotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.. Microtubules, isolated by successive cycles of assemblydisassembly, are composed of tubulin and several additional proteins designated microtubule-associated proteins or MAPs. Two major groups of MAPs have been identified in neutral tissues and in cultured cells. These include high-molecularmass components (350 and 270 kDa) termed MAP1 and MAP2, respectively [l -51 and a number of polypeptides in the molecular mass range of 55 -68 kDa, known collectively as z proteins [6 -8 1. Both MAP2 and z factors have been shown to promote nucleation and elongation of microtubules in vitro [I -7,9,10], but their role in the control of microtubule assembly and its interactions in the living cells is still uncertain.The most prominent MAP components in neuronal tissue are the high-molecular-mass proteins MAP1 and MAP2. The latter was found by electron microscopy to associate with microtubules at a fixed periodicity forming laterally projected arms [2,11,12]. The tissue and species distribution of MAP2 is still controversial at the present time. Immunocytochemical data suggest that this protein is associated with microtubules from a wide variety ofcells and tissues [I3 -171. Other groups, on the other hand, have used multispecific and monoclonal antibodies to show that MAP2 is present only in cells of neuronal origin [18], while other cells, such as HeLa, containAbbreviations. MAP1 and MAP2, microtubule-associated proteins 1 and 2; Mes, 4-morpholineethanesulfonic acid; SDS, sodium dodecyl su...

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Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins

Abstract: Detergent-extracted BSC-1 monkey cells have been used as a model system to study the Ca2+ sensitivity of in vivo polymerized microtubules under in vitro conditions. The effects of various experimental treatments were observed by immunofluorescence microscopy. Whereas

Cited by 220 publications

References 21 publications

NMDA Receptor Activation Suppresses Microtubule Growth and Spine Entry

NMDA Receptor Activation Suppresses Microtubule Growth and Spine Entry

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

Mapping of Distinct Structural Domains on Microtubule‐Associated Protein 2 by Monoclonal Antibodies

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