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 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 MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties.

1987 ◽  
Vol 105 (3) ◽  
pp. 1273-1282 ◽  
Author(s):  
B M Paschal ◽  
H S Shpetner ◽  
R B Vallee
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Sedimentation Coefficient ◽  
Minor Component ◽  
High Molecular Mass ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Axonemal Dynein ◽  
A Minor

We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP IC, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G.S., T. Schoenfeld, and R.B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent extraction of the microtubules with ATP. Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein.

scholarly journals MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons

2004 ◽  
Vol 167 (1) ◽  
pp. 99-110 ◽  
Author(s):  
Eva-Maria Mandelkow ◽  
Edda Thies ◽  
Bernhard Trinczek ◽  
Jacek Biernat ◽  
Eckard Mandelkow
Keyword(s):  
Cell Polarity ◽  
Tau Protein ◽  
Ganglion Cells ◽  
Intrinsic Activity ◽  
Active Movement ◽  
Retinal Ganglion ◽  
Microtubule Associated Proteins ◽  
Cell Components ◽  
Associated Proteins ◽  
Dependent Transport

Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

scholarly journals MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments

2002 ◽  
Vol 157 (7) ◽  
pp. 1187-1196 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Rachel S. Ozer ◽  
Daniel Safer ◽  
Shelley Halpain ◽  
Ronald A. Milligan
Keyword(s):  
Image Analysis ◽  
Strong Evidence ◽  
Ordered Structure ◽  
Microtubule Binding ◽  
Microtubule Associated Proteins ◽  
Specific Targeting ◽  
Close Proximity ◽  
Repeat Domain ◽  
Associated Proteins ◽  
Β Tubulin

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either α or β tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.

scholarly journals Protein Kinase MARK/PAR-1 Is Required for Neurite Outgrowth and Establishment of Neuronal Polarity

2002 ◽  
Vol 13 (11) ◽  
pp. 4013-4028 ◽  
Author(s):  
Jacek Biernat ◽  
Yong-Zhong Wu ◽  
Thomas Timm ◽  
Qingyi Zheng-Fischhöfer ◽  
Eckhard Mandelkow ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Tau Protein ◽  
Point Mutations ◽  
Dominant Negative ◽  
Neuronal Polarity ◽  
Dominant Negative Mutant ◽  
Cell Models ◽  
Alzheimer Brain ◽  
Repeat Domain ◽  
Cell Processes

Protein kinases of the microtubule affinity-regulating kinase (MARK) family were originally discovered because of their ability to phosphorylate certain sites in tau protein (KXGS motifs in the repeat domain). This type of phosphorylation is enhanced in abnormal tau from Alzheimer brain tissue and causes the detachment of tau from microtubules. MARK-related kinases (PAR-1 and KIN1) occur in various organisms and are involved in establishing and maintaining cell polarity. Herein, we report the ability of MARK2 to affect the differentiation and outgrowth of cell processes from neuroblastoma and other cell models. MARK2 phosphorylates tau protein at the KXGS motifs; this results in the detachment of tau from microtubules and their destabilization. The formation of neurites in N2a cells is blocked if MARK2 is inactivated, either by transfecting a dominant negative mutant, or by MARK2 inhibitors such as hymenialdisine. Alternatively, neurites are blocked if the target KXGS motifs on tau are rendered nonphosphorylatable by point mutations. The results suggest that MARK2 contributes to the plasticity of microtubules needed for neuronal polarity and the growth of neurites.

scholarly journals Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo.

1980 ◽  
Vol 87 (3) ◽  
pp. 792-801 ◽  
Author(s):  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Hela Cells ◽  
Cross Reactivity ◽  
Fiber Network ◽  
Reactive Material ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Associated Proteins ◽  
A Minor

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross-reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross-reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.

scholarly journals Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs.

1993 ◽  
Vol 121 (4) ◽  
pp. 823-835 ◽  
Author(s):  
J W Raff ◽  
D R Kellogg ◽  
B M Alberts
Keyword(s):  
Microtubule Organizing Center ◽  
Substantial Fraction ◽  
Beta Tubulin ◽  
Microtubule Associated Proteins ◽  
Organizing Center ◽  
Associated Proteins ◽  
Gamma Tubulin ◽  
A Minor ◽  
Drosophila Embryos

gamma-tubulin is a minor tubulin that is localized to the microtubule organizing center of many fungi and higher eucaryotic cells (Oakley, B. R., C. E. Oakley, Y. Yoon, and M. C. Jung. 1990. Cell. 61: 1289-1301; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836; Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823). Here we show that gamma-tubulin is a component of a previously isolated complex of Drosophila proteins that contains at least two centrosomal microtubule-associated proteins called DMAP190 and DMAP60. Like DMAP190 and DMAP60, the gamma-tubulin in extracts of early Drosophila embryos binds to microtubules, although this binding may be indirect. Unlike DMAP190 and DMAP60, however, only 10-50% of the gamma-tubulin in the extract is able to bind to microtubules. We show that gamma-tubulin binds to a microtubule column as part of a complex, and a substantial fraction of this gamma-tubulin is tightly associated with DMAP60. As neither alpha- nor beta-tubulin bind to microtubule columns, the gamma-tubulin cannot be binding to such columns in the form of an alpha:gamma or beta:gamma heterodimer. These observations suggest that gamma-tubulin, DMAP60, and DMAP190 are components of a centrosomal complex that can interact with microtubules.

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 Tau in Health and Neurodegenerative Diseases

2021 ◽  
Author(s):  
Dandan Chu ◽  
Fei Liu
Keyword(s):  
Neurodegenerative Diseases ◽  
Tau Protein ◽  
Dynamic Properties ◽  
Corticobasal Degeneration ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Argyrophilic Grain ◽  
The Brain ◽  
Filamentous Inclusions

Tau, one of the major microtubule-associated proteins, modulates the dynamic properties of microtubules in the mammalian nervous system. Tau is abundantly expressed in the brain, particularly in the hippocampus. Insoluble and filamentous inclusions of tau in neurons or glia are discovered in neurodegenerative diseases termed ‘tauopathies’, including Alzheimer’s disease (AD), argyrophilic grain disease (AGD), corticobasal degeneration (CBD), frontotemporal dementia (FTD), Pick’s disease (PiD) and progressive supranuclear palsy (PSP). Accumulation of intracellular neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated tau, is directly correlated with the degree of Alzheimer\'s dementia. This chapter reviews the role of tau protein in physiological conditions and the pathological changes of tau related to neurodegenerative diseases. The applications of tau as a therapeutic target are also discussed.

scholarly journals Modulation of Protein Kinases and Microtubule-associated Proteins and Changes in Ultrastructure in Female Rat Pituitary Cells: Effects of Estrogen and Bromocriptine

1997 ◽  
Vol 45 (6) ◽  
pp. 805-813 ◽  
Author(s):  
Akira Matsuno ◽  
Susumu Takekoshi ◽  
Naoko Sanno ◽  
Hirotoshi Utsunomiya ◽  
Yoshitaka Ohsugi ◽  
...  
Keyword(s):  
Tau Protein ◽  
Intracellular Transport ◽  
Structural Changes ◽  
Ultrastructural Changes ◽  
Pituitary Cells ◽  
Female Rat ◽  
Cam Kinase ◽  
Microtubule Associated Proteins ◽  
Rat Pituitary ◽  
Associated Proteins

This study focused on the intracellular signal transduction system and microtubule-associated proteins (MAPs), such as MAP-2 and Tau protein. The modulation of these proteins and their correlation with ultrastructural changes were investigated in rat pituitary prolactin (PRL) cells. Adult female Wistar rats were treated with estrogen and bromocriptine and their pituitary glands were removed for analysis of the expression of tubulin, MAP-2, Tau protein, protein kinase C (PKC), and calcium calmodulin (CaM) kinase. Western blot analysis showed that estrogen increased and bromocriptine decreased the expression of PKCα, β1, β2, CaM kinase α, β, MAP-2, and Tau protein. MAP-2 and Tau protein, which are cytosolic proteins, being translated on free ribosomes, were associated with the membrane of whirling rough endoplasmic reticulum (RER) in estrogen-treated cells and dissociated with vesiculated RER induced by bromocriptine. These results suggested that the modulation of MAP-2 and Tau protein may reflect changes of PKC and CaM kinase, and that the quantitative changes and intracellular modulation of MAPs induced by estrogen and bromocriptine, i.e., estrogen-induced association and bromocriptine-induced dissociation of MAP-2 and Tau protein with membrane of RER, may reflect the dynamics of microtubules and are associated with structural changes in the RER and changes in the synthesis and intracellular transport of PRL.

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