The tektin family of microtubule-stabilizing proteins (original) (raw)
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Putative involvement of a 49 kDa protein in microtubule assembly in vitro
European Journal of Cell Biology, 1999
In higher plant cells, thus far only a few molecules have been inferred to be involved in microtubule organizing centers (MTOCs). Examination of a 49 kDa tobacco protein, homologous to a 51 kDa protein involved in sea urchin MTOCs, showed that it also accumulated at the putative MTOC sites in tobacco BY-2 cells. In this report, we show that the 49 kDa protein is likely to play a significant role in microtubule organization in vitro. We have established a system prepared from BY-2 cells, capable of organizing microtubules in vitro. The fraction, which was partially purified from homogenized miniprotoplasts (evacuolated protoplasts) by salt extraction and subsequent ion exchange chromatography, contained many particles of diameters about 1 !J.IIl after desalting by dialysis. When this fraction was incubated with purified porcine brain tubulin, micro tubules were elongated radially from the particles and organized into structures similar to the asters observed in animal cells, and therefore also termed "asters" here. Since we could hardly detect BY-2 tubulin molecules in this fraction, the micro tubules in "asters" seemed to be solely composed of the added porcine tubulin. Tubulin molecules were newly polymerized at the ends of the microtnbnles distal to the particles, and the elongation rate of microtnbnles was more similar to the reported rate of the plusends than that of the minus-ends in vitro. By fluorescence microscopy, the 49 kDa protein was shown to be located at the particles. Thus, its location at the centers of the "asters" suggests that the protein plays a role in microtubule organization in vitro.
SnapShot: Microtubule Regulators II
Cell, 2009
Dynamic remodeling of the microtubule cytoskeleton is essential for many cell processes including division, migration and differentiation. Microtubules are dynamic polymers of α/β-tubulin dimers and transition stochastically between phases of growth (polymerization) and shortening (depolymerization). Intracellular microtubule organization is controlled by the activity and distribution of nucleation sites, proteins that directly influence polymerization dynamics, proteins that cut or bundle existing microtubules, and proteins that indirectly stabilize microtubules. Classical microtubule-associated proteins are mainly found in neuronal cells. Tau and MAP1B is specific for axons and MAP2 is predominantly localized to dendrites. These proteins bind along the length of microtubules and protect neurite microtubule arrays from depolymerization. Although many microtubule-associated proteins bundle microtubules when overexpressed in cells, true bundling activity has only been demonstrated for few protein families. Homotetrameric motor proteins of the kinesin-5 family slide antiparallel microtubules, and are required for spindle formation and spindle pole separation. MAP65-related proteins have been shown to promote antiparallel microtubule bundling, and yeast Ase1 is required for spindle midzone formation. MAP65 proteins are particularly numerous in plants. Together with additional plant-specific proteins such as WVD2, MAP65 proteins are involved in the formation of cortical microtubule bundles in plant cells. In addition to direct regulation of polymerization dynamics, microtubules can be stabilized by interactions with other intracellular structures. For example, CLASPs and spectraplakins mediate microtubule interactions with actin cables and adhesion sites. Because microtubules are the primary component of the mitotic spindle and essential for accurate chromosome segregation during cell division, it is not surprising that a number of microtubule-regulatory proteins function predominantly during spindle assembly. Many mitosis-specific microtubule stabilizers such as TPX2, NuMA, RHAMM, and HURP are segregated into the nucleus during interphase and are activated in a Ran•GTP dependent manner around mitotic chromatin. Tektins are a group of highly specialized microtubulestabilizing proteins necessary for the assembly of cilia and flagella in all eukaryotic cells. The specific functions of many other microtubule-associated and/or stabilizing proteins are poorly understood. Microtubule-based motor proteins that have no documented effects on microtubule dynamics are not included in this table.
European Journal of Biochemistry
Microtubule-assembly inhibitor protein (MIP) is an acidic protein with Mr 33,000 which inhibits microtubule assembly in vitro [Kotani, S., Murofushi, H., Nishida, E. & Sakai, H. (1984) J. Biochem. (Tokyo) 96, 959-969]. Anti-MIP antibody was affinity-purified from rabbit anti-MIP sera raised against chemically modified MIP. MIP was localized in the nucleus in interphase culture cells as revealed by immunofluorescent light microscopy. Immunoblotting experiments showed that MIP exists in a variety of mammalian cells and tissues. Kidney appeared to be a better source of MIP than brain, the original source. Kidney MIP was isolated by the same procedure as for brain MIP and proved to be indistinguishable from brain MIP in the inhibitory activity of microtubule assembly, molecular mass, immunoreactivity, and one-dimensional peptide mapping. Physico-chemical characteristics of MIP were studied using the kidney protein. It contained 20% aspartic acid and 25% glutamic acid, accounting for its...
The functional domain grouping of microtubule associated proteins
2008
Microtubules (MTs), which play crucial roles in normal cell function, are regulated by MT associated proteins (MAPs). Using a combinatorial approach that includes biochemistry, proteomics and bioinformatics, we have recently identified 270 putative MAPs from Drosophila embryos and characterized some of those required for correct progression through mitosis. Here we identify functional groups of these MAPs using a reciprocal hits sequence alignment technique and assign InterPro functional domains to 28 previously uncharacterized proteins. This approach gives insight into the potential functions of MAPs and how their roles may affect MTs.
We previously reported that the microtubule-stabilizing activity of a microtubule-associated protein (MAP) 4 variant, with a deletion in the Pro-rich region (MAP4-SP), was lower than that of a variant with a full length Pro-rich region (MAP4-LP). However, it remained unclear whether the deletion of the specific site in the Pro-rich region is responsible for the reduction of the microtubule-stabilizing activity. To answer this question, we examined the microtubule-stabilizing activities of four different MAP4 variants, MAP4-SP, MAP4-LP, and two additional MAP4-LP variants lacking a part of the Repeat region, and considered the correlation between the activity and the structure. When microtubules assembled in the presence of each of the MAP4 variants were treated with nocodazole for disassembly, the MAP4-SP-induced microtubules were significantly less stable than the other variant-induced microtubules. Another set of experiments, in which the microtubules were allowed to disassemble b...
European Journal of Biochemistry, 1988
Microtubule-assembly inhibitor protein (MIP) is an acidic protein with M , 33 000 which inhibits microtubule assembly in vitro [Kotani, S., Murofushi, H., J. Biochem. (Tokyo) 96, 959-9691. Anti-MIP antibody was affinity-purified from rabbit anti-MIP sera raised against chemically modified MIP. MIP was localized in the nucleus in interphase culture cells as revealed by immunofluorescent light microscopy.
MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments
Journal of Cell Biology, 2002
AP2 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 M 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 microtubuleassociated 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 ␣ or  tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces. *Abbreviations used in this paper: cf-MAP2c, cysteine-free MAP2c; cIR-MAP2c, cysteine-IR-MAP2c; H11, helix 11; H12, helix 12; IR, interrepeat; MAP, microtubule-associated protein; MTBR, microtubule binding repeat.
Journal of Cell Biology, 1985
We have developed a method to distinguish microtubule associated protein (MAP)containing regions from MAP-free regions within a microtubule, or within microtubule subpopulations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.