Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein

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Tubulin expression in trypanosomes

Microtubules in trypanosomes are the main component of the flagellar axoneme and of the subpellicular microtubule corset, whose relative positions determine the morphology of each cell stage of the life cycle of these parasites. Microtubules are polymers of tubulin, a protein dimer of two 55-kDa subunits, alpha- and beta-tubulin; in Trypanosoma brucei, the tubulin-coding sequences are clustered in a 40-kb fragment of tandemly repeated alpha- and beta-tubulin genes separated by a 170-bp intergenic zone. This cluster is transcribed in a unique RNA which is rapidly processed into mature mRNAs carrying the 5' 35-nucleotide leader sequence found in all trypanosome mRNAs. Although no heterogeneity has been found at the gene level, tubulin can be post-translationally modified in 2 ways: the C-terminal tyrosine of alpha-tubulin can be selectively cleaved and added again with 2 enzymes, tubulin carboxypeptidase and tubulin-tyrosine ligase; alpha-tubulin can also be acetylated on a lysine residue. Some molecular domains of tubulin are restricted to subpopulations of microtubules; for instance, the beta-tubulin form defined by the monoclonal antibody 1B41 is sequestered into a part of the subpellicular cytoskeleton limited to the flagellar adhesion zone, which might correspond to the group of 4 microtubules associated with a cisterna of the endoplasmic reticulum, forming the so-called "subpellicular microtubule quartet" (SFMQ). The early assembly of this zone in each daughter cell during the cell division of T. brucei, together with the alterations undergone by the domain defined by the monoclonal antitubulin 24E3 during the differentiation of Trypanosoma cruzi, suggest that specific tubulin forms are responsible for dynamic properties of SFMQ possibly involved in trypanosome morphogenesis.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (FA) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F _A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F _A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK _m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK _m value of about 3 µM. When using microtubules as substrates,F _A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, theF _A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF _A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF _A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF _A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F _A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.     

Microtubule assembly is required for the formation of the pronuclei,nuclear lamin acquisition,and DNA synthesis during mouse,but not sea urchin,fertillization

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.     

Doxorubicin affects tau protein metabolism in human neuroblastoma cells

The microtubule associated protein called tau, found primarily in neurons, was detected in a human neuroblastoma cell line, LAN-5. Cells treated with retinoic acid (2.0×10^–5M) differentiate and acquire processes similar to neurons. Differentiated and logarithmically growing undifferentiated cells were exposed to varying doses of doxorubicin (an anthracycline chemotherapeutic antibiotic). While doxorubicin was lethal to many undifferentiated dividing cells, it was not as damaging to differentiated cells. After 2 to 4 days of doxorubicin treatment, the cells were harvested, the protein concentration determined and SDS-PAGE performed. Proteins were blotted onto nitrocellulose paper and immunostained with either a rabbit antiserum or mouse monoclonal antibody to tau. Undifferentiated LAN-5 cells treated with 4.0×10^–8M doxorubicin for 4 days and cells treated with 8.0×10^–8M doxorubicin for 2 days displayed a distinct lower band (just below the 50kd marker) that was either absent or very faint in untreated controls.Special issue dedicated to Dr. Paola S. Timiras.     

Geitler's “Plattenband” in the diatomSynedra cf.ulna in the light of TEM investigations

The ultrastructure of the diatomSynedra cf.ulna was examined paying special attention to the Plattenband (platelet band). This structure was first described byGeitler in 1948 on the basis of LM observations and denotes a linear array of dictyosomes along the apical axis of the cell. The present investigation confirmsGeitler's observations in all essential details and demonstrates that the dictyosomes are arranged along polarized nuclear extensions running towards the cell poles. Laterally the extensions are accompanied by a number of microtubules. In large cells the total length of the nucleus thus may reach 400 µm and more. Since only the central part of the nucleus is DNA-positive with DAPI and acridine orange, the nuclear nature of the backbone of the Plattenband cannot be recognized by LM techniques. TEM investigation of serial apical and transapical sections, however, prove unambiguously the identity with extended parts of the nucleus.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.