Female genital mutilation: an overview

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

The effects of insulin, cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton x 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50-205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton x 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.     

Microtubule-associated protein 2 and the organization of cellular microtubules

Microtubule-associated proteins (MAPs) are prominent components of the neuronal cytoskeleton that can promote microtubule formation and whose expression is under strong developmental regulation. They are thought to be involved in organizing the structure of microtubule fascicles in axons and dendrites, although whether they form active cross-links between microtubules or serve as strut-like spacer elements has yet to be resolved. In the experiments reported here we explored their influence on microtubules by expressing them in non-neuronal cells using DNA transfection techniques. We confirm earlier reports that microtubule-associated proteins of the MAP2/tau class can induce bundling of microtubules. In addition we find that MAP2 causes the rearrangement of microtubules in the cytoplasm in a manner that is dependent on the length of the microtubule bundles. Short bundles are straight and run across the cytoplasm whereas long bundles form a marginal band-like array at the periphery. We suggest that the latter arrangement is produced when microtubule bundles that are too long to fit inside the diameter of the cell bend under the restraining influence of the cortical cytoskeleton. In confirmation of this, we show that when the cortical actin network is depolymerized by cytochalasin B the MAP2-containing microtubule bundles push out cylindrical extensions from the cell surface. These results suggest that the induction of stiff microtubules bundles by MAP2, coupled with a breach in the cortical actin network, can confer two of the properties characteristic of neuronal processes; their cylindrical form and the presence of fasciculated microtubules.     

Upregulation and redistribution of E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) in terminally differentiating keratinocytes is coincident with the formation of intercellular contacts

Microtubules are involved in the positioning and movement of organelles and vesicles and therefore play fundamental roles in cell polarization and differentiation. Their organization and properties are cell-type specific and are controlled by microtubule-associated proteins (MAP). E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) has been identified as a microtubule-stabilizing protein predominantly expressed in epithelial cells. We have used human skin and primary keratinocytes as a model to assess a putative function of E-MAP-115 in stabilizing and reorganizing the microtubule network during epithelial cell differentiation. Immunolabeling of skin sections indicated that E-MAP-115 is predominantly expressed in the suprabasal layers of the normal epidermis and, in agreement with this observation, is relatively abundant in squamous cell carcinomas but barely detectable in basal cell carcinomas. In primary keratinocytes whose terminal differentiation was induced by increasing the Ca2+ concentration of the medium, E-MAP-115 expression significantly increased during the first day, as observed by northern and western blot analysis. Parallel immunofluorescence studies showed an early redistribution of E-MAP-115 from microtubules with a paranuclear localization to cortical microtubules organized in spike-like bundles facing intercellular contacts. This phenomenon is transient and can be reversed by Ca2+ depletion. Treatment of cells with cytoskeleton-active drugs after raising the Ca2+ concentration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical localization of these microtubules is dependent on other microtubules but not on strong interactions with the actin cytoskeleton or the plasma membrane. The mechanism whereby E-MAP-115 would redistribute to and stabilize cortical microtubules used for the polarized transport of vesicles towards the plasma membrane, where important reorganizations take place upon stratification, is discussed.     

The role of Saccharomyces cerevisiae coronin in the actin and microtubule cytoskeletons

Coronin was originally identified as a cortical protein associated with the actin cytoskeleton in Dictyostelium [1]. More recent studies have revealed that coronin is involved in actin-based motility, cytokinesis and phagocytosis [2,3]. Here, we describe the identification of a single homolog of coronin in Saccharomyces cerevisiae, which we show localizes to cortical actin patches in an actin-dependent manner. Unlike Dictyostelium mutants that lack coronin, yeast strains lacking coronin had no detectable defects in actin-based processes. This may reflect differences in the functions of the actin cytoskeleton in these two organisms. Previous studies have shown that cortical actin may mediate astral microtubule-based movements of the mitotic spindle in S. cerevisiae [4,5] and that, during mitosis in Dictyostelium, the regions of the cell cortex that overlap with astral microtubules become enriched in actin and coronin [6]. We therefore examined whether yeast lacking coronin had defects in the microtubule cytoskeleton. The mutant strains had increased sensitivity to the microtubule-destabilizing drug benomyl and an increased number of large-budded cells with short spindles. Further examination of microtubule-related processes, including spindle formation, migration of the mitotic spindle to the bud neck, spindle elongation, and translocation of the elongating spindle through the bud neck, failed to reveal any defects in the coronin mutant. Taken together, these results suggest that S. cerevisiae coronin is a component of the actin cytoskeleton that may interact with the microtubule cytoskeleton.     

Characterization of the microtubule-binding domain of microtubule-associated protein 1A and its effects on microtubule dynamics

To determine how MAP1a interacts with microtubules we expressed several 6myc-tagged MAP1a fragments in P19 EC and HeLa cells. Confocal immunofluorescence microscopy showed that the fragment consisting of amino acids (aa) 1-281 of MAP1a did not bind while the fragment consisting of aa 1-630 did, indicating that the region of MAP1a between aa 281 and 630 contains a microtubule-binding domain. Deletion of the basic repeats from aa 336-540 did not result in loss of microtubule binding, suggesting that the regions flanking the basic repeats can bind MAP1a to microtubules. These observations were confirmed using an in vitro microtubule binding assay. The levels of acetylation and detyrosination of polymerized microtubules were assessed by quantitative dot blotting in cells expressing MAP1a fragments or MAP2c. Compared with untransfected cells, the polymerized tubulin in cells expressing full-length MAP1a was more acetylated and detyrosinated, but these increases were smaller than those seen in cells expressing MAP2c. Consistent with this, the microtubules in MAP2c expressing cells were more resistant to colchicine than those in cells overexpressing MAP1a. These data implicate aa 281-336 and/or 540-630 of MAP1a in microtubule binding and suggest that MAP1a is less able to stabilize microtubules than MAP2c.     

Effects of an antiallergic drug, pemirolast potassium on tyrosine phosphorylation and map kinase activation in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Aggregation of high affinity IgE Fc receptors (Fc epsilon RI) on RBL-2H3 cells results in tyrosine phosphorylation of 33-, 42-, 44-, 72-, 80-, 90-, 125-kDa proteins. The 42 and 44 kDa proteins were identified as mitogen-activated protein (MAP) kinases with immunoblotting of anti-MAP kinase antibody. The effects of an antiallergic drug, pemirolast potassium (TBX) on Ag-induced protein tyrosine phosphorylation and MAP kinase activation were investigated. When RBL-2H3 cells were stimulated with Ag in the presence of TBX, tyrosine phosphorylation of three proteins (33, 42 and 44 kDa) was inhibited concentration-dependently (0.1-10 micrograms/ml). Inhibition of Ag-induced tyrosine phosphorylation of 33 kDa protein, which could be a beta subunit of Fc epsilon RI, suggests that TBX may prevent the activation of Fc epsilon RI. TBX suppressed activation of MAP kinases (42 and 44 kDa) in response to Ag as well as phorbol myristate acetate (100 nM) or calcium ionophore A23187 (500 nM), implying that the drug acts on signal transduction component(s) between the second messengers and MAP kinases. However, TBX had no effects on protein tyrosine phosphorylation and MAP kinase activation in MC3T3-E1 osteoblastic cells. These results indicate that TBX may affect Fc epsilon RI and also may act as a step distal of Ca2+ mobilization and protein kinase C activation leading to MAP kinase activation in RBL-2H3 cells.     

Intracellular insulin binding in Tetrahymena pyriformis

Insulin, a classic vertebrate hormone, produces alterations in cellular metabolism and growth in the ciliate Tetrahymena pyriformis, as well as an increase in insulin binding upon subsequent exposure, a phenomenon known as hormonal imprinting. An antibody to a peptide corresponding to the alpha-subunit of the human insulin receptor (amino acid residues 657-670) was used to investigate the location and to partially characterize immunoreactive proteins in insulin-exposed and non-insulin-exposed cells (control). Confocal microscopy revealed immunofluorescent labeling of cilia, nuclei, vesicles and an oblong structure of unknown nature. Labeling of nuclei, mitochondria and ciliary microtubules was seen with immunoelectron microscopy. Labeling was absent on the cell and ciliary membranes by immunoelectron microscopy. Polyacrylamide gel electrophoresis revealed several differences in protein composition between control and insulin-exposed ciliary membrane extracts, especially in the 30-50 kDa range. Immunoblotting revealed 2 reactive proteins in whole cell lysates but none were detected in ciliary membrane extracts or wheat germ agglutinin affinity column eluates of T. pyriformis whole cell preparations. Based on these findings it is unlikely that a cell surface structure similar to a mammalian insulin receptor exists in T. pyriformis.     

In situ synthesis of beta-glucan microfibrils on tobacco plasma membrane sheets

A major concern in plant morphogenesis is whether cortical microtubules are responsible for the arrangement and action of beta-glucan synthases in the plasma membrane. We prepared isolated plasma membrane sheets with cortical microtubules attached and tested whether beta-glucan synthases penetrated through the membrane to form microfibrils and whether these synthases moved in the fluid membrane along the cortical microtubules. This technique enabled us to examine synthesis of beta-glucan as a fiber with a two-dimensional structure. The synthesis of beta-glucan microfibrils was directed in arrays by cortical microtubules at many loci on the membrane sheets. The microfibrils were mainly arranged along the microtubules, but the distribution of microfibrils was not always parallel to that of the microtubules. The rate of beta-glucan elongation as determined directly on the exoplasmic surface was 620 nm per min. When the assembly of microtubules was disrupted by treatment with propyzamide, the beta-glucans were not deposited in arrays but in masses. This finding shows that the arrayed cortical microtubules are not required for beta-glucan synthesis but are required for the formation of arranged microfibrils on the membrane sheet.     

Microtubule-associated proteins and the stimulation of tubulin assembly in vitro

Microtubules, purified by cycles of assembly and disassembly in vitro, are composed of tubulin and several microtubule-associated proteins (MAPs). When the MAPs were separated from the tubulin by phosphocellulose chromatography, the tubulin by phosphocellulose chromatography, the tubulin no longer assembled at 37 degrees C as measured by turbidity. If the MAPs and tubulin were recombined and warmed to 37 degrees C, microtubules assembled. MAPs stimulated tubulin assembly by affecting both the initiation and elongation processes. The effect on initiation was indicated by results showing an increase in initial rate and a decrease in average microtubule length as the MAP:tubulin ratio was increased. The initiation and elongation activities of the MAPs at 4 degrees C during which time the initiating activity decreased while the ability to affect the total amount of assembly remained constant. The decrease in initiating ability was correlated with the loss of the two major components of the MAP fraction, MAPs 1 and 2.     

Characterization of p59fyn-mediated signal transduction on T cell activation

Protein tyrosine kinase p59fyn is associated with the TCR-CD3 complex and is suggested to play a role in T cell activation. To determine the molecular mechanism of p59fyn-mediated signal transduction in T cell activation, we established murine T cell hybridoma lines that expressed an elevated amount of wild-type or mutant fyns. Clones that expressed high levels of normal p59fyn and active p59fyn, encoded by wild-type and f-14 mutant fyn respectively, showed enhanced IL-2 production upon stimulation by anti-CD3 antibodies or natural antigen. On the other hand, clones that expressed kinase negative p59fyn and p59fyn with an SH2 (Src-homology 2) deletion encoded by t-1 mutant fyn showed little induction of IL-2 production upon stimulation. These data suggest that p59fyn is important in T cell signaling and that the SH2 sequence plays a critical role in the reaction. Induction of tyrosine phosphorylation of multiple proteins upon antigenic stimulation was augmented similarly in the cells that respectively expressed wild-type and f-14 mutant fyns at elevated levels. The proteins that became highly tyrosine-phosphorylated included phospholipase C (PLC-gamma 1), p95vav, ZAP-70, the MAP kinase, CD3 zeta and unidentified proteins of 120, 100 and 80 kDa. Tyrosine phosphorylation of the 120, 95 and 68 kDa proteins associated with PLC-gamma 1 was also observed in these cells upon stimulation. In contrast, only the 100 kDa protein and the MAP kinase were increasingly tyrosine phosphorylated in the antigen-stimulated cells expressing t-1 fyn. These data suggest that PLC-gamma 1, PLC-gamma 1 associated molecules, p95vav, the 80 kDa protein, ZAP-70 and the CD3 zeta chain may be substrates of p59fyn or of other tyrosine kinases regulated by p59fyn and be important in T cell signaling.     

A quantitative analysis of microtubule elongation

Methods have been developed for differentially inhibiting microtubule nucleation and elongation in vitro. By use of polyanions, assembly-competent tubulin solutions of several milligrams/milliliter can be prepared which do not exhibit appreciable spontaneous assembly during the time-course of an experiment. Microtubule elongation can be initiated by the addition of known numbers of microtubule fragments. A detailed analysis of the resulting process demonstrates that: (a) rings are not obligatory intermediates in the nucleation sequence, and neither rings nor protofilament sheets are obligatory intermediates in the elongation reaction. (b) The end of an elongating microtubule often has a short region of open protofilament sheet or "C-microtubule" similar to that observed in vivo. (c) The development of turbidity follows a simple exponential approach to an equilibrium value. (d) The final equilibrium values are independent of the number of added nucleating fragments, while the initial growth rates and half-times to reach equilibrium are dependent on the number of added nuclei. (e) The final lengths of the microtubules at equilibrium are inversely proportional to the number of added fragments. (f) The equilibrium constants are independent of microtubule length. (g) The number of assembly and disassembly sites per microtubule is not a function of microtubule length. (h) The forward rate constants, the final polymer concentrations, and growth rates of microtubules are dependent upon the concentration of polyanion present. These results are strongly supportive of the idea that microtubule assembly is a "condensation-polymerization" and provide basic information on the kinetics and length distributions of the elongation in vitro.     

Lanthanides inhibit the human noradrenaline, 5-hydroxytryptamine and dopamine transporters

The spermatozoon of the monopisthocotylean monogenean Pseudodactylogyrus sp. (a gill parasite of eels) has a single axoneme showing a 9 + '1' pattern, a nucleus and a mitochondrion, but has no cortical microtubules. This species thus provides a very simple model for the study of tubulin in the 9 + '1' axonemes of the Platyhelminthes, in contrast with digenean sperm which have a more complex spermatozoon with two such axonemes and cortical microtubules. Indirect immunofluorescence labelling of tubulin shows that the elongating spermatids, initially lying in all directions in the early stages, are arranged as parallel elements in further stages. The number of spermatids in an isogenic group could also be precisely counted and equals 32. Nuclear labelling with fluorescent dyes shows that the nuclei, first located in the common mass of the spermatids, later elongate and migrate into the growing spermatids, and that the nucleus is located in the central part of the mature spermatozoon, with the two extremities devoid of nucleus. Labelling with antibodies directed against acetylated, tyrosinated, and polyglutamylated tubulin gave positive results, thus indicating that these post-translational modifications of tubulin are present in the axoneme of spermatids and spermatozoa of monopisthocotylean monogeneans.     

Localization of neuronal and glial glutamate transporters

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.     

Microtubule-associated protein 2 expressing COS7 cells are resistant to argyrophilia under oxygen- and glucose-free condition

Argyrophil III silver impregnation is a very sensitive method to detec t the early damage to neurons following brain ischemia. The argyrophil III staining and microtubule-associated protein 2 (MAP2) immunocytochemistry were performed on PC12D cells and MAP2C cDNA-transfected COS7 cells to detect the changes cytoskeletal proteins (microtubules/MAP2). After exposing these cells to simulated ischemic condition (oxygen and/or glucose free), the correlation between the appearance of the argyrophilia and the disappearance of MAP2 was investigated. The PC12D cells expressed very low MAP2 and became argyrophilic very easily depending on the degree of the ischmeia-like insult, whereas MAP2C cDNA-transfected COS7 cells expressed a higher level of MAP2C and were resistant to argyrophilia, although the diameter of their immunoreactive processes became thinner. Thus, when MAP2C is expressed at a higher level, cells became resistant to argyrophilia, suggesting a correlation between the argyrophilia and the damage on microtubules and MAPs.     

A newly identified membrane protein localized exclusively in intracellular organelles of neurons

We report here cloning of the cDNA of a novel membrane protein, termed p24, which, of the eight mouse tissues tested, was found only in brain where it is localized exclusively in neurons. The cDNA encodes 196 amino acids with a molecular weight of approximately 24000. P24 contains two putative membrane spanning domains and a sequence in the hydrophilic tail homologous to the microtubule-binding domain of microtubule-associated proteins, such as TAU and MAP-2. We prepared antibodies to p24 and demonstrated that the protein is rich in nerve fibers of the cerebral cortex, anterior cerebral nuclei and hypothalamus. When neuroblastoma Neuro 2a cells were treated with retinoic acid to induce differentiation, p24 mRNA increased but the p24 protein was not detected. The protein expressed from the p24 cDNA in non-neuronal Cos-7 cells was 24 kDa in size and were localized only in lysosomes. These findings indicate that p24 is a neuron-specific membrane protein localized in intracellular organelles of highly differentiated neural cells and suggest that it may play a role in the neural organelle transport system.     

Passive mechanical behavior of human neutrophils: effects of colchicine and paclitaxel

The role of microtubules in determining the mechanical rigidity of neutrophils was assessed. Neutrophils were treated with colchicine to disrupt microtubules, or with paclitaxel to promote formation of microtubules. Paclitaxel caused an increase in the number of microtubules in the cells as assessed by immunofluorescence, but it had no effect on the presence or organization of actin filaments or on cellular mechanical properties. Colchicine at concentrations <1.0 microM caused disruption of microtubular structures, but had little effect on either F-actin or on cellular mechanical properties. Higher concentrations of colchicine disrupted microtubular structure, but also caused increased actin polymerization and increases in cell rigidity. Treatment with 10 microM colchicine increased F-actin content by 17%, the characteristic cellular viscosity by 30%, the dependence of viscosity on shear rate by 10%, and the cortical tension by 18%. At 100 microM colchicine the corresponding increases were F-actin, 25%; characteristic viscosity, 50%; dependence of viscosity on shear rate, 20%; and cortical tension, 21%. These results indicate that microtubules have little influence on the mechanical properties of neutrophils, and that increases in cellular rigidity caused by high concentrations of colchicine are due to a secondary effect that triggers actin polymerization. This study supports the conclusion that actin filaments are the primary structural determinants of neutrophil mechanical properties.     

Thyroid hormone regulates protein expression in C6 glioma cells

Thyroid hormone (T3) is essential to normal brain development. Previously, we have shown that T3 induces cerebellar astrocyte proliferation. This effect is accompanied by alteration in glial fibrillary acidic protein (GFAP) and fibronectin organization. In the present study, we report that the C6 glioma cell line, which expresses GFAP and is classified as an undifferentiated astrocytic cell type, is a target for T3 action. The C6 monolayers were treated with 50 nM T3 for 3 days, after which the cells were maintained for 2 days without medium changes. In C6 cells, T3 induced the expression of proteins of 107, 73 and 62 kDa. The hormone also up-regulated protein bands of 100 (+50%), 37 (+50%) and 25.5 kDa (+50%) and down-regulated proteins of 94 (-100%), 86.5 (-100%), 68 (-100%), 60 (-100%), 54 (-33%), 51 (-33%) and 43.5 kDa (-33%). We suggest, on the basis of molecular mass, that the 54-, 51- and 43.5-kDa proteins could be the cytoskeletal proteins vimentin, GFAP and actin, respectively. The down-regulation of these proteins may be involved in the effects of thyroid hormone on C6 differentiation.     

Domains of neuronal microtubule-associated proteins and flexural rigidity of microtubules

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 approximately 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.