Cargo binding and regulatory sites in the tail of fungal conventional kinesin.

Here, using a quantitative in vivo assay, we map three regions in the carboxy terminus of conventional kinesin that are involved in cargo association, folding and regulation, respectively. Using C-terminal and internal deletions, point mutations, localization studies, and an engineered 'minimal' kinesin, we identify five heptads of a coiled-coil domain in the kinesin tail that are necessary and sufficient for cargo association. Mutational analysis and in vitro ATPase assays highlight a conserved motif in the globular tail that is involved in regulation of the motor domain; a region preceding this motif participates in folding. Although these sites are spatially and functionally distinct, they probably cooperate during activation of the motor for cargo transport.

Molecular components of the centrosome.

The centrosome organizes microtubules during both interphase and mitosis and therefore governs fundamental processes in the life of a eukaryotic cell. The past few years have seen a substantial increase in the identification of potential components localized at the centrosome. Although we are still far from achieving a coherent picture of the workings of the centrosome, these recent discoveries are promising first steps towards an understanding of centrosomal functions at the molecular level.

The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187.

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.

Microtubular apparates of melanophores. Three-dimensional organization.

Microtubular organization in the melanophores of the angelfish, Pterophyllum scalare, has been studied by serial thin sectioning. The course of microtubules has been followed in sets of transverse serial sections taken from the centrosphere and a segment of a cell process, respectively. Microtubules arise from a prominent zone in the cell center, the central apparatus, which is composed of numerous, small, electron-dense aggregates. the number of these loosely distributed densities is highest in the center of the centrosphere, but they may also be found at its periphery. Microtubules insert into, or becomes part of, the dense material, or at least start in its vicinity. Dense aggregates may be separated from centrioles by several micrometers rather than only being closely associated with these organelles. At some distance from the organizing zone, most of the microtubules gradually assume a cortical arrangement, i.e., take a course within about 100 nm of the limiting membrane. Serial sections were used to trace all microtubules within a 6mum-long segment of a cell process. 94 percent of the microtubules observed in this segment run its entire length; it is conceivable, therefore that a considerable number of microtubules extend between the initiating site in the centrosphere and the outermost cell region. A three-dimensional model of the 6mum-long segment reveals that, despite changes in the cell process outline, microtubules maintain a strictly cortical arrangement which gives the impression of a microtubule "palisade" lining the cortex of the cell process. The features of the microtubular apparatus of angelfish melanophores are discussed in relation to factors controlling microtubule initiation and distribution.

Action of cytochalasin D on cytoskeletal networks.

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end-to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.

Motility of cultured fish epidermal cells in the presence and absence of direct current electric fields.

The motile behavior and cytoskeletal structures of fish epidermal cells (keratocytes) in the presence and absence of direct current (DC) electric fields were examined. These cells spontaneously show highly directional locomotion in culture, migrating at rates of up to 1 micron/s. When DC electric fields between 0.5 and 15 V/cm are applied, single epidermal cells as well as cell clusters and cell sheets migrate towards the cathode. Cell clusters and sheets break apart into single migratory cells in the upper range of these field strengths. Cell shape and morphology are unaltered when the keratocytes are guided by an electric field. Neither the spontaneous locomotion nor the electrically guided motility were found to be microtubule dependent. 1 mM La3+, 10 mM Co2+, 50 microM verapamil, and 50 microM nitrendipine (calcium channel antagonists) reversibly inhibited lamellipod formation and cell locomotion in both spontaneously migrating and electrically guided cells. Ciba-Geigy Product 28392, which stimulates the opening of calcium channels, and is a competitive inhibitor of nitrendipine, has no effect on the locomotion of keratocytes. Cell motility was also unaffected by hyperpolarizing and depolarizing (low and high K+) media. It is argued that while a tissue cell may accommodate changes in resting membrane potential without becoming more or less motile, the cell may not be able to counterbalance the effects of depolarization and hyperpolarization simultaneously. In this context, a gradient of membrane potential, which is induced by an external DC electric field, will serve as a persistent stimulus for cell locomotion.

Reactivation of organelle movements along the cytoskeletal framework of a giant freshwater ameba.

The peripheral feeding network of the giant freshwater ameba Reticulomyxa can be easily and rapidly lysed to produce an extensive, stable, and completely exposed cytoskeletal framework of colinear microtubules and microfilaments. Most of the organelles that remain attached to this framework resume rapid saltatory movements at rates of up to 20 micron/s if ATP is added. This lysed model system is also capable of other forms of motility, namely an active splaying of microtubule bundles and bulk streaming. Reactivation does not occur with other nucleoside triphosphates, requires Mg ions, is insensitive to even high concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine, is sensitive to vanadate only at concentrations of approximately 100 microM, and is inhibited by N-ethylmaleimide at concentrations greater than 100 microM. The physiology of this reactivation suggests an organelle transport motor distinct from cytoplasmic dynein and possibly the recently described kinesin. This system can serve as a model for elucidating the mechanisms of intracellular transport and, in addition, provides a unique opportunity to examine associations between microtubules and microfilaments.

Bidirectional organelle transport can occur in cell processes that contain single microtubules.

Intracellular organelle transport was studied in a new model system, the giant freshwater ameba Reticulomyxa. The ameba extends a large reticulate network of cytoplasmic strands in which various phase-dense organelles can be seen to move at a rate of up to 25 microns/s. This combined light and high voltage electron microscopic study shows that organelles move bidirectionally in even the finest network strands that contain only a single microtubule. In terms of microtubule-associated intracellular transport, this observation defines a minimum set of conditions necessary for such movement. The implications of this finding for possible models of force generation are discussed.

Evidence for an involvement of actin in the positioning and motility of centrosomes.

Cultured human polymorphonuclear leukocytes exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) spread on the substratum and undergo centrosome splitting. The two centrioles may separate by a distance of several micrometers, each being surrounded by an aster of microtubules. Here we show that the centriole/aster complexes are in constant, rapid motion through the cytoplasm, carrying with them some of the cytoplasmic granules while pushing aside others, or deforming and displacing the nucleus. An analysis of this unique motility phenomenon was undertaken. We show that intact microtubules are required for TPA-induced centrosome splitting and aster motility, but not for cell spreading. More importantly, disruption of the actin network inhibits both centrosome splitting and cell spreading, and even reverses splitting (induces convergence and fusion of asters) in polymorphonuclear leukocytes pretreated with TPA alone. These observations indicate the existence of a dynamic relationship between microtubules and actin networks and provide evidence for a role of actin in determining the position of the centrosome by way of interaction with the microtubules radiating from it.

Structural interaction of cytoskeletal components.

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament "branches" are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the "microtrabecular network" (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).

Mechanism of centrosome positioning during the wound response in BSC-1 cells.

Locomoting cells are characterized by a pronounced external and internal anterior-posterior polarity. One of the events associated with cell polarization at the onset of locomotion is a shift of the centrosome, or MTOC, ahead of the nucleus. This position is believed to be of strategic importance for directional cell movement and cell polarity. We have used BSC-1 cells at the edge of an in vitro wound to clarify the causal relationship between MTOC position and the initiation of cell polarization. We find that pronounced cell polarization (the extension of a lamellipod) can take place in the absence of MTOC repositioning or microtubules. Conversely, MTOCs will reposition even after lamellar extension and cell polarization have occurred. Repositioning requires microtubules that extend to the cell periphery and is independent of selective detyrosination of microtubules extending towards the cell front. Significantly, MTOCs maintain, or at least attempt to maintain, a position at the cell's centroid. This is most clearly demonstrated in wounded monolayers of enucleated cells where the MTOC closely follows the centroid position. We suggest that the primary response to the would is the biased extension of a lamellipod, which can occur in the absence of microtubules and MTOC repositioning. Lamellipod extension leads to a shift of the cell's centroid towards the wound. The MTOC, in an attempt to maintain a position near the cell center, will follow. This will automatically put the MTOC ahead of the nucleus in the vast majority of cells. The nucleus as a reference for MTOC position may not be as meaningful as previously thought.

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements.

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

Microtubule system of isolated fish melanophores as revealed by immunofluorescence microscopy.

The microtubule system of melanophores of the angelfish, Pterophyllum scalare, has been studied using antibodies prepared against purified porcine brain tubulin in indirect immunofluorescence microscopy. Melanophores were freed from the surrounding tissue components of isolated scales by mild enzymatic digestion and then allowed to settle on a glass cover slip. In both the dispersed and the aggregated states large numbers of fluorescent fibers are seen. The number and the astral arrangement of these fibers, which run from the central region to the periphery of the cell, are striking. The system of fluorescent fibers is replaced by diffuse fluorescence of moderate intensity after cold treatment, but is restored after rewarming the cells. Differences in the immunofluorescence profiles between cells with dispersed and aggregated pigment are discussed in relation to electron microscopic data available for this system.

Localization and organization of actin in melanophores.

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.

Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable.

Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.

Viscoelastic properties of vimentin compared with other filamentous biopolymer networks.

The cytoplasm of vertebrate cells contains three distinct filamentous biopolymers, the microtubules, microfilaments, and intermediate filaments. The basic structural elements of these three filaments are linear polymers of the proteins tubulin, actin, and vimentin or another related intermediate filament protein, respectively. The viscoelastic properties of cytoplasmic filaments are likely to be relevant to their biologic function, because their extreme length and rodlike structure dominate the rheologic behavior of cytoplasm, and changes in their structure may cause gel-sol transitions observed when cells are activated or begin to move. This paper describes parallel measurements of the viscoelasticity of tubulin, actin, and vimentin polymers. The rheologic differences among the three types of cytoplasmic polymers suggest possible specialized roles for the different classes of filaments in vivo. Actin forms networks of highest rigidity that fluidize at high strains, consistent with a role in cell motility in which stable protrusions can deform rapidly in response to controlled filament rupture. Vimentin networks, which have not previously been studied by rheologic methods, exhibit some unusual viscoelastic properties not shared by actin or tubulin. They are less rigid (have lower shear moduli) at low strain but harden at high strains and resist breakage, suggesting they maintain cell integrity. The differences between F-actin and vimentin are optimal for the formation of a composite material with a range of properties that cannot be achieved by either polymer alone. Microtubules are unlikely to contribute significantly to interphase cell rheology alone, but may help stabilize the other networks.

A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells.

Treatment of epithelial African green monkey kidney (BSC-1) cells with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid and reversible redistribution of actin and vinculin that is detectable after only 2 min of treatment. Within 20-40 min, stress fibers disappear, while at the same time large actin-containing ribbons resembling ruffles develop both at the cell periphery and in more central regions. Vinculin is associated with these actin ribbons or bands in a punctate or patchy staining pattern. Adhesion to the substratum is changed from predominantly focal contacts associated with stress fiber ends in untreated cells to broad zones of close contact after TPA treatment. High voltage electron microscopic observations disclose the ribbons to consist of highly cross-linked actin filament networks. Thus, association of vinculin with filament networks, rather than (the ends of) filament bundles, is demonstrated. The integrity of microtubules and vimentin filaments is not affected by TPA treatment, but their distribution is altered to conform with the highly distorted cell shape. The response to TPA is neither prevented nor modified by nocodazole-induced depolymerization or taxol-induced stabilization of microtubules. An intact intermediate filament network seems not required either since colcemid-induced collapse of vimentin filaments towards the nucleus does not affect the cell's response to TPA. Rapid redistribution of actin and vinculin also takes place in enucleated cells and in the presence of cycloheximide, but is prevented by dinitrophenol or oligomycin. TPA-induced cytoskeletal alterations are independent of fibronectin expression and not mimicked, modified, or prevented by calmodulin inhibitors or experimentally elevated levels of calcium and cyclic AMP. Thus the morphological response to TPA involves rapid redistribution of actin and vinculin independent of transcription and translation, fluctuations in the levels of calcium or cyclic AMP, or changes in the organization of microtubules, intermediate filaments, and fibronectin.

Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: direct measurement by fluorescence ratio imaging.

Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694-697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility.

Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses.

A cellular protein of 36,000 daltons becomes phosphorylated at tyrosine in chicken embryo fibroblasts transformed with avian sarcoma viruses. We have used cellular fractionation and immunofluorescence to locate the 36-kdalton protein in virus-transformed and uninfected chicken fibroblasts. The 36-kdalton protein in transformed cells fractionated mainly with high-speed particulate material, and in density gradient separations, the 36-kdalton protein was found in association with light density membranes together with most of the plasma membrane marker. Increasing the concentration of salt or adding ion chelators solubilized some of the 36-kdalton protein that otherwise was pelletable with high g forces. Based on these data, we conclude that this protein is peripherally or indirectly attached to light density membranes, including plasma membranes. Indirect immunofluorescent staining of the 36-kdalton protein in fixed cells revealed that it was located inside the cell in an extensive reticulum apposed to surface membranes. The same pattern of staining was found in both uninfected and virus-transformed cells. Pretreatment of cells with nonionic detergents before fixation altered or abolished 36-kdalton staining. The 36-kdalton protein appeared to be excluded from regions of the cells where actin cables were present. The pattern of staining observed with the anti-36-kdalton antibody was similar, but not identical, to that observed with antiserum against nonerythroid spectrin. Thus, the data obtained by biochemical fractionation and by immunofluorescent staining indicate that the 36-kdalton protein is found in a reticulum at the inner surface of the plasma membrane, possibly in association with cytoskeletal proteins.

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.