Cellular factors tightly regulate the architecture of bundles of filamentous cytoskeletal proteins, giving rise to assemblies with distinct morphologies and physical properties, and a similar control of the supramolecular organization of nanotubes and nanorods in synthetic materials is highly desirable. However, it is unknown what principles determine how macromolecular interactions lead to assemblies with defined morphologies. In particular, electrostatic interactions between highly charged polyelectrolytes, which are ubiquitous in biological and synthetic self-assembled structures, are poorly understood. We have used a model system consisting of microtubules (MTs) and multivalent cations to examine how microscopic interactions can give rise to distinct bundle phases in biological polyelectrolytes. The structure of these supramolecular assemblies was elucidated on length scales from subnanometer to micrometer with synchrotron x-ray diffraction, transmission electron microscopy, and differential interference contrast microscopy. Tightly packed hexagonal bundles with controllable diameters were observed for large trivalent, tetravalent, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of 2D dynamic assemblies of MTs with linear, branched, and loop topologies. This new bundle phase is an experimental example of nematic membranes. The morphologically distinct MT assemblies give insight into general features of bundle formation and may be used as templates for miniaturized materials with applications in nanotechnology and biotechnology.cation ͉ like-charge attraction ͉ x-ray I n this article, we present our findings on the assembly behavior of microtubules (MTs), a model nanoscale tubule. MTs are hollow, cylindrical protein polymers, with inner and outer diameters of Ϸ15 and 25 nm, respectively, involved in a variety of cellular functions, including cell division, intracellular transport, and cell morphology. MTs often assemble into arrays and bundles as in axostyles in protozoa, the cortical array in plants, the mitotic spindle, and neuronal processes (1). MT-associated proteins (MAPs) regulate the interactions between MTs, giving rise to MT bundles with various degrees of order and different MT-MT spacings, both in native biological structures such as axons and dentrites and in in vivo overexpression experiments (2). In vitro experiments show that subtle mutations in MAPs can lead to bundles with radically different structures, converting hexagonally packed bundles into linear chains of MTs (3). However, it is unclear how MAP-controlled, MT-MT interactions lead to bundles with the observed architectures.Understanding the fundamental mechanisms underlying the nature of the self-assembly of nanometer-scale tubules and rods is also important from a technological perspective. Nanotubes are currently being developed as miniaturized materials with applications as circuitry components, templates for nanosized wires and optic...

Microtubules are hollow cylinders composed of tubulin heterodimers that stack into linear protofilaments that interact laterally to form the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows they transition to rectangular bundles with noncircular buckled cross sections, followed by hexagonally packed bundles. This new technique probes the strength of interprotofilamen bonds, yielding insight into the mechanism by which associated proteins and the chemotherapy drug taxol stabilize microtubules.

Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.

Bundles of taxol-stabilized microtubules (MTs) – hollow tubules comprised of assembled αβ-tubulin heterodimers – spontaneously assemble above a critical concentration of tetravalent spermine and are stable over long times at room temperature. Here we report that at concentrations of spermine several-fold higher the MT bundles (BMT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (BITT), the outside surface of which corresponds to the inner surface of the BMT tubules. Using transmission electron microscopy and synchrotron small-angle x-ray scattering, we quantitatively determined both the nature of the BMT to BITT transformation pathway, which results from a spermine-triggered conformation switch from straight to curved in the constituent taxol-stabilized tubulin oligomers, and the structure of the BITT phase, which is formed of tubules of helical tubulin oligomers. Inverted tubulin tubules provide a platform for studies requiring exposure and availability of the inside, luminal surface of MTs to MT-targeted-drugs and MT-associated-proteins.