Abstract. We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37°C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 I.tM.Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 ~tM -t s -t for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s -t. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 lxM. The rate of rapid shortening was similar at the two ends (plus = 733 s-t; minus = 915 s-l), and did not vary with tubulin concentration.Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.T HE term "dynamic instability" describes microtubule assembly in which individual microtubules exhibit alternating phases of elongation and rapid shortening. Transitions between these phases are abrupt, stochastic, and infrequent in comparison to the rates of tubulin association and dissociation at the microtubule ends (17, 28). Substantial evidence has accumulated to indicate that dynamic instability is the basic mechanism of microtubule assembly in vitro (17, 28), and in both the mitotic spindle and the cyoplasmic microtubule complex (CMTC) l (10,11,34,35,37). Although microtubule dynamics within the cell may be regulated by various microtubule-associated proteins (MAPs) and other intracellular regulatory molecules, it is important 1. Abbreviations used in this paper: CMTC, cytoplasmic microtubule complex; DIC, differential interference contrast; MAP(s), microtubule-associated protein(s).first to understand the details of the inherent behavior of microtubules assembled from tubulin alone.Th...

An important part of characterizing any protein molecule is to determine its size and shape. Sedimentation and gel filtration are hydrodynamic techniques that can be used for this medium resolution structural analysis. This review collects a number of simple calculations that are useful for thinking about protein structure at the nanometer level. Readers are reminded that the Perrin equation is generally not a valid approach to determine the shape of proteins. Instead, a simple guideline is presented, based on the measured sedimentation coefficient and a calculated maximum S, to estimate if a protein is globular or elongated. It is recalled that a gel filtration column fractionates proteins on the basis of their Stokes radius, not molecular weight. The molecular weight can be determined by combining gradient sedimentation and gel filtration, techniques available in most biochemistry laboratories, as originally proposed by Siegel and Monte. Finally, rotary shadowing and negative stain electron microscopy are powerful techniques for resolving the size and shape of single protein molecules and complexes at the nanometer level. A combination of hydrodynamics and electron microscopy is especially powerful.

SUMMARY FtsZ, a bacterial homolog of tubulin, is well established as forming the cytoskeletal framework for the cytokinetic ring. Recent work has shown that purified FtsZ, in the absence of any other division proteins, can assemble Z rings when incorporated inside tubular liposomes. Moreover, these artificial Z rings can generate a constriction force, demonstrating that FtsZ is its own force generator. Here we review light microscope observations of how Z rings assemble in bacteria. Assembly begins with long-pitch helices that condense into the Z ring. Once formed, the Z ring can transition to short-pitch helices that are suggestive of its structure. FtsZ assembles in vitro into short protofilaments that are ∼30 subunits long. We present models for how these protofilaments might be further assembled into the Z ring. We discuss recent experiments on assembly dynamics of FtsZ in vitro, with particular attention to how two regulatory proteins, SulA and MinC, inhibit assembly. Recent efforts to develop antibacterial drugs that target FtsZ are reviewed. Finally, we discuss evidence of how FtsZ generates a constriction force: by protofilament bending into a curved conformation.

We have determined the 2.0 A crystal structure of a fragment of human fibronectin encompassing the seventh through the RGD-containing tenth type III repeats (FN7-10). The structure reveals an extended rod-like molecule with a long axis of approximately 140 A and highly variable relationships between adjacent domains. An unusually small rotation between domains 9 and 10 creates a distinctive binding site, in which the RGD loop from domain 10 and the "synergy" region from domain 9 are on the same face of FN7-10 and thus easily accessible to a single integrin molecule. The cell-binding RGD loop is well-ordered in this structure and extends approximately 10 A away from the FN7-10 core.

Extracellular matrix proteins are thought to provide a rigid mechanical anchor that supports and guides migrating and rolling cells. Here we examine the mechanical properties of the extracellular matrix protein tenascin by using atomic-force-microscopy techniques. Our results indicate that tenascin is an elastic protein. Single molecules of tenascin could be stretched to several times their resting length. Force-extension curves showed a saw-tooth pattern, with peaks of force at 137pN. These peaks were approximately 25 nm apart. Similar results have been obtained by study of titin. We also found similar results by studying recombinant tenascin fragments encompassing the 15 fibronectin type III domains of tenascin. This indicates that the extensibility of tenascin may be due to the stretch-induced unfolding of its fibronectin type III domains. Refolding of tenascin after stretching, observed when the force was reduced to near zero, showed a double-exponential recovery with time constants of 42 domains refolded per second and 0.5 domains per second. The former speed of refolding is more than twice as fast as any previously reported speed of refolding of a fibronectin type III domain. We suggest that the extensibility of the modular fibronectin type III region may be important in allowing tenascin-ligand bonds to persist over long extensions. These properties of fibronectin type III modules may be of widespread use in extracellular proteins containing such domain.

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation.

The bacterial cell division protein FtsZ is a homolog of tubulin, but it has not been determined whether FtsZ polymers are structurally related to the microtubule lattice. In the present study, we have obtained high-resolution electron micrographs of two FtsZ polymers that show remarkable similarity to tubulin polymers. The first is a twodimensional sheet of protofilaments with a lattice very similar to that of the microtubule wall. The second is a miniring, consisting of a single protofilament in a sharply curved, planar conformation. FtsZ minirings are very similar to tubulin rings that are formed upon disassembly of microtubules but are about half the diameter. This suggests that the curved conformation occurs at every FtsZ subunit, but in tubulin rings the conformation occurs at either f3-or a-tubulin subunits but not both. We conclude that the functional polymer of FtsZ in bacterial cell division is a long thin sheet of protofilaments. There is sufficient FtsZ in Escherichia coli to form a protofilament that encircles the cell 20 times. The similarity of polymers formed by FtsZ and tubulin implies that the protofilament sheet is an ancient cytoskeletal system, originally functioning in bacterial cell division and later modified to make microtubules.FtsZ is an abundant bacterial protein essential for cell division in all bacterial species investigated (1). Outside bacteria, FtsZ has recently been discovered in Arabidopsis as a nuclearencoded gene that is transported into chloroplasts (2). A second Arabidopsis FtsZ (partial) sequence is in the dbEST data base (GenBank accession no. Z48464). FtsZ may form the cytoskeletal framework for cell division in all bacteria, as well as chloroplasts and mitochondria.FtsZ has been identified as a homolog of tubulin on the basis of limited but convincing sequence similarity and its binding and hydrolysis of reviewed in ref. 6). Two laboratories have reported assembly of FtsZ into protofilaments and into larger rods or tubular polymers (7,8). The subunit lattice of these polymers was not clearly resolved but appeared different from that of microtubules. Thus, the structural relation of FtsZ polymers to tubulin remained unclear.The most important place to look for structural similarity is at the level of the subunit lattice because it is this lattice that is dictated by the repeating protein-protein contacts. The gross structure of the polymer is less important as an indicator of conserved structure. An open microtubule wall is shown in Fig. IA, and a computer-reconstructed image of the subunit lattice is shown in Fig. 2A. The two-dimensional (2D) lattice consists of subunits, alternating a-and ,3-tubulin, in a parallel array of longitudinal protofilaments. Subunits are 4.0 nm apart along the protofilament, and the protofilaments are 5.1 nm apart laterally (9). If FtsZ polymers are structurally homologous to a microtubule, we should expect to see long, straightThe publication costs of this article were defrayed in part by page charge payment. This article must ther...

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