Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast

Using a monoclonal antibody (MAb1.1ASML) raised against a surface glycoprotein of the metastasizing rat pancreatic carcinoma cell line BSp73ASML, cDNA clones have been isolated that encode glycoproteins with partial homology to CD44, a presumed adhesion molecule. In one of the clones, pMeta-1, the epitope marks an additional extracellular domain of 162 amino acids inserted into the rat CD44 protein between amino acid positions 223 and 247 (by analogy to human and murine CD44). The new variants are expressed only in the metastasizing cell lines of two rat tumors, the pancreatic carcinoma BSp73 and the mammary adenocarcinoma 13762NF; they are not expressed in the non-metastasizing tumor cell lines nor in most normal rat tissues. Overexpression of pMeta-1 in the nonmetastasizing BSp73AS cells suffices to establish full metastatic behavior.

XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.C ells assemble and disassemble actin filaments and microtubules to carry out a vast array of functions, such as defining cell shape, directing cellular movement, and mediating chromosome segregation and cell division. Although these polymeric filaments have different structures and display different dynamics, the cell regulates their assembly and disassembly in related ways. Polymer growth is polar in both cases and occurs at the plus ends of microtubules and the barbed ends of actin filaments. Both polymers have specific nucleating proteins, assemble with the help of polymerases, and disassemble with the aid of severing proteins and depolymerases (1-4). How these various activities coordinate to create the cytoskeleton is a central question in cell biology (5). This work focuses on assembly.The main promoters of polymer growth are the XMAP215/Dis family for microtubules and the formins for actin (4,(6)(7)(8)(9). The function of formins in actin polymerization is well characterized. Formins have two key domains that are important for their activity, FH1 and FH2 (8,10). While the FH2 domain is necessary for binding to the barbed end of actin, repeats of polyproline in the FH1 domain are required to interact with actin/profilin complexes and recruit them to the barbed end (4,11,12).Much less is known about how the regions of XMAP215 coordinate in promoting microtubule growth (13). Recent work has shown that XMAP215 acts as a classic catalyst (14). At physiological tubulin concentrations, XMAP215 is a tubulin polymerase that promotes incorporation of tubulin into the growing plus end. However, in the absence of free tubulin, XMAP215 accelerates depolymerization of GMPCPP-stabili...

Metaphase spindles are microtubule-based structures that use a multitude of proteins to modulate their morphology and function. Today, we understand many details of microtubule assembly, the role of microtubule-associated proteins, and the action of molecular motors. Ultimately, the challenge remains to understand how the collective behaviour of these nanometre-scale processes gives rise to a properly sized spindle on the micrometre scale. By systematically engineering the enzymatic activity of XMAP215, a processive microtubule polymerase, we show that Xenopus laevis spindle length increases linearly with microtubule growth velocity, whereas other parameters of spindle organization, such as microtubule density, lifetime and spindle shape, remain constant. We further show that mass balance can be used to link the global property of spindle size to individual microtubule dynamic parameters. We propose that spindle length is set by a balance of non-uniform nucleation and global microtubule disassembly in a liquid-crystal-like arrangement of microtubules.