Hoenger Lab
University of Colorado at Boulder
Department of Molecular, Cellular and Developmental Biology

Structural and Functional Investigations
into Cytoskeletal Assemblies by
Cryo Electron Microscopy
and 3D Image Analysis

 
Research:: Microtubule Associated Proteins

Microtubules associated proteins, MAPs, mediate the dynamic processes of microtubules. MAPs stabilize and destabilize tubules, are associated with the coupling of molecular motors and microtubules, and play critical roles in nuclear spindle formation.

End Binding 1 proteins such as Malp3 binds at microtubule seams, TAU fortifies microtubules in nerve axons. HURP, NuSAP, and TPX2 are other MAPs that mediate spindle formation.
The examples above are of end binding proteins EB1, this particular example is Mal3p from yeast cells. This protein stabilizes microtubules along the microtubule lattice seam. Occasionally microtubules can be found where Malp3 aligns in two adjacent rows indicating that there are two seams side by side. Most likely there are adjacent to one another as modeled in H. A 13 protofilament microtubule would need to compensate this double seam with a third seam as shown above.
High-resolution model of the kinesin–tubu­lin complex in ribbon representation and the proposed major interaction site of htau40 (red wire frame). A, View from outside the MT cylinder. B, Tangential view from a point between two protofilaments. According to cross-correlation studies with reconstructions obtained from MTs decorated with kinesin, the major tau-binding site locates to tubulin, in close proximity to helices 11 and 12. However, the volume indicated in red accounts for less than 5% of the mass of htau40, the remainder is distributed non-periodically. The exposed position of this site, near the outer protofilament ridge and almost between two protofilaments, suggests that tau may also bind across protofilaments (see also Figures 5 and 6B).

 

Selected Publications:

 

NuSAP, a Mitotic RanGTP Target That Stabilizes and Cross-links Microtubules (pdf)

Mol Biol Cell. 2006 Jun;17(6):2646-60

Ribbeck K, Groen AC, Santarella R, Bohnsack MT, Raemaekers T, Kocher T, Gentzel M, Gorlich D, Wilm M, Carmeliet G, Mitchison TJ, Ellenberg J, Hoenger, A, Mattaj IW.

Abstract

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) α, Impβ, and Imp7. While Impα and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impβ specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.

The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam

Cell. 2006 Dec 29;127(7):1415-24.

Sandblad L, Busch KE, Tittmann P, Gross H, Brunner D, Hoenger A.

Abstract

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

HURP wraps microtubule ends with an additional tubulin sheet that has a novel conformation of tubulin.

J Mol Biol. 2007 Feb 2;365(5):1587-95.

Santarella RA, Koffa MD, Tittmann P, Gross H, Hoenger A.

Abstract

HURP is a newly discovered microtubule-associated protein (MAP) required for correct spindle formation both in vitro and in vivo. HURP protein is highly charged with few predicted secondary and tertiary folding domains. Here we explore the effect of HURP on pure tubulin, and describe its ability to induce a new conformation of tubulin sheets that wrap around the ends of intact microtubules, thereby forming two concentric tubes. The inner tube is a normal microtubule, while the outer one is a sheet composed of tubulin protofilaments that wind around the inner tube with a 42.5 degrees inclination. We used cryo-electron microscopy and unidirectional surface shadowing to elucidate the structure and conformation of HURP-induced tubulin sheets and their interaction with the inner microtubule. These studies clarified that HURP-induced sheets are composed of anti-parallel protofilaments exhibiting P2 symmetry. HURP is a unique MAP that not only stabilizes and bundles microtubules, but also polymerizes free tubulin into a new configuration.

Surface-decoration of microtubules by human tau

J Mol Biol. 2004 Jun 4;339(3):539-53.

Santarella RA, Skiniotis G, Goldie KN, Tittmann P, Gross H, Mandelkow EM, Mandelkow E, Hoenger A.

Abstract

Tau is a neuronal, microtubule-associated protein that stabilizes microtubules and promotes neurite outgrowth. Tau is largely unfolded in solution and presumably forms mostly random coil. Because of its hydrophilic nature and flexible structure, tau complexed to microtubules is largely invisible by standard electron microscopy methods. We applied a combination of high-resolution metal-shadowing and cryo-electron microscopy to study the interactions between tau and microtubules. We used recombinant tau variants with different domain compositions, (1) full length tau, (2) the repeat domain that mediates microtubule binding (K19), and (3) two GFP-tau fusion proteins that contain a globular marker (GFP) attached to full-length tau at either end. All of these constructs bind exclusively to the outside of microtubules. Most of the tau-related mass appears randomly distributed, creating a "halo" of low-density mass spread across the microtubule surface. Only a small fraction of tau creates a periodic signal at an 8 nm interval, centered on alpha-tubulin subunits. Our data suggest that tau retains most of its disordered structure even when bound to the microtubule surface. Hence, it binds along, as well as across protofilaments. Nevertheless, even minute concentrations of tau have a strong stabilizing effect and effectively scavenge unpolymerized tubulin.

 

Importin α-regulated nucleation of microtubules by TPX2 (pdf)

EMBO J. 2003 May 1;22(9):2060-70

Christoph A. Schatz, Rachel Santarella, Andreas Hoenger, Eric Karsenti, Iain W. Mattaj,a Oliver J. Gruss, and Rafael E. Carazo-Salasa

Abstract

The importin α-regulated microtubule-associated protein TPX2 is known to be critical for meiotic and mitotic spindle formation in vertebrates, but its detailed mechanism of action and regulation is not understood. Here, the site of interaction on TPX2 for importin α is mapped. A TPX2 mutant that cannot bind importin α is constitutively active in the induction of microtubule-containing aster-like structures in Xenopus egg extract, demonstrating that no other importin α or RanGTPase target is required to mediate microtubule assembly in this system. Further, recombinant TPX2 is shown to induce the formation and bundling of microtubules in dilute solutions of pure tubulin. In this purified system, importin α prevents TPX2-induced microtubule formation, but not TPX2–tubulin interaction or microtubule bundling. This demonstrates that TPX2 has more than one mode of interaction with tubulin and that only one of these types of interaction is abolished by importin α. The data suggest that the critical early function in spindle formation regulated by importin α is TPX2-mediated microtubule nucleation.
 

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