Structural and biophysical investigation of +TIPs in yeast and -TIPs in higher eukaryotes

In eukaryotic cells, microtubules represent a highly dynamic protein filament system that is involved in cellular processes as cell division or transport of cargo. Microtubules oscillate between growth and shrinking, and the switch between these states is caused by catastrophe and rescue events. The building block of microtubules is the heterodimer tubulin, which polymerizes into tubular structures and switches from a curved state in the soluble form to a straight state in microtubules. Due to the polarity of tubulin, microtubules feature a plus-end and a minus-end. The highly dynamic plus-end is regulated by the plus-end tracking proteins (+TIPs). Certain +TIPs can function as a microtubule polymerase or rescue shrinking microtubules. Since budding yeast contains only a small number of microtubules, this organism is predestinated to study +TIPs and microtubule dynamics by microscopy on the system level. The exact function and mechanism of yeast +TIPs such as Bik1 remain unresolved. In addition, it is unexplained how kinesins such as Kip2 or Kip3 can act as a microtubule polymerase or rescue factor. In my thesis, the budding yeast +TIPs Bik1, Kip2 and Kip3 were investigated to understand the role of these proteins in the formation of the +TIP network and how these proteins are capable of influencing microtubule dynamics. Recently, it has been discovered that minus-end tracking proteins (-TIPs) recognize the minus-end in cells such as neuronal cells. However, it is enigmatic how -TIPs target the microtubule minus-end. In order to elucidate the mechanism how -TIPs track the minus-end, my work focused on the discovered first -TIP class of CAMSAPs. In all projects, biophysical methods were applied, and besides for Kip2 crystal structures were determined to unravel mechanistic details of the proteins. In budding yeast, Bik1 plays an important role especially in the dynein pathway, which is one of two major pathways for spindle positioning. Bim1 localizes Bik1 to the microtubule plus-end because Bik1 cannot autonomously track the plus-end. Here, we biophysically and structurally describe the interaction of the Bik1 CAP-Gly domain with the C-terminal tail of the +TIP Bim1. The crystal structure of the complex showed that Bik1 CAP-Gly binds specifically to C-terminal phenylalanine residues with a different binding mode compared to CAP-Gly domains of higher eukaryotes. Based on the structure, two different mutants were conceived to perturb the Bik1-Bim1 interaction. Then, the effect of this perturbation on Bik1 localization, microtubule length and Kar9 function was analyzed in yeast cells. Besides, we proved that the coiled-coil of Bik1 interacts with the C-terminal tail of microtubule polymerase Stu2, establishing Bik1 as an adaptor protein between Bim1 and Stu2. Apart from Bim1, the budding yeast kinesin Kip2 also has the ability to transport Bik1 to the plusend. We biophysically characterized the interaction of the Bik1 coiled-coil with the Kip2 coiledcoil. The C-terminal unstructured part of Kip2 turned out to be essential for the Bik1-Kip2 interaction, allowing an elegant way to disrupt this interaction without removing the Kip2 coiledcoil. In addition, Kip2 functions as a microtubule polymerase. By studying the interaction of the Kip2 motor domain with soluble tubulin, we were able to postulate a mechanism how Kip2 can polymerize microtubules. Furthermore, we identified the importance of the Bik1-Kip2 interaction for the polymerase activity. The budding yeast kinesin Kip3 can depolymerize microtubules but exhibits the ability to rescue them as well. The N-terminal motor domain of Kip3 is responsible for the depolymerization activity. We discovered that Kip3 possesses a C-terminal tubulin-binding domain (TBD), followed by a weak microtubule-binding domain. The crystal structure of the Kip3 TBD was solved, and a sophisticated assembly of alpha-helices was revealed. Furthermore, the combination of the Kip3 motor domain together with the Kip3 TBD was identified as the minimal construct that can rescue microtubules. Therefore, we proposed that the Kip3 motor domain can also act as an anchor at the microtubule plus-end so that the Kip3 TBD can fulfill its rescue function by either increasing the tubulin concentration or facilitating the exchange of tubulin. Most microtubules minus-ends are attached to the centrosome. However, some microtubules can occur with free minus-ends because not all microtubules are attached to the centrosome or cells such as neuronal cells entirely lack the centrosome. Thus, -TIPs like CAMSAPs can stabilize these free minus-ends. CAMSAP proteins have a CKK domain that can autonomously track the microtubule minus-end. In this study, we determined the crystal structure of this CKK domain. Our collaborator used this structure for fitting into a cryo-EM map of microtubules decorated by the CKK domain. Combined with other experimental results, we found that the CKK domain recognizes a unique curved state of tubulin that only occurs at the microtubule minus-end. Overall, important insights into the mechanisms of Bik1 Kip2, Kip3 and CAMSAP were obtained. In the +TIP network, the understanding of Bik1 as a critical adaptor protein was considerably increased. Furthermore, we revealed new insights into the function of Kip2 as a microtubule polymerase. For Kip3, a mechanism for its microtubule rescue function was postulated. In the case of CAMSAP, it was discovered how this protein can recognize the microtubule minus-end. This represents the first described mechanism of a -TIP

Remote control of microtubule plus-end dynamics and function from the minus-end

In eukaryotes, the organization and function of the microtubule cytoskeleton depend on the allocation of different roles to individual microtubules. For example, many asymmetrically dividing cells differentially specify microtubule behavior at old and new centrosomes. Here we show that yeast spindle pole bodies (SPBs, yeast centrosomes) differentially control the plus-end dynamics and cargoes of their astral microtubules, remotely from the minus-end. The old SPB recruits the kinesin motor protein Kip2, which then translocates to the plus-end of the emanating microtubules, promotes their extension and delivers dynein into the bud. Kip2 recruitment at the SPB depends on Bub2 and Bfa1, and phosphorylation of cytoplasmic Kip2 prevents random lattice binding. Releasing Kip2 of its control by SPBs equalizes its distribution, the length of microtubules and dynein distribution between the mother cell and its bud. These observations reveal that microtubule organizing centers use minus to plus-end directed remote control to individualize microtubule function.ISSN:2050-084

The motor domain of the kinesin Kip2 promotes microtubule polymerization at microtubule tips

Kinesins are microtubule-dependent motor proteins, some of which moonlight as microtubule polymerases, such as the yeast protein Kip2. Here, we show that the CLIP-170 ortholog Bik1 stabilizes Kip2 at microtubule ends where the motor domain of Kip2 promotes microtubule polymerization. Live-cell imaging and mathematical estimation of Kip2 dynamics reveal that disrupting the Kip2-Bik1 interaction aborts Kip2 dwelling at microtubule ends and abrogates its microtubule polymerization activity. Structural modeling and biochemical experiments identify a patch of positively charged residues that enables the motor domain to bind free tubulin dimers alternatively to the microtubule shaft. Neutralizing this patch abolished the ability of Kip2 to promote microtubule growth both in vivo and in vitro without affecting its ability to walk along microtubules. Our studies suggest that Kip2 utilizes Bik1 as a cofactor to track microtubule tips, where its motor domain then recruits free tubulin and catalyzes microtubule assembly.ISSN:0021-9525ISSN:1540-814

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends

Author Correction: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

Correction to: Nature Structural and Molecular Biology https://doi-org.proxy.library.uu.nl/10.1038/nsmb.3483, published online 9 October 2017

Author Correction: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

Correction to: Nature Structural and Molecular Biology https://doi-org.proxy.library.uu.nl/10.1038/nsmb.3483, published online 9 October 2017

A structural model for microtubule minus-end recognition and protection by CAMSAP proteins

CAMSAP and Patronin family members regulate microtubule minus-end stability and localization and thus organize noncentrosomal microtubule networks, which are essential for cell division, polarization and differentiation. Here, we found that the CAMSAP C-terminal CKK domain is widely present among eukaryotes and autonomously recognizes microtubule minus ends. Through a combination of structural approaches, we uncovered how mammalian CKK binds between two tubulin dimers at the interprotofilament interface on the outer microtubule surface. In vitro reconstitution assays combined with high-resolution fluorescence microscopy and cryo-electron tomography suggested that CKK preferentially associates with the transition zone between curved protofilaments and the regular microtubule lattice. We propose that minus-end-specific features of the interprotofilament interface at this site serve as the basis for CKK's minus-end preference. The steric clash between microtubule-bound CKK and kinesin motors explains how CKK protects microtubule minus ends against kinesin-13-induced depolymerization and thus controls the stability of free microtubule minus ends