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

Stangier, Marcel. Structural and biophysical investigation of +TIPs in yeast and -TIPs in higher eukaryotes. 2018, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_12977

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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.
Advisors:Steinmetz, Michel and Maier, Timm
Faculties and Departments:05 Faculty of Science > Departement Chemie > Former Organization Units Chemistry > Physikalische Chemie (Maier)
UniBasel Contributors:Maier, Timm
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12977
Thesis status:Complete
Number of Pages:1 Online-Ressource (VIII, 176 Seiten)
Identification Number:
edoc DOI:
Last Modified:01 Jan 2022 02:30
Deposited On:25 Mar 2019 13:55

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