%0 Journal Article %J Nat Cell Biol %D 2022 %T Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours. %A Jijumon, A S %A Bodakuntla, Satish %A Genova, Mariya %A Bangera, Mamata %A Sackett, Violet %A Besse, Laetitia %A Maksut, Fatlinda %A Henriot, Veronique %A Magiera, Maria M %A Sirajuddin, Minhajuddin %A Janke, Carsten %X

The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.

%B Nat Cell Biol %V 24 %P 253-267 %8 2022 Feb %G eng %N 2 %R 10.1038/s41556-021-00825-4 %0 Journal Article %J J Cell Biol %D 2020 %T Genetically encoded live-cell sensor for tyrosinated microtubules. %A Kesarwani, Shubham %A Lama, Prakash %A Chandra, Anchal %A Reddy, P Purushotam %A Jijumon, A S %A Bodakuntla, Satish %A Rao, Balaji M %A Janke, Carsten %A Das, Ranabir %A Sirajuddin, Minhajuddin %X

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

%B J Cell Biol %V 219 %8 2020 Oct 05 %G eng %N 10 %R 10.1083/jcb.201912107