Tubulin modifications and cytoskeleton dynamics
Final Report Abstract
Except for the detyrosination/tyrosination of α-tubulin, post-translational modifications of tubulin remain poorly understood in respect to their contribution to microtubule function and physiological significance. The tubulin tyrosination cycle involves the enzymatic removal of the encoded C-terminal tyrosine of α-tubulin by a so far unknown tubulin carboxypeptidase and its re-addition by the tubulin tyrosine ligase (TTL). Based on our finding that TTL-deficient mice die immediately after birth due to severe disorganization of the brain, we investigated conventional and conditional TTL knockout mice and studied the consequences of TTL deficiency in various tissues and in pre- and post-mitotic cells. Furthermore, by focusing on microtubule plus-end tracking protein (+TIP) complexes, we aimed to identify proteins and protein complexes that are involved in the dynamics of the microtubule cytoskeleton and influence the interplay/crosstalk between the actin and the microtubule systems at the cell cortex. Overexpression of fluorescent-labeled tubulin followed by real-time video microscopy was used to analyze “standard” microtubule dynamics in wild type and TTL-deficient MEFs. Socalled history plots were generated from single microtubules which were used to determine the time that microtubules spent in growth, shrinking and pausing phases, growth- and shrinking speed as well as catastrophe- and rescue frequencies. In short, our results underscore the idea that detyrosinated microtubules are more stable, since microtubules in TTL-deficient cells spent more time in pausing and less time in growing as compared to wild type cells, 55.9% to 35.6% and 33.6% to 57.3%, respectively. Furthermore, microtubules in TTL-deficient cells showed higher rescue- and lower catastrophe frequencies than wild type cells, corroborating the view of higher stability of detyrosinated microtubules versus tyrosinated microtubules. Multiple mouse lines in which TTL expression is ablated were generated. To analyse the influence of TTL activity in vivo, we used the Cre/lox strategy for targeting the TTL gene followed by spatial and temporal deletion in different cell types. In contrast to the conventional TTL knock-out mouse, TTL deletion outside the brain, by using the Mx1-Cre mouse line, did not result in an obvious phenotype. Also TTL ablation in oligodendrocytes did not reveal obvious phenotypes. In contrast, using NexCre-reporter mice, a time resolved loss of tyrosinated α-tubulin in projecting neurons resulted in a developmental arrest of axonal processes and failure in cortical layer formation. Although TTL was absent in the cortex and the hippocampus, these mice were able form a normal thalamocortical loop, showing that extrinsic signals most probably derived from the thalamus are able to overcome growth insufficiencies of TTL-deficient cortical neurons. However, these mice showed reduced commissural fibre formation and defects in cortical layering revealing the necessity of TTL in axonal outgrowth and neuronal migration in vivo. Furthermore, we could show differences in axonal outgrowth mechanisms between in vitro cultured neurons and neurogenesis in vivo. Finally, we could reveal indications that kinesin1-mediated anterograde cargo transport is enhanced in cortical neuronal cells deficient for TTL. Our results suggest that kinesin1acts as a down-stream effector in the observed TTL knock-out phenotypes. A strategy combining in vitro microtubule polymerization and highly accurate mass spectrometry has been established and is now applied to cell lysates and different tissues derived from wild type and TTL-deficient mice.
Publications
- (2006) Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends. J Cell Biol 174: 839-849
Peris L, Thery M, Faure J, Saoudi Y, Lafanechere L, Chilton JK, Gordon-Weeks P, Galjart N, Bornens M, Wordeman L, Wehland J, Andrieux A, Job D
(See online at https://doi.org/10.1083/jcb.200512058) - (2010) History-dependent catastrophes regulate axonal microtubule behavior. Curr Biol 20: 1023-1028
Stepanova T, Smal I, van Haren J, Akinci U, Liu Z, Miedema M, Limpens R, van Ham M, van der Reijden M, Poot R, Grosveld F, Mommaas M, Meijering E, Galjart N
(See online at https://doi.org/10.1016/j.cub.2010.04.024) - (2010) N-WASP is a novel regulator of hair-follicle cycling that controls antiproliferative TGF{beta} pathways. J Cell Sci 123: 128-140
Lefever T, Pedersen E, Basse A, Paus R, Quondamatteo F, Stanley AC, Langbein L, Wu X, Wehland J, Lommel S, Brakebusch C
(See online at https://doi.org/10.1242/jcs.053835)
