Efficient axonal transport of endolysosomes relies on the balanced ratio of microtubule tyrosination and detyrosination.

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.

Regulation of microtubule detyrosination by Ca2+ and conventional calpains.

Detyrosination is a major post-translational modification of microtubules (MTs), which has significant impact on MT function in cell division, differentiation, growth, migration and intracellular trafficking. Detyrosination of α-tubulin occurs mostly via the recently identified complex of vasohibin 1 or 2 (VASH1 and VASH2, respectively) with small vasohibin binding protein (SVBP). However, there is still remaining detyrosinating activity in the absence of VASH1 and/or VASH2 and SVBP, and little is known about the regulation of detyrosination. Here, we found that intracellular Ca2+ is required for efficient MT detyrosination. Furthermore, we show that the Ca2+-dependent proteases calpains 1 and 2 (CAPN1 and CAPN2, respectively) regulate MT detyrosination in VASH1- and SVBP-overexpressing human embryonic kidney (HEK293T) cells. We identified new calpain cleavage sites in the N-terminal disordered region of VASH1. However, this cleavage did not affect the enzymatic activity of vasohibins. In conclusion, we suggest that the regulation of VASH1-mediated MT detyrosination by calpains could occur independently of vasohibin catalytic activity or via another yet unknown tubulin carboxypeptidase. Importantly, the Ca2+ dependency of calpains could allow a fine regulation of MT detyrosination. Thus, identifying the calpain-regulated pathway of MT detyrosination can be of major importance for basic and clinical research.

Direct and indirect effects of tubulin post-translational modifications on microtubule stability: Insights and regulations.

Microtubules (MTs) mediate various cellular functions such as structural support, chromosome segregation, and intracellular transport. To achieve this, the pivotal properties of MTs have to be changeable and tightly controlled. This is enabled by a high variety of tubulin posttranslational modifications, which influence MT properties directly, via altering the MT lattice structurally, or indirectly by changing MT interaction partners. Here, the distinction between these direct and indirect effects of MT PTMs are exemplified by acetylation of the luminal α-tubulin K40 resulting in decreased rigidity of MTs, and by MT detyrosination which decreases interaction with depolymerizing proteins, thus causing more stable MTs. We discuss how these PTMs are reversed and regulated, e.g. on the level of enzyme transcription, localization, and activity via various signalling pathways including the conventional calcium-dependent proteases calpains and how advances in microscopy techniques and development of live-sensors facilitate the understanding of MT PTM interaction and effects.

The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domain.

In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our in vitro results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.