RESUMEN
Fidgetin, a microtubule-severing enzyme, regulates neurite outgrowth, axonal regeneration, and cell migration by trimming off the labile domain of microtubule polymers. Because maintenance of the microtubule labile domain is essential for axon initiation, elongation, and navigation, it is of interest to determine whether augmenting the microtubule labile domain via depletion of fidgetin serves as a therapeutic approach to promote axonal regrowth in spinal cord injury. In this study, we constructed rat models of spinal cord injury and sciatic nerve injury. Compared with spinal cord injury, we found that expression level of tyrosinated microtubules in the labile portion of microtubules continuously increased, whereas fidgetin decreased after peripheral nerve injury. Depletion of fidgetin enhanced axon regeneration after spinal cord injury, whereas expression level of end binding protein 3 (EB3) markedly increased. Next, we performed RNA interference to knockdown EB3 or fidgetin. We found that deletion of EB3 did not change fidgetin expression. Conversely, deletion of fidgetin markedly increased expression of tyrosinated microtubules and EB3. Deletion of fidgetin increased the amount of EB3 at the end of neurites and thereby increased the level of tyrosinated microtubules. Finally, we deleted EB3 and overexpressed fidgetin. We found that fidgetin trimmed tyrosinated tubulins by interacting with EB3. When fidgetin was deleted, the labile portion of microtubules was elongated, and as a result the length of axons and number of axon branches were increased. These findings suggest that fidgetin can be used as a novel therapeutic target to promote axonal regeneration after spinal cord injury. Furthermore, they reveal an innovative mechanism by which fidgetin preferentially severs labile microtubules.
RESUMEN
Microtubule (MT) organization is essential for many cellular events, including mitosis, migration, and cell polarity. Fidgetin (Fign), an ATP-dependent, MT-severing protein, contributes to the regulation of MT configuration by cutting and trimming MT polymers. Functions of Fign have been indicated in neurite outgrowth, mitosis, meiosis, and cellular migration. Here we focus on migration of astrocytes. We find that Fign plays an essential role in cultured astrocyte migration by preferentially targeting MTs (or regions of MTs) that are rich in tyrosinated tubulin, a marker for especially dynamic MTs or especially dynamic regions of MTs. Inhibition of cellular migration induced by Fign knockdown can be rescued with concomitant knockdown of kinesin-12, a motor protein best known for its role in mitosis. We propose a novel working model for MT reconfiguration underlying cellular migration elicited by the functional cooperation of two distinct MT-related proteins.
