Microtubule Minus-End Binding Protein CAMSAP2 Controls Axon Specification and Dendrite Development

Abstract

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development.

Microtubule nucleation and organization in dendrites

“…23,24 Loss of g-tubulin or g-TuRC factors reduces microtubule polymerization frequency in both dendrites and axons of hippocampal neuron cultures and in terminal dendrite branches of Drosophila class IV sensory neurons. 34,35 Pre-existing microtubule fragments can also act as seeds. In this case nucleation occurs by binding at the b-tubulin surface on the pre-existing fragment; this event is independent of g-tubulin.…”

“…The centrosome also supports nucleation in nascent neurons 47 ; however, this activity is rapidly lost, and dendrite elaboration occurs after the centrosome has stopped functioning as a microtubule nucleation site. 34,47,48 The centrosome maintains a second function as a signal transduction hub, which also influences dendrite growth. 49 In the differentiating dendrite, microtubule nucleation and polymerization events occur away from the centrosome and throughout the growing arbor.…”

“…51 Endosomes and mitochondria, other common membrane-rich organelles in dendrites, were not found to be sites of nucleation. 35 Notably, while g-tubulin is used in both dendrites and axons to seed microtubules, 34 Golgi outposts have only been detected in the dendrites. 51 Whether there are regulatory mechanisms to control the positions of microtubule nucleation in the elongating axon is as yet unclear.…”

Microtubule Organization and Microtubule-Associated Proteins (MAPs)

“…Although the centrosome is the main nucleating point in non-polarized neurons, it loses its function as MTOC during early neuronal development, and only a few MTs emanate from the centrosome in mature neurons (Stiess et al 2010). Consistently, it has been shown that noncentrosomal MTs are abundantly present in neurons (Yau et al 2014). Recently, CAMSAP/Patronin/ Nezha family proteins have been characterized and found to specifically recognize MT minus ends and stabilize free minus ends against depolymerization (Akhmanova and Hoogenraad 2015).…”

“…By forming stable CAMSAP stretches at the MT minus ends, these short MT fragments may also serve as "seeds" for new MT regrowth. Indeed, repetitive MT plus-end growth was observed from CAMSAP2 stretches in neurons (Yau et al 2014). CAMSAP2 is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo (Yau et al 2014).…”