CAMSAPs organize an acentrosomal microtubule network from basal varicosities in radial glial cells

Abstract

Neurons of the neocortex are generated by stem cells called radial glial cells. These polarized cells extend a short apical process toward the ventricular surface and a long basal fiber that acts as a scaffold for neuronal migration. How the microtubule cytoskeleton is organized in these cells to support long-range transport is unknown. Using subcellular live imaging within brain tissue, we show that microtubules in the apical process uniformly emanate for the pericentrosomal region, while microtubules in the basal fiber display a mixed polarity, reminiscent of the mammalian dendrite. We identify acentrosomal microtubule organizing centers localized in varicosities of the basal fiber. CAMSAP family members accumulate in these varicosities, where they control microtubule growth. Double knockdown of CAMSAP1 and 2 leads to a destabilization of the entire basal process. Finally, using live imaging of human fetal cortex, we reveal that this organization is conserved in basal radial glial cells, a related progenitor cell population associated with human brain size expansion.

γ-TuRCs are required for asymmetric microtubule nucleation from the somatic Golgi of Drosophila neurons

“…Strikingly, while depletion of Grip91 GCP3 does not prevent microtubule nucleation from the Golgi, it does randomise the direction in which microtubules initially grow from the Golgi (discussed further below). The ability of microtubules to originate from the Golgi in the apparent absence of γ-TuRCs was somewhat surprising to us, but there is accumulating evidence that microtubules can be nucleated independently of γ-TuRCs both in vitro and in vivo (Coquand et al, 2021;Flor-Parra et al, 2018;Hannak et al, 2002;Hussmann et al, 2016;King et al, 2020;Llamazares et al, 1999;Nashchekin et al, 2016;Popov et al, 2002;Roostalu et al, 2015;Sampaio et al, 2001;Slep and Vale, 2007;Tanaka et al, 2012;Thawani et al, 2018;Tsuchiya and Goshima, 2021;Wang et al, 2015;Wieczorek et al, 2015;Woodruff et al, 2017;Zheng et al, 2020).…”

“…Similar varicosities have been observed in class I Drosophila da neurons (Mukherjee et al, 2020), but these are filled with γ-tubulin and other centrosomal proteins and it remains to be determined if they organise microtubules. Disrupting Augmin/HAUS-mediated microtubule nucleation in hippocampal neurons and CAMSAP-mediated microtubule organisation in radial glial cells disrupts microtubule polarity (Coquand et al, 2021;Cunha-Ferreira et al, 2018;Qu et al, 2019;Sánchez-Huertas et al, 2016). In summary, various modes of microtubule nucleation have been discovered both across species and within the same neuron of the same species and play a role, to differing degrees, in regulating microtubule polarity within neurons.…”

“…Despite a clear role for γ-TuRCs in microtubule nucleation, other microtubule associated proteins, particularly TOG-domain and CAMSAP proteins, can be important for efficient microtubule nucleation and can also promote microtubule nucleation independently of γ-TuRCs both in vitro and in vivo (Coquand et al, 2021;Flor-Parra et al, 2018;Hannak et al, 2002;Hussmann et al, 2016;King et al, 2020;Llamazares et al, 1999;Nashchekin et al, 2016;Popov et al, 2002;Roostalu et al, 2015;Sampaio et al, 2001;Slep and Vale, 2007;Tanaka et al, 2012;Thawani et al, 2018;Tsuchiya and Goshima, 2021;Wang et al, 2015;Wieczorek et al, 2015;Woodruff et al, 2017;Zheng et al, 2020). These proteins bind directly to a/b-tubulin dimers and can either catalyse their assembly into microtubule polymers or stabilise nascent microtubule polymers.…”

“…These proteins bind directly to a/b-tubulin dimers and can either catalyse their assembly into microtubule polymers or stabilise nascent microtubule polymers. The extent to which γ-TuRC-independent nucleation occurs within cells remains unclear, but examples of γ-TuRC independent nucleation occurring both naturally (Coquand et al, 2021;Nashchekin et al, 2016;Tanaka et al, 2012;Wang et al, 2015;Zheng et al, 2019) and after γ-TuRC depletion (Hannak et al, 2002;Llamazares et al, 1999;Sampaio et al, 2001;Tsuchiya and Goshima, 2021) have been reported.…”

“…Augmin/HAUS-mediated nucleation generates new microtubules that have the same polarity as the original 'mother' microtubule (Kamasaki et al, 2013;Petry et al, 2013), explaining why it can be important for maintaining microtubule polarity within neurons (Cunha-Ferreira et al, 2018;Qu et al, 2019;Sánchez-Huertas et al, 2016). In the basal processes of mammalian and human radial glial cells, microtubules are generated at neurite expansions (varicosities) that lack γ-tubulin but that are filled with the minus end binding CAMSAP proteins (Coquand et al, 2021). Similar varicosities have been observed in class I Drosophila da neurons (Mukherjee et al, 2020), but these are filled with γ-tubulin and other centrosomal proteins and it remains to be determined if they organise microtubules.…”