A role for the Golgi matrix protein giantin in ciliogenesis through control of the localization of dynein-2

Abstract

SummaryThe correct formation of primary cilia is central to the development and function of nearly all cells and tissues. Cilia grow from the mother centriole by extension of a microtubule core, the axoneme, which is then surrounded with a specialized ciliary membrane that is continuous with the plasma membrane. Intraflagellar transport moves particles along the length of the axoneme to direct assembly of the cilium and is also required for proper cilia function. The microtubule motor, cytoplasmic dynein-2 mediates retrograde transport along the axoneme from the tip to the base; dynein-2 is also required for some aspects of cilia formation. In most cells, the Golgi lies adjacent to the centrioles and key components of the cilia machinery localize to this organelle. Golgi-localized proteins have also been implicated in ciliogenesis and in intraflagellar transport. Here, we show that the transmembrane Golgi matrix protein giantin (GOLGB1) is required for ciliogenesis. We show that giantin is not required for the Rab11-Rabin8-Rab8 pathway that has been implicated in the early stages of ciliary membrane formation. Instead we find that suppression of giantin results in mis-localization of WDR34, the intermediate chain of dynein-2. Highly effective depletion of giantin or WDR34 leads to an inability of cells to form primary cilia. Partial depletion of giantin or of WDR34 leads to an increase in cilia length consistent with the concept that giantin acts through dynein-2. Our data implicate giantin in ciliogenesis through control of dynein-2 localization.

Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control

“…Our hypothesis was that RCAN2 could be suppressing the phenotypes seen following acute loss of giantin in RNAi experiments, namely an increase in cilia length. Consistent with this, we see that reducing the levels of RCAN2 in giantin KO cells recapitulates the longer cilia phenotypes seen on giantin knockdown in WT cells (Asante et al, 2013). Depletion of RCAN2 has no effect on the integrity of the Golgi, nor on targeting of the glycosyltransferase galactosyltransferase T (GalT) (Supplemental Fig.…”

“…The phenotypes we observe following acute and partial depletion of giantin in mammalian cells (Asante et al, 2013) and zebrafish embryos (Bergen et al, 2017) are not seen following permanent loss of function of giantin. RCAN2 has been characterised as a negative regulator of Nuclear Factor of Activated T-cells (NFAT)-dependent transcriptional activation by calcineurin (Cao et al, 2002;Loh et al, 1996).…”

“…Using RNAi, we have shown previously that a reduction in expression of giantin is associated with a ciliogenesis defect in cells (Asante et al, 2013). Giantin-depleted cells produce fewer cilia and those that remain are longer suggesting a defect in length control.…”

“…We have shown previously using RNAi that the golgin giantin is required for ciliogenesis in vitro (Asante et al, 2013). This was linked to the accumulation of the dynein-2 motor complex around the ciliary base.…”

Clarin-1 acts as a modulator of mechanotransduction activity and presynaptic ribbon assembly

“…As a second approach, we analyzed the distribution of the recycling endosomal marker rab11a (Ullrich et al, 1996). Rab11a is mainly expressed at the apical aspect of zebrafish hair cells (Clark et al, 2011), and its involvement in ciliogenesis has been demonstrated (Westlake et al, 2011;Asante et al, 2013). Fig.…”