Centriole Number and the Accumulation of Microtubules Modulate the Timing of Apical Insertion during Radial Intercalation

Abstract

Highlights d Radial intercalation of multiciliated cells and ionocytes is temporally separable d Timing of apical insertion correlates with centriole number and microtubules d Modulating centriole numbers influences the timing of apical insertion d Ectopic accumulation of microtubules accelerates apical insertion

Tubulin Acetylation Promotes Penetrative Capacity of Cells Undergoing Radial Intercalation

“…Importantly, co-injection with HDAC6 significantly lowered the fluorescent intensity of acetylated MTs but not the overall amount of MTs (Figure 1G-J). In addition, the precocious apical insertion observed with CAMSAP1-Par6 is abrogated by the addition of HDAC6 and insertion rates revert to wild type levels (Figure 1K-L) (Collins et al, 2020a). This suggests that MTs in general are not sufficient to promote intercalation but that those MTs must be acetylated.…”

“…MTs are important for the intercalation of both MCCs and ICs. Increasing centriole number results in an increase in MTs and precocious apical insertion (Collins et al, 2020a). In contrast, treatment with the MT depolymerizing drug Nocodazole leads to partial intercalation where cells do not achieve normal apical size (Werner et al, 2014).…”

“…Here we have built on these results by performing an analysis of the timing of apical insertion in the presence of Nocodazole. Radial intercalation is a progressive process but we have previously defined the initial apical insertion event as the point when cells achieve a small apical size of 35µm 2 , equivalent to the average size of the apical domain at the earliest stage observed (Collins et al, 2020a). Here we show that cells treated with Nocodazole are delayed specifically in apical insertion such that at each developmental stage (ST) significantly fewer MCCs or ICs had successfully penetrated the epithelial barrier (Supplemental Figure 1).…”

“…pCS2+ plasmids containing the Pendrin (Pen) promoter (Quigley et al, 2011) with an N-terminal GFP or RFP were used to drive expression of some constructs specifically in ionocytes (Collins et al, 2020a). The GFP-tricellulin construct was described previously, and the GFP-CAMSAP1-Par6 construct has been previously reported (Collins et al, 2020a). HDAC6-FLAG (#13823) and pEF5B-FRT-GFP-aTAT1 (#27099) were purchased from Addgene.…”

“…Actin-based forces, as well as small small GTPase activity, drive apical expansion of MCCs (Ioannou et al, 2013;Kim et al, 2012;Kulkarni et al, 2018;Sedzinski et al, 2016Sedzinski et al, , 2017. Additionally, we have previously shown that centriole number and the subsequent changes to microtubule (MT) accumulation can regulate the timing of apical insertion, such that cells with more centrioles or more MTs insert earlier than cells with less (Collins et al, 2020a). While these results clearly establish a critical role for MTs during intercalation, the functional mechanism of how MTs promote apical insertion remains poorly understood.…”