XB-ART-51304Nat Commun September 18, 2015; 6 8386.
miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways.
Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways.
PubMed ID: 26381333
PMC ID: PMC4595761
Article link: Nat Commun
Genes referenced: arhgap1 arhgdib gnl3 igf2bp3 mcc mcidas mmut notch1 odc1 rho rho.2 rhoa rras tcirg1 tub
Morpholinos: dll1 miR-449 protector MO1 dll1 miR-449 protector MO2 miR-449 MO1 miR-449 MO2 miR-449 MO3 rras MO1 rras miR-449 protector MO
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|Figure 8. MiR-34/449 promote FLNA relocalization during MCC differentiation.(a) Apical layer of differentiated HAECs (LC stage) were stained for the basal bodies marker centrin-2 (in red) and the actin-binding protein filamin-A (FLNA, in green). FLNA labelling was enriched in the apical layer near basal bodies (a1–3). Basal layer of differentiated HAECs (LC stage) were stained for FLNA (in green) and for nuclei with 4,6-diamidino-2-phenylindole (DAPI; in blue) (a4). (b) Modulation of protein levels of FLNA and R-Ras in total fraction (Total), membrane fraction (Membrane) and cytoskeletal fraction (Cytoskeleton) isolated from proliferating HAECs transfected for 72 h with miR-Neg or miR-449a as indicated each below corresponding band. Protein levels were normalized against HSP60 as an internal control for the total fraction and normalized fold changes are indicated above the corresponding bands. Data were representative of three independent experiments. (c) In the epidermis of stage 25 Xenopus embryos, FLNA labelling (in green, c1) is apically enriched in acetylated tubulin-positive MCCs (in magenta, c2,3). (d) Model illustrating the roles of miR-449 and other interconnected actors in MCC differentiation. Early cues required to trigger MCC differentiation involve the inhibition of BMP and Notch pathway. The expression of Multicilin (MCIDAS), CCNO and miR-449 is controlled by the Notch pathway activity. Notch repression is associated with the increase in MCIDAS, CCNO and miR-449 expression. Then, miR-449 miRNAs repress the Notch pathway inducing a double-negative feedback loop increasing miR-449 expression. MiR-449 inhibit cell cycle-related genes to stop proliferation and promote entry in differentiation. In addition, MCIDAS drives expression of centriole multiplication-related genes including CCNO, which then participates to centriole assembly and amplification. MCIDAS also contributes to increase FOXJ1 expression, which in turn controls ciliogenesis-related genes and apical actin remodelling through a RhoA-dependent mechanism. In parallel, miR-449 also control apical actin network remodelling by repressing R-Ras, promoting FLNA redistribution and modulating RhoA activity. Finally, miR-449 favour basal body maturation and anchoring by downregulating CP110. All these events are key pre-requisites for axoneme elongation and motile cilia formation during MCC differentiation. Plain lines indicate direct interactions; dotted lines identify pathways that may or may not be direct.|