CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia

Abstract

<jats:p>Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a “9 + 2” microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3),<jats:italic>Camsap3</jats:italic><jats:sup><jats:italic>tm1a/tm1a</jats:italic></jats:sup>, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of<jats:italic>Camsap3</jats:italic><jats:sup><jats:italic>tm1a/tm1a</jats:italic></jats:sup>mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in<jats:italic>Camsap3</jats:italic><jats:sup><jats:italic>tm1a/tm1a</jats:italic></jats:sup>, MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of<jats:italic>Xenopus</jats:italic>embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.</jats:p>

Celsr1 and CAMSAP3 differently regulate intercellular and intracellular cilia orientation in oviduct multiciliated cells

“…We found that CAMSAP3, a non-centrosomal microtubule minus-end binding protein, localizes to the base of the cilia of MCCs, in addition to γ-tubulin, a centrosomal microtubule minus-end binding protein (Clare et al, 2014). The localization of CAMSAP3 to BB of the trachea was also recently reported (Robinson et al, 2020). In contrast to the prevailing view that a connection of microtubules to a BF controls cilia orientation, we found that CAMSAP3 was not localized to BF.…”

Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3

“…In the case of Camsap3 mutation, formation of primary cilia seemed to be unaffected. A recent study showed that loss of CAMSAP3 in nasal multi-ciliated epithelial cells affected the formation of the central pairs of microtubules in the axoneme, causing aberrant motility in motile cilia 49 . Although the primary cilia on epithelial cells are nonmotile and lack the central pairs of microtubules 50 , it remains uncertain whether the primary cilium in the Camsap3 mutant mice is still functional.…”

Tracheal motile cilia in mice require CAMSAP3 for formation of central microtubule pair and coordinated beating

“…To begin investigating how CAMSAP3 contributes to microtubule assembly in axonemes, we observed its distribution in airway epithelial cells of adult trachea by immunofluorescence (IF) microscopy, using samples fixed with paraformaldehyde (PFA). The results showed that CAMSAP3 was concentrated around the apical plasma membranes (Figure 3A), as assessed by co-immunostaining for EZRIN, which is known to associate with the apical membranes or microvilli (Berryman et al, 1993), as observed in other epithelial cells (Toya et al, 2016;Robinson et al, 2020;Kimura et al, 2021;Usami et al, 2021). This position of CAMSAP3 corresponded to the proximal edge of strong a-tubulin IF signals that should represent ciliary microtubules (Figure 3B).…”

“…Whichever the case is, the CAMSAP3 dot in P30 nasal cilia may correspond to the #2 or #3 puncta in tracheal cilia, considering their distance from gtubulin of the BB. In nasal motile cilia, an additional dot overlapping with g-tubulin was also observed in P3 mice (Robinson et al, 2020), but we did not examine tracheal motile cilia in such young mice.…”