The primary cilium maintains a well-regulated complement of soluble and membrane proteins, allowing it to mediate a variety of signaling pathways that are essential for development and tissue homeostasis [1-3]. Entry into the cilium is regulated at the base, where a complex containing nucleoporins, referred to as the "ciliary pore complex" (CPC), has been proposed to set a size-exclusion limit for soluble molecule diffusion into the cilium [4-6]. Here, using a fluorescence-based diffusion trap system, we demonstrate that NUP98, a component of the phenylalanine-glycine (FG) hydrogel permeability barrier at the nuclear pore complex [7, 8], limits the diffusion of soluble molecules >70 kDa into the cilium in cultured mammalian cells. Small interfering RNA (siRNA)-mediated knockdown of NUP98 increases the rate of diffusion of molecules >100 kDa into the cilium. The tubulin heterodimer, the building block of the axoneme [9, 10], is approximately 100 kDa in size. After knockdown of NUP98, cilia become shorter, and their length is more sensitive to changes in cytoplasmic soluble tubulin levels. These data indicate a novel function of the ciliary pore complex, limiting diffusion of soluble tubulin between the ciliary matrix and the cytosol, allowing the cilium to regulate its length independently of cytosolic microtubule dynamics.

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMAlike serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry.

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Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, where individual peptides, recognized by a consensus motif, are translocated directly across the lysosomal membrane. CMA regulates the abundance of many disease-related proteins, with causative roles in neoplasia, neurodegeneration, hepatosteatosis, and other pathologies relevant to human health and aging. At the lysosomal membrane, CMA is inhibited by Akt-dependent phosphorylation of the CMA regulator GFAP. The INS-PI3K-PDPK1 pathway regulates Akt, but its role in CMA is unclear. Here, we report that inhibition of class I PI3K or PDPK1 activates CMA. In contrast, selective inhibition of class III PI3Ks does not activate CMA. Isolated liver lysosomes from mice treated with either of two orally bioavailable class I PI3K inhibitors, pictilisib or buparlisib, display elevated CMA activity, and decreased phosphorylation of lysosomal GFAP, with no change in macroautophagy. The findings of this study represent an important first step in repurposing class I PI3K inhibitors to modulate CMA in vivo.

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