Microtubule Motors Drive Hedgehog Signaling in Primary Cilia

Abstract

The mammalian Hedgehog (Hh) signaling pathway is required for development and for maintenance of adult stem cells, and overactivation of the pathway can cause tumorigenesis. All responses to Hh family ligands in mammals require the primary cilium, an ancient microtubule-based organelle that extends from the cell surface. Genetic studies in mice and humans have defined specific functions for cilium-associated microtubule motor proteins: they act in the construction and disassembly of the primary cilium, they control ciliary length and stability, and some have direct roles in mammalian Hh signal transduction. These studies highlight how integrated genetic and cell biological studies can define the molecular mechanisms that underlie cilium-associated health and disease.

Keywords: Hedgehog pathway; ciliopathies; dynein; kinesin; microtubules; primary cilia.

Figure 1. Primary cilia are required for mammalian Sonic hedgehog signaling

a. Primary cilia on progenitor cells the mouse neuroepithelium at embryonic day 10.5 (E10.5) visualized by scanning electron microscopy. b. Primary cilium of cultured mouse embryonic fibroblast formed during Go/G1 of the cell cycle. Staining of acetylated α-tubulin (green) labels the axonemal microtubule, and γ-tubulin staining (red) labels the basal body. Nucleus is labeled by DAPI in blue. Scale bars = 5 μm. c. Core Hedgehog components localize to the primary cilium. Hh ligand inhibits Patched localization to the cilium and promotes Smoothened accumulation in the ciliary membrane. GLI transcription factors, Suppressor of Fused (SUFU) and KIF7 are enriched in the cilia tip compartment upon Hh stimulation [3]. Dissociation of the GLI-SUFU complex is required for maximal activation of the Hh pathway [145,146]. Protein Kinase A (PKA) is required for phosphorylation and proteolytic processing to GLI to form transcriptional repressors [147,148]. PKA resides at the base of cilia in neuroepithelial cells [148], but is found within the ciliary compartment in mIMCD3 cells and cholangiocytes [149,150]. The activity of PKA may be coupled to an orphan G-protein coupled receptor GPR161, which can increase intracellular cAMP when activated. Like PTCH1, GPR161 localization to the ciliary membrane is negatively controlled by Hh ligand [88]. Correct localizations of membrane-bound cilia proteins depend on both active transport through IFT trains/BBSome and diffusion.

Figure 2. Vertebrate cilia-associated kinesins and IFT dynein

a. Four families of kinesins and one dynein are known to be associated with the structure and IFT of mammalian cilia. Heterotrimeric kinesin-2 and homodimeric KIF17 belong to the Kinesin-2 family. Homodimeric KIF7 and KIF27 belong to the Kinesin-4 family. Homodimeric kinesin KIF19 belongs to the Kinesin-8 family. The motor domains of those kinesins are located at the N-terminus of the protein. KIF24 and KIF2A belong to the kinesin-13 family, with motor domains in the central region of the protein. Cytoplasmic dynein-2 is the only dynein required for intraflagellar transport. b. Both kinesin-2 and KIF17 are anterograde kinesins that move along the axoneme from the cilia base, the minus end, to the cilia tip, the plus end [56]. KIF7 lacks motility along the microtubule lattice, but can bind preferentially to GTP-bound tubulin at the growing ends of microtubule and promote catastrophe. Other members of the Kinesin-4 family, including KIF4 and KIF21A, exhibit plus-end directed motility to reach microtubule ends, where these kinesins regulate microtubule dynamics [151,152]. KIF19 is a motile kinesin and exhibits microtubule depolymerization activity towards the plus-ends of microtubules and appears to act specifically in motile cilia [132]. KIF24 and KIF2A lack motility and show microtubule-depolymerizing activity towards both ends of microtubules. Although both Kinesin- 8 and Kinesin-13 family members can shorten microtubules, the underlying actions are different [153]. Cytoplasmic dynein-2 is required for microtubule-based cargo transport from plus ends to the minus ends of microtubules within cilia. The motor activity resides in the heavy chain, which acts as a homodimer, and a large number of smaller chains mediate cargo loading and specificity of the motor (see Figure 4c).

Figure 3. Cell-type dependent functions of ciliary kinesins

a. In primary cilia, kinesin-2 is required for the anterograde transport of IFT complexes, BBSome and ciliary cargo along the axoneme, which is composed of nine microtubule doublets. Kinesin-2 dependent transport is essential for cilia formation in all kinds of cilia (a–d). KIF7, which can autonomously track the plus-ends of growing microtubules, localizes to the tip of cilia, where axonemal microtubule plus ends are located. KIF7 controls the structure and length of primary cilia. KIF24 and KIF2A exhibit microtubule-depolymerizing activities and are thought to act on centrosomal microtubules to provide temporal control of cilia formation during the cell cycle. b. While kinesin-2 is required for motile cilia formation, the length of motile ciliary axonemes is controlled by KIF19. KIF19 relies on its plus-end directed motility to reach the axonemal plus ends at the motile cilia tip, where the kinesin plays a role in shortening the length of the axoneme. KIF27 localizes to the base of motile cilia and is required for motility. c. Olfactory receptor cilia are much longer than motile cilia and primary cilia. The proximal segment of the axoneme consists of doublet microtubules, whereas the distal segment has only singlet microtubules. KIF17 is present along the axoneme, but it is unclear whether the motor activity of KIF17 is required for its targeting to the distal end or if KIF17 is necessary for IFT. d. The connecting cilium of the photoreceptor does not directly project into the extracellular environment but bridges the inner segment and outer segment of the photoreceptor cells, such as rod and cone cells. Kinesin-2, IFT proteins and KIF17 appear to form a complex that moves along the connecting ciliary axoneme. KIF17 is required for the morphogenesis of the outer segment during an early stage of photoreceptor development in zebrafish.

Figure 4. Mutations that disrupt function of the mouse cytoplasmic dynein-2

a. Schematic of the domains of cytoplasmic dynein heavy chain 2 (DYNC2H1). The N-terminal regulatory domain is responsible for dynein oligomerization, cargo and non-catalytic subunits binding, and is not part of the catalytic motor domain that generates motility. The motor domain consists of the linker, six non-identical AAA+ modules (1–6), the coiled-coil stalk and strut/buttress (not depicted), as well as the C-terminal region. b. All mutations found in mouse Dync2h1 cause disruption in cilia structure and Hh signaling. For mutations in the human DYNC2H1 that cause ciliopathies, see [77]. c. Subunits of mammalian cytoplasmic dynein-2 complex. Mutations in the human genes encoding these subunits, including TCTEX1D2, DYNC2LI1, WDR34, WDR60 and DYNC2LI1, cause ciliopathies and presumably disrupt retrograde ciliary trafficking and Hh signaling.

Figure 5. Kif7 mutations cause ciliopathies in humans and embryonic lethality in mice

a. The catalytic motor head of KIF7 is located near the N-terminus and possesses microtubule-stimulated ATPase and microtubule-binding activities. Two KIF7 motor heads of a kinesin motor are coupled by the neck linker and the coiled-coil domain to form homodimers. The neck linker is important for the coordination of two motor heads as well as for the amplification of motility; the linker region of KIF7 is 100 amino acids long. This is longer than seen in most motile N-kinesins, which correlates with the lack of processive motility of the KIF7 motor. The globular domain at the C-terminus is believed to bind to cargo or adaptor proteins. Homozygous and compound heterozygous human KIF7 mutations are present in patients with Acrocallosal syndrome (ACLS), fetal hydrolethalus, agenesis of the corpus callosum, Joubert syndrome as well as mild epiphyseal dysplasia. The majority of human KIF7 mutations are found in the motor domain. b. The mouse KIF7 protein is highly homologous to human KIF7. Two ENU induced missense mutations are present in the motor domain, while a third ENU induced nonsense mutation is found in the coiled-coil region. All characterized mouse Kif7 alleles, including two targeted null alleles of Kif7 (not depicted) in which the first 2 exons were deleted, show very similar developmental defects, associated with perinatal lethality.