Cilia, organelles that move to execute functions like fertilization and signal to execute functions like photoreception and embryonic patterning, are composed of a core of nine-fold doublet microtubules overlain by a membrane. Distinct types of cilia display distinct membrane morphologies, ranging from simple domed cylinders to the highly ornate invaginations and membrane disks of photoreceptor outer segments. Critical for the ability of cilia to signal, both the protein and the lipid compositions of ciliary membranes are different from those of other cellular membranes. This specialization presents a unique challenge for the cell as, unlike membrane-bounded organelles, the ciliary membrane is contiguous with the surrounding plasma membrane. This distinct ciliary membrane is generated in concert with multiple membrane remodeling events that comprise the process of ciliogenesis. Once the cilium is formed, control of ciliary membrane composition relies on discrete molecular machines, including a barrier to membrane proteins entering the cilium at a specialized region of the base of the cilium called the transition zone and a trafficking adaptor that controls G protein-coupled receptor (GPCR) localization to the cilium called the BBSome. The ciliary membrane can be further remodeled by the removal of membrane proteins by the release of ciliary extracellular vesicles that may function in intercellular communication, removal of unneeded proteins or ciliary disassembly. Here, we review the structures and transport mechanisms that control ciliary membrane composition, and discuss how membrane specialization enables the cilium to function as the antenna of the cell.

Due to low labeling efficiency and structural heterogeneity in fluorescence-based single-molecule localization microscopy (SMLM), image alignment and quantitative analysis is often required to make accurate conclusions on the spatial relationships between proteins. Cryo-electron microscopy (EM) image alignment procedures have been applied to average structures taken with super-resolution microscopy. However, unlike cryo-EM, the much larger cellular structures analyzed by super-resolution microscopy are often heterogeneous, resulting in misalignment. And the light-microscopy image library is much smaller, which makes classification challenging. To overcome these two challenges, we developed a method to deform semi-flexible ring-shaped structures and then align the 3D structures without classification. These algorithms can register semi-flexible structures with an accuracy of several nanometers in short computation time and with greatly reduced memory requirements. We demonstrated our methods by aligning experimental Stochastic Optical Reconstruction Microscopy (STORM) images of ciliary distal appendages and simulated structures. Symmetries, dimensions, and locations of protein complexes in 3D are revealed by the alignment and averaging for heterogeneous, tilted, and under-labeled structures.

Septins are conserved guanosine triphosphate-binding cytoskeletal proteins involved in membrane remodeling. In budding yeast, five mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1), which are essential for cytokinesis, transition during bud growth from a patch to a collar, which splits into two rings in cytokinesis and is disassembled before the next cell cycle. Cdc3, Cdc10, Cdc11, and Cdc12 form an apolar octameric rod with Cdc11 at each tip, which polymerizes into straight paired filaments. We show that Shs1 substitutes for Cdc11, resulting in octameric rods that do not polymerize into filaments but associate laterally, forming curved bundles that close into rings. In vivo, half of shs1Δ mutant cells exhibit incomplete collars and disrupted neck filaments. Importantly, different phosphomimetic mutations in Shs1 can either prevent ring formation or promote formation of a gauzelike meshwork. These results show that a single alternative terminal subunit is sufficient to confer a distinctive higher-order septin ultrastructure that can be further regulated by phosphorylation.

Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling.

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

Ciliopathies, including nephronophthisis (NPHP), Meckel syndrome (MKS) and Joubert syndrome (JBTS), can be caused by mutations affecting components of the transition zone, a domain near the base of the cilium that controls the protein composition of its membrane. We defined the three-dimensional arrangement of key proteins in the transition zone using two-colour stochastic optical reconstruction microscopy (STORM). NPHP and MKS complex components form nested rings comprised of nine-fold doublets. JBTS-associated mutations in RPGRIP1L or TCTN2 displace certain transition-zone proteins. Diverse ciliary proteins accumulate at the transition zone in wild-type cells, suggesting that the transition zone is a waypoint for proteins entering and exiting the cilium. JBTS-associated mutations in RPGRIP1L disrupt SMO accumulation at the transition zone and the ciliary localization of SMO. We propose that the disruption of transition-zone architecture in JBTS leads to a failure of SMO to accumulate at the transition zone and cilium, disrupting developmental signalling in JBTS.

Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences.

Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28-Spr3-Cdc3-Cdc10-Cdc10-Cdc3-Spr3-Spr28 hetero-octamer. Only coexpressed Spr3 and Spr28 colocalize with Cdc3 and Cdc10 in mitotic cells, indicating that incorporation requires a Spr28-Spr3 protomer. Unlike their mitotic counterparts, Spr28-Spr3-capped rods are unable to form higher-order structures in solution but assemble to form long paired filaments on lipid monolayers containing phosphatidylinositol-4,5-bisphosphate, mimicking presence of this phosphoinositide in the prospore membrane. Spr28 and Spr3 fail to rescue the lethality of a cdc11Δ cdc12Δ mutant, and Cdc11 and Cdc12 fail to restore sporulation proficiency to spr3Δ/spr3Δ spr28Δ/spr28Δ diploids. Thus, specific meiotic and mitotic subunits endow septin complexes with functionally distinct properties.

Vertebrate Hedgehog signals are transduced through the primary cilium, a specialized lipid microdomain that is required for Smoothened activation. Cilia-associated sterol and oxysterol lipids bind to Smoothened to activate the Hedgehog pathway, but how ciliary lipids are regulated is incompletely understood. Here we identified DHCR7, an enzyme that produces cholesterol, activates the Hedgehog pathway, and localizes near the ciliary base. We found that Hedgehog stimulation negatively regulates DHCR7 activity and removes DHCR7 from the ciliary microenvironment, suggesting that DHCR7 primes cilia for Hedgehog pathway activation. In contrast, we found that Hedgehog stimulation positively regulates the oxysterol synthase CYP7A1, which accumulates near the ciliary base and produces oxysterols that promote Hedgehog signaling in response to pathway activation. Our results reveal that enzymes involved in lipid biosynthesis in the ciliary microenvironment promote Hedgehog signaling, shedding light on how ciliary lipids are established and regulated to transduce Hedgehog signals.

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.