T. Tony Yang

Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone.

The transition zone (TZ) of primary cilia serves as a diffusion barrier to regulate ciliogenesis and receptor localization for key signaling events such as sonic hedgehog signaling. Its gating mechanism is poorly understood due to the tiny volume accommodating a large number of ciliopathy-associated molecules. Here we performed stimulated emission depletion (STED) imaging of collective samples and recreated superresolved relative localizations of eight representative species of ciliary proteins using position averages and overlapped with representative electron microscopy (EM) images, defining an architectural foundation at the ciliary base. Upon this framework, transmembrane proteins TMEM67 and TCTN2 were accumulated at the same axial level as MKS1 and RPGRIP1L, suggesting that their regulation roles for tissue-specific ciliogenesis occur at a specific level of the TZ. CEP290 is surprisingly localized at a different axial level bridging the basal body (BB) and other TZ proteins. Upon this molecular architecture, two reservoirs of intraflagellar transport (IFT) particles, correlating with phases of ciliary growth, are present: one colocalized with the transition fibers (TFs) while the other situated beyond the distal edge of the TZ. Together, our results reveal an unprecedented structural framework of the TZ, facilitating our understanding in molecular screening and assembly at the ciliary base.

SAS-6 Assembly Templated by the Lumen of Cartwheel-less Centrioles Precedes Centriole Duplication

Centrioles are 9-fold symmetric structures duplicating once per cell cycle. Duplication involves self-oligomerization of the centriolar protein SAS-6, but how the 9-fold symmetry is invariantly established remains unclear. Here, we found that SAS-6 assembly can be shaped by preexisting (or mother) centrioles. During S phase, SAS-6 molecules are first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall, rather than via their self-oligomerization activity. The removal or release of luminal SAS-6 requires Plk4 and the cartwheel protein STIL. Abolishing either the recruitment or the removal of luminal SAS-6 hinders SAS-6 (or centriole) assembly at the outside wall of mother centrioles. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication. These results lead to a proposed model that centrioles may duplicate via a template-based process to preserve their geometry and copy number.

Superresolution STED microscopy reveals differential localization in primary cilia

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the cAMP, Wnt, and hedgehog signaling pathways. Adenylyl cyclase type III (ACIII) is enriched in primary cilia and acts as a marker that is involved in cAMP signaling, while also playing an important role in regulating ciliogenesis and sensory functions. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). The detailed localization and interactions of these important proteins remain unclear due to the limited resolution of conventional microscopy. We conducted superresolution imaging of immunostained ACIII and IFT88 in human fibroblasts using stimulated emission depletion (STED) microscopy. Instead of a homogeneous distribution along a primary cilium, our STED images revealed that ACIII formed a periodic punctate pattern with a roughly equal spacing between groups of puncta. Superresolution imaging of IFT88, an important protein of the IFT complexes, demonstrated two novel distinct distribution patterns at the basal end: a triangle of three puncta with similar fluorescence intensities, and a Y‐shaped configuration of a bright punctum connected to two branches. We also performed STED imaging of IFT88 in mouse inner medullary collecting duct cells and mouse embryonic fibroblasts. The similar three‐puncta and Y‐shape patterns were observed in these cells, suggesting that these distribution patterns are common among primary cilia of different cell types. Our results demonstrate the ability of superresolution STED microscopy to reveal novel structural characteristics in primary cilia.

TTBK2: a tau protein kinase beyond tau phosphorylation.

Tau tubulin kinase 2 (TTBK2) is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1) kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.

Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.Distal appendages (DAPs) at the cilia base mediate membrane docking during ciliogenesis. Here the authors use super-resolution microscopy to map 16 centriole distal end components, revealing the structure of the backbone of the DAP, as well as a previously undescribed distal appendage matrix.

Architecture of mammalian centriole distal appendages accommodates distinct blade and matrix functional elements

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here, we determined a superresolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we termed the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164 which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs.

Super-Resolution Imaging Reveals TCTN2 Depletion-Induced IFT88 Lumen Leakage and Ciliary Weakening

The primary cilium is an essential organelle mediating key signaling activities, such as sonic hedgehog signaling. The molecular composition of the ciliary compartment is distinct from that of the cytosol, with the transition zone (TZ) gated the ciliary base. The TZ is a packed and organized protein complex containing multiple ciliopathy-associated protein species. Tectonic 2 (TCTN2) is one of the TZ proteins in the vicinity of the ciliary membrane, and its mutation is associated with Meckel syndrome. Despite its importance in ciliopathies, the role of TCTN2 in ciliary structure and molecules remains unclear. Here, we created a CRISPR/Cas9 TCTN2 knockout human retinal pigment epithelial cell line and conducted quantitative analysis of geometric localization using both wide-field and super-resolution microscopy techniques. We found that TCTN2 depletion resulted in partial TZ damage, loss of ciliary membrane proteins, leakage of intraflagellar transport protein IFT88 toward the basal body lumen, and cilium shortening and curving. The basal body lumen occupancy of IFT88 was also observed in si-RPGRIP1L cells and cytochalasin-D-treated wild-type cells, suggesting varying lumen accessibility for intraflagellar transport proteins under different perturbed conditions. Our findings support two possible models for the lumen leakage of IFT88, i.e., a tip leakage model and a misregulation model. Together, our quantitative image analysis augmented by super-resolution microscopy facilitates the observation of structural destruction and molecular redistribution in TCTN2-/- cilia, shedding light on mechanistic understanding of TZ-protein-associated ciliopathies.

STED and STORM Superresolution Imaging of Primary Cilia

The characteristic lengths of molecular arrangement in primary cilia are below the diffraction limit of light, challenging structural and functional studies of ciliary proteins. Superresolution microscopy can reach up to a 20 nm resolution, significantly improving the ability to map molecules in primary cilia. Here we describe detailed experimental procedure of STED microscopy imaging and dSTORM imaging, two of the most powerful superresolution imaging techniques. Specifically, we emphasize the use of these two methods on imaging proteins in primary cilia.

Superresolution STED imaging reveals a periodic punctate pattern of adenylyl cyclase type III on primary cilia

Adenylyl cyclases type III (ACIII) is a primary cilia marker involved in cAMP signaling, playing important roles in regulating ciliogenesis and sensory function. Despite its importance, detailed ACIII localization and their interactions with other proteins remain unclear due to the limited resolution of conventional microscopy. To determine the morphological characteristics of ACIII in primary cilia, we conducted superresolution imaging of immunostained ACIII in fibroblasts and neurons using stimulated emission depletion (STED) microscopy, which allows us to resolve the localization of ACIII achieving a resolution of 50 nm. In contrast to the previous understanding that ACIII distributes uniformly along a primary cilium, our STED images revealed that ACIII formed a periodic punctate pattern with a roughly equal spacing between groups of puncta. These puncta occupied less than 50% of the area, with the size of 137±20 nm in the axial direction along the primary cilia. The spacing between puncta was 250±67 nm. Some primary cilia even showed two rows of periodic puncta along the axial direction, with a tilted angle of about 12° to 35° between the two rows. The spacing between the two rows was 195±19 nm. In some cells, ACIII was only localized in the basal body, where the periodic punctate pattern was absent. In summary, based on our superresolution studies, we found that ACIII can be transported into a primary cilium, but would only occupy regions approximately equally spaced along the cilium.