Meng-Fu Bryan Tsou

Centriole distal appendages promote membrane docking, leading to cilia initiation

The distal appendages (DAPs) of centrioles have been proposed to anchor cilia to the plasma membrane, but their molecular composition, assembly, and exact function in ciliogenesis remain poorly understood. Using quantitative centrosome proteomics and superresolution microscopy, we identified five DAP components, including one previously described (CEP164), one partially characterized (CEP89 [ccdc123]), and three novel (CEP83 [ccdc41], SCLT1, and FBF1) DAP proteins. Analyses of DAP assembly revealed a hierarchy. CEP83 recruits both SCLT1 and CEP89 to centrioles. Subsequent recruitment of FBF1 and CEP164 is independent of CEP89 but mediated by SCLT1. All five DAP components are essential for ciliogenesis; loss of CEP83 specifically blocks centriole-to-membrane docking. Undocked centrioles fail to recruit TTBK2 or release CP110, the two earliest modifications found on centrioles prior to cilia assembly, revealing centriole-to-membrane docking as a temporal and spatial cue promoting cilia initiation.

The conversion of centrioles to centrosomes: essential coupling of duplication with segregation.

Plk1-dependent modification of centrioles early in mitosis is necessary for accurate centriole duplication and segregation.

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.

CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base

The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly. CEP162 interacts with core transition zone components, and mediates their association with microtubules. Loss of CEP162 arrests ciliogenesis at the stage of transition zone assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme and ectopically assemble transition zone components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein pre-tethered at centriole distal ends before ciliogenesis to promote and restrict transition zone formation specifically at the cilia 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.

53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis

Mitosis occurs efficiently, but when it is disturbed or delayed, p53-dependent cell death or senescence is often triggered after mitotic exit. To characterize this process, we conducted CRISPR-mediated loss-of-function screens using a cell-based assay in which mitosis is consistently disturbed by centrosome loss. We identified 53BP1 and USP28 as essential components acting upstream of p53, evoking p21-dependent cell cycle arrest in response not only to centrosome loss, but also to other distinct defects causing prolonged mitosis. Intriguingly, 53BP1 mediates p53 activation independently of its DNA repair activity, but requiring its interacting protein USP28 that can directly deubiquitinate p53 in vitro and ectopically stabilize p53 in vivo. Moreover, 53BP1 can transduce prolonged mitosis to cell cycle arrest independently of the spindle assembly checkpoint (SAC), suggesting that while SAC protects mitotic accuracy by slowing down mitosis, 53BP1 and USP28 function in parallel to select against disturbed or delayed mitosis, promoting mitotic efficiency. DOI: http://dx.doi.org/10.7554/eLife.16270.001

Spatial Control of Primary Ciliogenesis by Subdistal Appendages Alters Sensation-Associated Properties of Cilia

Vertebrate cells can initiate ciliogenesis from centrioles at the cell center, near the Golgi, forming primary cilia confined or submerged in a deep narrow pit created by membrane invagination. How or why cells maintain submerged cilia is unclear. Here, by characterizing centriole subdistal appendages (sDAP) in cells exclusively growing submerged cilia, we found that a group of sDAP components localize to the centriole proximal end through the cohesion factor C-Nap1 and that sDAP function redundantly with C-Nap1 for submerged cilia maintenance. Loss of sDAP and C-Nap1 has no effect on cilia assembly, but it disrupts stable Golgi-cilia association and allows normally submerged cilia to fully surface, losing the deep membrane invagination. Intriguingly, unlike submerged cilia (stationary), surfaced cilia actively respond to mechanical stimuli with motions and can ectopically recruit Hedgehog signaling components in the absence of agonist. We propose that spatial control of ciliogenesis uncouples or specifies sensory properties of cilia.

De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly

Vertebrate centrioles normally propagate through duplication, but in the absence of preexisting centrioles, de novo synthesis can occur. Consistently, centriole formation is thought to strictly rely on self-assembly, involving self-oligomerization of the centriolar protein SAS-6. Here, through reconstitution of de novo synthesis in human cells, we surprisingly found that normal looking centrioles capable of duplication and ciliation can arise in the absence of SAS-6 self-oligomerization. Moreover, whereas canonically duplicated centrioles always form correctly, de novo centrioles are prone to structural errors, even in the presence of SAS-6 self-oligomerization. These results indicate that centriole biogenesis does not strictly depend on SAS-6 self-assembly, and may require preexisting centrioles to ensure structural accuracy, fundamentally deviating from the current paradigm. DOI: http://dx.doi.org/10.7554/eLife.10586.001

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.

Promotion and Suppression of Centriole Duplication Are Catalytically Coupled through PLK4 to Ensure Centriole Homeostasis.

SUMMARY PLK4 is the major kinase driving centriole duplication. Duplication occurs only once per cell cycle, forming one new (or daughter) centriole that is tightly engaged to the preexisting (or mother) centriole. Centriole engagement is known to block the reduplication of mother centrioles, but the molecular identity responsible for the block remains unclear. Here, we show that the centriolar cartwheel, the geometric scaffold for centriole assembly, forms the identity of daughter centrioles essential for the block, ceasing further duplication of the mother centriole to which it is engaged. To ensure a steady block, we found that the cartwheel requires constant maintenance by PLK4 through phosphorylation of the same substrate that drives centriole assembly, revealing a parsimonious control in which “assembly” and “block for new assembly” are linked through the same catalytic reaction to achieve homeostasis. Our results support a recently deduced model that the cartwheel-bound PLK4 directly suppresses centriole reduplication.