Pair Of Centrioles Research Articles

Hybrid incompatibility emerges at the one-cell stage in interspecies Caenorhabditis embryos.

Intrinsic reproductive isolation occurs when genetic differences between populations disrupt the development of hybrid organisms, preventing gene flow and enforcing speciation. 1-4 While prior studies have examined the genetic origins of hybrid incompatibility, 5-18 the effects of incompatible factors on development remain poorly understood. Here, we investigate the mechanistic basis of hybrid incompatibility in Caenorhabditis nematodes by capitalizing on the ability of C. brenneri females to produce embryos after mating with males from several other species. Contrary to expectations, hybrid incompatibility was evident immediately after fertilization, suggesting that post-fertilization barriers to hybridization originate from physical incompatibility between sperm and oocyte-derived factors rather than from zygotic transcription, which starts after the 4-cell stage. 19-22 Sperm deliver chromatin, which expands to form a pronucleus, and a pair of centrioles, which form centrosomes that attach to the sperm-derived pronucleus and signal to establish the embryo's anterior-posterior axis. 23,24 In C. brenneri oocytes fertilized with C. elegans sperm, sperm pronuclear expansion was compromised, frequent centrosome detachment was observed, and cortical polarity was disrupted. Live imaging revealed that defective polar body extrusion contributes to defects in mitotic spindle morphology. C. brenneri oocytes fertilized with C. remanei or C. sp. 48 sperm showed similar defects, and their severity and frequency increased with phylogenetic distance. Defective expansion of the sperm-derived pronucleus and unreliable polar body extrusion immediately after fertilization generally underlie the inviability of hybrid embryos in this clade. These results indicate that physical mismatches between sperm and oocyte-derived structures may be a primary mechanism of hybrid incompatibility.

Maternal inheritance of functional centrioles in two parthenogenetic nematodes

Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus, centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys, the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species.

Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

Developmental and tissue‐specific roles of mammalian centrosomes

Centrosomes are dominant microtubule organizing centers in animal cells with a pair of centrioles at their core. They template cilia during interphase and help organize the mitotic spindle for a more efficient cell division. Here, we review the roles of centrosomes in the early developing mouse and during organ formation. Mammalian cells respond to centrosome loss‐of‐function by activating the mitotic surveillance pathway, a timing mechanism that, when a defined mitotic duration is exceeded, leads to p53‐dependent cell death in the descendants. Mouse embryos without centrioles are highly susceptible to this pathway and undergo embryonic arrest at mid‐gestation. The complete loss of the centriolar core results in earlier and more severe phenotypes than that of other centrosomal proteins. Finally, different developing tissues possess varying thresholds and mount graded responses to the loss of centrioles that go beyond the germ layer of origin.

Aneuploidy is frequent in heterozygous diploid and triploid hydatidiform moles

Hydatidiform moles are abnormal conceptuses. Many hydatidiform moles are diploid androgenetic, and of these, most are homozygous in all loci. Additionally, most hydatidiform moles are euploid. Using Single Nucleotide Polymorphism (SNP) array analysis, in two studies a higher frequency of aneuploidy was observed in diploid androgenetic heterozygous conceptuses, than in their homozygous counterparts. In the Danish Mole Project, we analyze conceptuses suspected to be hydatidiform moles due to the clinical presentation, using karyotyping and Short Tandem Repeat (STR) analysis. Among 278 diploid androgenetic conceptuses, 226 were homozygous in all loci and 52 (18.7%) were heterozygous in several loci. Among 142 triploid diandric conceptuses, 141 were heterozygous for paternally inherited alleles in several loci. Here we show that the frequencies of aneuploidy in diploid androgenetic heterozygous and triploid diandric heterozygous conceptuses were significantly higher than the frequency of aneuploidy in diploid androgenetic homozygous conceptuses. In diploid androgenetic and triploid diandric conceptuses that are heterozygous for paternally inherited alleles, the two paternally inherited sets of genomes originate in two spermatozoa. Each spermatozoon provides one pair of centrioles to the zygote. The presence of two pairs of centrioles may cause an increased risk of aneuploidy.

A membrane reticulum, the centriculum, affects centrosome size and function in Caenorhabditis elegans

Centrosomes are cellular structures that nucleate microtubules. At their core is a pair of centrioles that recruit pericentriolar material (PCM). Although centrosomes are considered membraneless organelles, in many cell types, including human cells, centrosomes are surrounded by endoplasmic reticulum (ER)-derived membranes of unknown structure and function. Using volume electron microscopy (vEM), we show that centrosomes in the Caenorhabditis elegans (C.elegans) early embryo are surrounded by a three-dimensional (3D), ER-derived membrane reticulum that we call the centriculum, for centrosome-associated membrane reticulum. The centriculum is adjacent to the nuclear envelope in interphase and early mitosis and fuses with the fenestrated nuclear membrane at metaphase. Centriculum formation is dependent on the presence of an underlying centrosome and on microtubules. Conversely, increasing centriculum size by genetic means led to the expansion of the PCM, increased microtubule nucleation capacity, and altered spindle width. The effect of the centriculum on centrosome function suggests that in the C.elegans early embryo, the centrosome is not membraneless. Rather, it is encased in a membrane meshwork that affects its properties. We provide evidence that the centriculum serves as a microtubule "filter," preventing the elongation of a subset of microtubules past the centriculum. Finally, we propose that the fusion between the centriculum and the nuclear membrane contributes to nuclear envelope breakdown by coupling spindle elongation to nuclear membrane fenestration.

Ultrastructural characteristics of oligodendrocyte precursor cells in the early postnatal mouse optic nerve observed by serial block-face scanning electron microscopy.

Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs.

Self-assembly of pericentriolar material in interphase cells lacking centrioles.

The major microtubule-organizing center (MTOC) in animal cells, the centrosome, comprises a pair of centrioles surrounded by pericentriolar material (PCM), which nucleates and anchors microtubules. Centrosome assembly depends on PCM binding to centrioles, PCM self-association and dynein-mediated PCM transport, but the self-assembly properties of PCM components in interphase cells are poorly understood. Here, we used experiments and modeling to study centriole-independent features of interphase PCM assembly. We showed that when centrioles are lost due to PLK4 depletion or inhibition, dynein-based transport and self-clustering of PCM proteins are sufficient to form a single compact MTOC, which generates a dense radial microtubule array. Interphase self-assembly of PCM components depends on γ-tubulin, pericentrin, CDK5RAP2 and ninein, but not NEDD1, CEP152, or CEP192. Formation of a compact acentriolar MTOC is inhibited by AKAP450-dependent PCM recruitment to the Golgi or by randomly organized CAMSAP2-stabilized microtubules, which keep PCM mobile and prevent its coalescence. Linking of CAMSAP2 to a minus-end-directed motor leads to the formation of an MTOC, but MTOC compaction requires cooperation with pericentrin-containing self-clustering PCM. Our data reveal that interphase PCM contains a set of components that can self-assemble into a compact structure and organize microtubules, but PCM self-organization is sensitive to motor- and microtubule-based rearrangement.

Upstream open reading frames control PLK4 translation and centriole duplication in primordial germ cells.

Centrosomes are microtubule-organizing centers comprised of a pair of centrioles and the surrounding pericentriolar material. Abnormalities in centriole number are associated with cell division errors and can contribute to diseases such as cancer. Centriole duplication is limited to once per cell cycle and is controlled by the dosage-sensitive Polo-like kinase 4 (PLK4). Here, we show that PLK4 abundance is translationally controlled through conserved upstream open reading frames (uORFs) in the 5' UTR of the mRNA. Plk4 uORFs suppress Plk4 translation and prevent excess protein synthesis. Mice with homozygous knockout of Plk4 uORFs (Plk4 Δu/Δu ) are viable but display dramatically reduced fertility because of a significant depletion of primordial germ cells (PGCs). The remaining PGCs in Plk4 Δu/Δu mice contain extra centrioles and display evidence of increased mitotic errors. PGCs undergo hypertranscription and have substantially more Plk4 mRNA than somatic cells. Reducing Plk4 mRNA levels in mice lacking Plk4 uORFs restored PGC numbers and fully rescued fertility. Together, our data uncover a specific requirement for uORF-dependent control of PLK4 translation in counterbalancing the increased Plk4 transcription in PGCs. Thus, uORF-mediated translational suppression of PLK4 has a critical role in preventing centriole amplification and preserving the genomic integrity of future gametes.

Signed, sealed, and delivered: RNA localization and translation at centrosomes.

Protein localization is intrinsic to cellular function and specialized activities, such as migration or proliferation. Many localized proteins enrich at defined organelles, forming subdomains of functional activity further specified by interacting protein assemblies. One well-studied organelle showing dynamic, functional changes in protein composition is the centrosome. Centrosomes are microtubule-organizing centers with diverse cellular functions largely defined by the composition of the pericentriolar material, an ordered matrix of proteins organized around a central pair of centrioles. Also localizing to the pericentriolar material are mRNAs. Although RNA was identified at centrosomes decades ago, the characterization of specific RNA transcripts and their functional contributions to centrosome biology remained largely unstudied. While the identification of RNA localized to centrosomes accelerated with the development of high-throughput screening methods, this discovery still outpaces functional characterization. Recent work indicates RNA localized to centrosomes is biologically significant and further implicates centrosomes as sites for local protein synthesis. Distinct RNA localization and translational activities likely contribute to the diversity of centrosome functions within cells.

Modeling Human Primary Microcephaly With hiPSC-Derived Brain Organoids Carrying CPAP-E1235V Disease-Associated Mutant Protein.

The centrosome is composed of a pair of centrioles and serves as the major microtubule-organizing center (MTOC) in cells. Centrosome dysfunction has been linked to autosomal recessive primary microcephaly (MCPH), which is a rare human neurodevelopmental disorder characterized by small brain size with intellectual disability. Recently, several mouse models carrying mutated genes encoding centrosomal proteins have been generated to address the genotype–phenotype relationships in MCPH. However, several human-specific features were not observed in the mouse models during brain development. Herein, we generated isogenic hiPSCs carrying the gene encoding centrosomal CPAP-E1235V mutant protein using the CRISPR-Cas9 genome editing system, and examined the phenotypic features of wild-type and mutant hiPSCs and their derived brain organoids. Our results showed that the CPAP-E1235V mutant perturbed the recruitment of several centriolar proteins involved in centriole elongation, including CEP120, CEP295, CENTROBIN, POC5, and POC1B, onto nascent centrioles, resulting in the production of short centrioles but long cilia. Importantly, our wild-type hiPSC-derived brain organoid recapitulated many cellular events seen in the developing human brain, including neuronal differentiation and cortical spatial lamination. Interestingly, hiPSC-CPAP-E1235V-derived brain organoids induced p53-dependent neuronal cell death, resulting in the production of smaller brain organoids that mimic the microcephaly phenotype. Furthermore, we observed that the CPAP-E1235V mutation altered the spindle orientation of neuronal progenitor cells and induced premature neuronal differentiation. In summary, we have shown that the hiPSC-derived brain organoid coupled with CRISPR/Cas9 gene editing technology can recapitulate the centrosome/centriole-associated MCPH pathological features. Possible mechanisms for MCPH with centriole/centrosome dysfunction are discussed.

α-/γ-Taxilin are required for centriolar subdistal appendage assembly and microtubule organization.

The centrosome composed of a pair of centrioles (mother and daughter) and pericentriolar material, and is mainly responsible for microtubule nucleation and anchorage in animal cells. The subdistal appendage (SDA) is a centriolar structure located at the mother centriole's subdistal region, and it functions in microtubule anchorage. However, the molecular composition and detailed structure of the SDA remain largely unknown. Here, we identified α-taxilin and γ-taxilin as new SDA components that form a complex via their coiled-coil domains and that serve as a new subgroup during SDA hierarchical assembly. The taxilins' SDA localization is dependent on ODF2, and α-taxilin recruits CEP170 to the SDA. Functional analyses suggest that α- and γ-taxilin are responsible for SDA structural integrity and centrosomal microtubule anchorage during interphase and for proper spindle orientation during metaphase. Our results shed light on the molecular components and functional understanding of the SDA hierarchical assembly and microtubule organization.

Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos.

Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new 'daughter' centriole is built orthogonally on one side of each radially symmetric 'mother' centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.

PCMD-1 bridges the centrioles and the pericentriolar material scaffold in C. elegans.

Correct cell division relies on the formation of a bipolar spindle. In animal cells, microtubule nucleation at the spindle poles is facilitated by the pericentriolar material (PCM), which assembles around a pair of centrioles. Although centrioles are essential for PCM assembly, the proteins that anchor the PCM to the centrioles are less known. Here, we investigate the molecular function of PCMD-1 in bridging the PCM and the centrioles in Caenorhabditis elegans. We demonstrate that the centrosomal recruitment of PCMD-1 is dependent on the outer centriolar protein SAS-7. The most C-terminal part of PCMD-1 is sufficient to target it to the centrosome, and the coiled-coil domain promotes its accumulation by facilitating self-interaction. We reveal that PCMD-1 interacts with the PCM scaffold protein SPD-5, the mitotic kinase PLK-1 and the centriolar protein SAS-4. Using an ectopic translocation assay, we show that PCMD-1 can selectively recruit downstream PCM scaffold components to an ectopic location in the cell, indicating that PCMD-1 is able to anchor the PCM scaffold proteins at the centrioles. Our work suggests that PCMD-1 is an essential functional bridge between the centrioles and the PCM.

Abstract P351: The Post-transcriptional Regulations Of Centrosomal Plp Mrna In Drosophila

Centrosomes, functioning as microtubule organizing centers, are composed of a proteinaceous matrix of pericentriolar material (PCM) that surrounds a pair of centrioles. Drosophila Pericentrin (Pcnt)-like protein (PLP) is a key component of the centrosome that serves as a scaffold for PCM assembly. The disruption of plp in Drosophila results in embryonic lethality, while the deregulation of Pcnt in humans is associated with MOPD II and Trisomy 21.We recently found plp mRNA localizes to Drosophila embryonic centrosomes. While RNA is known to associate with centrosomes in diverse cell types, the elements required for plp mRNA localization to centrosomes remains completely unknown. Additionally, how plp translation is regulated to accommodate rapid cell divisions during early embryogenesis is unclear. RNA localization coupled with translational control is a conserved mechanism that functions in diverse cellular processes. Control of mRNA localization and translation is mediated by RNA-binding proteins (RBPs). We find PLP protein expression is specifically promoted by an RNA-binding protein, Orb, during embryogenesis; moreover, plp mRNA interacts with Orb. Importantly, we find overexpression of full-length PLP can rescue cell division defects and embryonic lethality caused by orb depletion. We aim to uncover the mechanisms underlying embryonic plp mRNA localization and function and how Orb regulates plp translation.

The Transformation of the Centrosome into the Basal Body: Similarities and Dissimilarities between Somatic and Male Germ Cells and Their Relevance for Male Fertility.

The sperm flagellum is essential for the transport of the genetic material toward the oocyte and thus the transmission of the genetic information to the next generation. During the haploid phase of spermatogenesis, i.e., spermiogenesis, a morphological and molecular restructuring of the male germ cell, the round spermatid, takes place that includes the silencing and compaction of the nucleus, the formation of the acrosomal vesicle from the Golgi apparatus, the formation of the sperm tail, and, finally, the shedding of excessive cytoplasm. Sperm tail formation starts in the round spermatid stage when the pair of centrioles moves toward the posterior pole of the nucleus. The sperm tail, eventually, becomes located opposed to the acrosomal vesicle, which develops at the anterior pole of the nucleus. The centriole pair tightly attaches to the nucleus, forming a nuclear membrane indentation. An articular structure is formed around the centriole pair known as the connecting piece, situated in the neck region and linking the sperm head to the tail, also named the head-to-tail coupling apparatus or, in short, HTCA. Finally, the sperm tail grows out from the distal centriole that is now transformed into the basal body of the flagellum. However, a centriole pair is found in nearly all cells of the body. In somatic cells, it accumulates a large mass of proteins, the pericentriolar material (PCM), that together constitute the centrosome, which is the main microtubule-organizing center of the cell, essential not only for the structuring of the cytoskeleton and the overall cellular organization but also for mitotic spindle formation and chromosome segregation. However, in post-mitotic (G1 or G0) cells, the centrosome is transformed into the basal body. In this case, one of the centrioles, which is always the oldest or mother centriole, grows the axoneme of a cilium. Most cells of the body carry a single cilium known as the primary cilium that serves as an antenna sensing the cell’s environment. Besides, specialized cells develop multiple motile cilia differing in substructure from the immotile primary cilia that are essential in moving fluids or cargos over the cellular surface. Impairment of cilia formation causes numerous severe syndromes that are collectively subsumed as ciliopathies. This comparative overview serves to illustrate the molecular mechanisms of basal body formation, their similarities, and dissimilarities, in somatic versus male germ cells, by discussing the involved proteins/genes and their expression, localization, and function. The review, thus, aimed to provide a deeper knowledge of the molecular players that is essential for the expansion of clinical diagnostics and treatment of male fertility disorders.

The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles

Centrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes assemble centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, we show that, during spermatogenesis of the bryophyte Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. Interestingly, we observe that these centrioles are twisted in opposite orientations. Microtubules emanate from the bicentrioles, which localize to the spindle poles during cell division. After their separation, the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two motile cilia are assembled that appear to alternate between different motility patterns. We further show that centriolar components SAS6, Bld10, and POC1, which are conserved across eukaryotes, are expressed during spermatogenesis and required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis, while driven by conserved molecular modules, is more diverse than previously thought.

Multifaceted roles of centrosomes in development, health, and disease.

The centrosome is a membrane-less organelle consisting of a pair of barrel-shaped centrioles and pericentriolar material and functions as the major microtubule-organizing center and signaling hub in animal cells. The past decades have witnessed the functional complexity and importance of centrosomes in various cellular processes such as cell shaping, division, and migration. In addition, centrosome abnormalities are linked to a wide range of human diseases and pathological states, such as cancer, reproductive disorder, brain disease, and ciliopathies. Herein, we discuss various functions of centrosomes in development and health, with an emphasis on their roles in germ cells, stem cells, and immune responses. We also discuss how centrosome dysfunctions are involved in diseases. A better understanding of the mechanisms regulating centrosome functions may lead the way to potential therapeutic targeting of this organelle in disease treatment.

WDR62 is required for centriole duplication in spermatogenesis and manchette removal in spermiogenesis

WDR62 is a scaffold protein involved in centriole duplication and spindle assembly during mitosis. Mutations in WDR62 can cause primary microcephaly and premature ovarian insufficiency. We have generated a genetrap mouse model deficient in WDR62 and characterised the developmental effects of WDR62 deficiency during meiosis in the testis. We have found that WDR62 deficiency leads to centriole underduplication in the spermatocytes due to reduced or delayed CEP63 accumulation in the pericentriolar matrix. This resulted in prolonged metaphase that led to apoptosis. Round spermatids that inherited a pair of centrioles progressed through spermiogenesis, however, manchette removal was delayed in WDR62 deficient spermatids due to delayed Katanin p80 accumulation in the manchette, thus producing misshapen spermatid heads with elongated manchettes. In mice, WDR62 deficiency resembles oligoasthenoteratospermia, a common form of subfertility in men that is characterised by low sperm counts, poor motility and abnormal morphology. Therefore, proper WDR62 function is necessary for timely spermatogenesis and spermiogenesis during male reproduction.

Structure and dynamics of photoreceptor sensory cilia.

The rod and cone photoreceptor cells of the vertebrate retina have highly specialized structures that enable them to carry out their function of light detection over a broad range of illumination intensities with optimized spatial and temporal resolution. Most prominent are their unusually large sensory cilia, consisting of outer segments packed with photosensitive disc membranes, a connecting cilium with many features reminiscent of the primary cilium transition zone, and a pair of centrioles forming a basal body which serves as the platform upon which the ciliary axoneme is assembled. These structures form a highway through which an enormous flux of material moves on a daily basis to sustain the continual turnover of outer segment discs and the energetic demands of phototransduction. After decades of study, the details of the fine structure and distribution of molecular components of these structures are still incompletely understood, but recent advances in cellular imaging techniques and animal models of inherited ciliary defects are yielding important new insights. This knowledge informs our understanding both of the mechanisms of trafficking and assembly and of the pathophysiological mechanisms of human blinding ciliopathies.