Higher-order Organization Research Articles

Background-suppressed live visualization of genomic loci with an improved CRISPR system based on a split fluorophore.

The higher-order structural organization and dynamics of the chromosomes play a central role in gene regulation. To explore this structure–function relationship, it is necessary to directly visualize genomic elements in living cells. Genome imaging based on the CRISPR system is a powerful approach but has limited applicability due to background signals and nonspecific aggregation of fluorophores within nuclei. To address this issue, we developed a novel visualization scheme combining tripartite fluorescent proteins with the SunTag system and demonstrated that it strongly suppressed background fluorescence and amplified locus-specific signals, allowing long-term tracking of genomic loci. We integrated the multicomponent CRISPR system into stable cell lines to allow quantitative and reliable analysis of dynamic behaviors of genomic loci. Due to the greatly elevated signal-to-background ratio, target loci with only small numbers of sequence repeats could be successfully tracked, even under a conventional fluorescence microscope. This feature enables the application of CRISPR-based imaging to loci throughout the genome and opens up new possibilities for the study of nuclear processes in living cells.

Myosin-18B Regulates Higher-Order Organization of the Cardiac Sarcomere through Thin Filament Cross-Linking and Thick Filament Dynamics

MYO18B loss-of-function mutations and depletion significantly compromise the structural integrity of striated muscle sarcomeres. The molecular function of the encoded protein, myosin-18B (M18B), within the developing muscle is unknown. Here, we demonstrate that recombinant M18B lacks motor ATPase activity and harbors previously uncharacterized N-terminal actin-binding domains, properties that make M18B an efficient actin cross-linker and molecular brake capable of regulating muscle myosin-2 contractile forces. Spatiotemporal analysis of M18B throughout cardiomyogenesis and myofibrillogenesis reveals that this structural myosin undergoes nuclear-cytoplasmic redistribution during myogenic differentiation, where its incorporation within muscle stress fibers coincides with actin striation onset. Furthermore, this analysis shows that M18B is directly integrated within the muscle myosin thick filament during myofibril maturation. Altogether, our data suggest that M18B has evolved specific biochemical properties that allow it to define and maintain sarcomeric organization from within the thick filament via its dual actin cross-linking and motor modulating capabilities.

Optimized Stochastic Optical Reconstruction Microscopy for Imaging Chromatin Structure in Pathological Tissue.

Direct visualization of higher-order chromatin structure at the molecular scale is of great importance for understanding the impact of chromatin organization on gene expression in many biological processes. Understanding the changes in chromatin structure during pathological processes requires the use of in vivo models and clinical samples, and formalin-fixed, paraffin-embedded (FFPE) tissue is the most widespread form of preservation. Here we describe the details of PathSTORM, an optimized stochastic optical reconstruction microscopy (STORM) protocol for high-quality super-resolution imaging of densely packed higher-order chromatin organization in pathological tissue. We discuss detailed methods for fluorescence staining of DNA and histone proteins, as well as the key technical factors for obtaining high-quality STORM images in pathological tissue samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Fluorescence staining of chromatin in pathological tissue Basic Protocol 2: STORM data processing Support Protocol 1: Drift correction Support Protocol 2: Image reconstruction Support Protocol 3: Hematoxylin & eosin (H&E) staining.

FreeHi-C spike-in simulations for benchmarking differential chromatin interaction detection.

High-throughput genome-wide chromatin conformation capture assay (Hi-C) is routinely used to profile long-range genomic interactions and three-dimensional organization of genomes. A key application of Hi-C is the comparative analysis of genomic interactions across different time points, cellular conditions, or multiple stimuli. While operating characteristics of methods for Hi-C data processing such as normalization, pairwise interaction and higher-order organization detection have been relatively well studied, properties of methods for differential chromatin interaction detection are less investigated. We have recently developed FreeHi-C to enable data-driven non-parametric simulations from Hi-C experiments. Here, we extend FreeHi-C with a user/data-driven spike-in module to facilitate comparisons of differential chromatin interaction detection methods where the ground truth differential chromatin interactions are known under a wide variety of settings. We use FreeHi-C to benchmark four differential chromatin interaction detection methods, namely HiCcompare, multiHiCcompare, diffHic, and Selfish, using three comparative analysis settings with different sequencing depths and spike-in proportions. This comparison reveals distinguished performances in terms of the standard metrics such as the false discovery rate control, detection power, significance order, precision-recall curve, and receiver operating characteristic curve as well as overall genomic properties of the types of differential chromatin interactions detectable by each method. Furthermore, it highlights the lack of power for all methods in small replication settings.

Abstract A42: Development and validation of diagnostic biomarkers for B-cell lymphoma using EpiSwitchTM profiling of whole blood: From humans to canines

Abstract Backgound: Changes in chromosome conformation are strongly associated with different clinical phenotypes and outcomes for a variety of diseases. Here, we used the EpiSwitchTM platform technology to evaluate chromosome conformation signatures (CCS) as biomarkers for detection of canine diffuse large B-cell lymphoma (DLBCL). We examined whether established, systemic liquid biopsy biomarkers previously characterized in human DLBCL patients by EpiSwitchTM would translate to dogs with the homologous disease. Orthologous sequence conversion of CCS from humans to dogs was first verified and validated in control and lymphoma canine cohorts. Methods: Blood samples from dogs with DLBCL and from apparently healthy dogs were obtained as part of the comparative cancer genomics and veterinary clinical trials programs of the Animal Cancer Care and Research Program of the University of Minnesota. Dog owners provided signed informed consent, and all studies were done under protocols approved by the Institutional Animal Care Committee of the University of Minnesota. All of the dogs diagnosed with DLBCL were part of the LICKing Lymphoma trial. Blood samples were obtained from each dog prior to initiating treatment at day+5 after the experimental intervention, but prior to initiating doxorubicin chemotherapy. EpiSwitchTM technology (Oxford Biodynamics) was used to monitor systemic epigenetic biomarkers for CCS. This application was based on published methodology for validated predictive biomarkers for response to treatment, systemic blood-based monitoring of oncologic conditions, and proprietary programs in collaboration with Roche/Genentech for liquid biopsy development for DLBCL prognosis. Results: A 11-marker classifier was generated with whole blood from 28 dogs, 14 diagnosed with DLBCL and 14 controls with no apparent disease, from a pool of 75 EpiSwitchTM CCSs identified in human DLBCL. Validation of the developed diagnostic markers was performed on a second cohort of 10 dogs: 5 with DLBCL and 5 controls. The classifier delivered stratifications for DLBCL vs. non-DLBCL with 80% accuracy, 80% sensitivity, 80% specificity, 80% positive predictive value (PPV), and 80% negative predictive value (NPV) on the second cohort. Conclusions: The established EpiSwitchTM classifier contains strong systemic binary markers of epigenetic deregulation with features normally attributed to genetic markers: the binary status of these classifying markers is statistically significant for diagnosis, and it is translatable across species. The data suggest that certain features of the higher-order structural chromatin organization associated with DLBCL, and perhaps other homologous cancers, may be evolutionarily conserved. Altogether, these findings highlight the potential of the EpiSwitchTM approach for disease diagnosis and further applications using liquid biopsies in humans and dogs. Citation Format: Lauren J. Mills, Ewan Hunter, Aaron L. Sarver, Davis Seelig, Jennifer Back, Louis James, Navin Jandor, Polina Brayer, Thomas Lavin, Ryan Powell, Matthew Salter, Jayne Green, Aroul Ramadass, Alexandre Akoulitchev, Jaime F. Modiano. Development and validation of diagnostic biomarkers for B-cell lymphoma using EpiSwitchTM profiling of whole blood: From humans to canines [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A42.

Emerging views of genome organization in Archaea.

Over the past decade, advances in methodologies for the determination of chromosome conformation have provided remarkable insight into the local and higher-order organization of bacterial and eukaryotic chromosomes. Locally folded domains are found in both bacterial and eukaryotic genomes, although they vary in size. Importantly, genomes of metazoans also possess higher-order organization into A- and B-type compartments, regions of transcriptionally active and inactive chromatin, respectively. Until recently, nothing was known about the organization of genomes of organisms in the third domain of life - the archaea. However, despite archaea possessing simple circular genomes that are morphologically reminiscent of those seen in many bacteria, a recent study of archaea of the genus Sulfolobus has revealed that it organizes its genome into large-scale domains. These domains further interact to form defined A- and B-type compartments. The interplay of transcription and localization of a novel structural maintenance of chromosomes (SMC) superfamily protein, termed coalescin, defines compartment identity. In this Review, we discuss the mechanistic and evolutionary implications of these findings.

MATCHA: Probing multi-way chromatin interaction with hypergraph representation learning.

Recent advances in ligation-free, genome-wide chromatin interaction mapping such as SPRITE and ChIA-Drop have enabled the identification of simultaneous interactions involving multiple genomic loci within the same nuclei, which are informative to delineate higher-order genome organization and gene regulation mechanisms at single-nucleus resolution. Unfortunately, computational methods for analyzing multi-way chromatin interaction data are significantly underexplored. Here we develop an algorithm, called MATCHA, based on hypergraph representation learning where multi-way chromatin interactions are represented as hyperedges. Applications to SPRITE and ChIA-Drop data suggest that MATCHA is effective to denoise the data and make de novo predictions, which greatly enhances the data quality for analyzing the properties of multi-way chromatin interactions. MATCHA provides a promising framework to significantly improve the analysis of multi-way chromatin interaction data and has the potential to offer unique insights into higher-order chromosome organization and function. MATCHA is freely available for download here: https://github.com/ma-compbio/MATCHA.

Liquid-liquid phase separation in biology: mechanisms, physiological functions and human diseases.

Cells are compartmentalized by numerous membrane-enclosed organelles and membraneless compartments to ensure that a wide variety of cellular activities occur in a spatially and temporally controlled manner. The molecular mechanisms underlying the dynamics of membrane-bound organelles, such as their fusion and fission, vesicle-mediated trafficking and membrane contactmediated inter-organelle interactions, have been extensively characterized. However, the molecular details of the assembly and functions of membraneless compartments remain elusive. Mounting evidence has emerged recently that a large number of membraneless compartments, collectively called biomacromolecular condensates, are assembled via liquid-liquid phase separation (LLPS). Phase-separated condensates participate in various biological activities, including higher-order chromatin organization, gene expression, triage of misfolded or unwanted proteins for autophagic degradation, assembly of signaling clusters and actin- and microtubule-based cytoskeletal networks, asymmetric segregations of cell fate determinants and formation of pre- and post-synaptic density signaling assemblies. Biomacromolecular condensates can transition into different material states such as gel-like structures and solid aggregates. The material properties of condensates are crucial for fulfilment of their distinct functions, such as biochemical reaction centers, signaling hubs and supporting architectures. Cells have evolved multiple mechanisms to ensure that biomacromolecular condensates are assembled and disassembled in a tightly controlled manner. Aberrant phase separation and transition are causatively associated with a variety of human diseases such as neurodegenerative diseases and cancers. This review summarizes recent major progress in elucidating the roles of LLPS in various biological pathways and diseases.

Weak interactions in higher-order chromatin organization.

The detailed principles of the hierarchical folding of eukaryotic chromosomes have been revealed during the last two decades. Along with structures composing three-dimensional (3D) genome organization (chromatin compartments, topologically associating domains, chromatin loops, etc.), the molecular mechanisms that are involved in their establishment and maintenance have been characterized. Generally, protein–protein and protein–DNA interactions underlie the spatial genome organization in eukaryotes. However, it is becoming increasingly evident that weak interactions, which exist in biological systems, also contribute to the 3D genome. Here, we provide a snapshot of our current understanding of the role of the weak interactions in the establishment and maintenance of the 3D genome organization. We discuss how weak biological forces, such as entropic forces operating in crowded solutions, electrostatic interactions of the biomolecules, liquid-liquid phase separation, DNA supercoiling, and RNA environment participate in chromosome segregation into structural and functional units and drive intranuclear functional compartmentalization.

Intercellular communication between artificial cells by allosteric amplification of a molecular signal

Multicellular organisms rely on intercellular communication to coordinate the behaviour of individual cells, which enables their differentiation and hierarchical organization. Various cell mimics have been developed to establish fundamental engineering principles for the construction of artificial cells displaying cell-like organization, behaviour and complexity. However, collective phenomena, although of great importance for a better understanding of life-like behaviour, are underexplored. Here, we construct collectives of giant vesicles that can communicate with each other through diffusing chemical signals that are recognized and processed by synthetic enzymatic cascades. Similar to biological cells, the Receiver vesicles can transduce a weak signal originating from Sender vesicles into a strong response by virtue of a signal amplification step, which facilitates the propagation of signals over long distances within the artificial cell consortia. This design advances the development of interconnected artificial cells that can exchange metabolic and positional information to coordinate their higher-order organization.

Exploring Uncharted Territories of Plant Specialized Metabolism in the Postgenomic Era.

For millennia, humans have used plants for food, raw materials, and medicines, but only within the past two centuries have we begun to connect particular plant metabolites with specific properties and utilities. Since the utility of classical molecular genetics beyond model species is limited, the vast specialized metabolic systems present in the Earth's flora remain largely unstudied. With an explosion in genomics resources and a rapidly expanding toolbox over the past decade, exploration of plant specialized metabolism in nonmodel species is becoming more feasible than ever before. We review the state-of-the-art tools that have enabled this rapid progress. We present recent examples of de novo biosynthetic pathway discovery that employ various innovative approaches. We also draw attention to the higher-order organization of plant specialized metabolism at subcellular, cellular, tissue, interorgan, and interspecies levels, which will have important implications for the future design of comprehensive metabolic engineering strategies.

Rearrangement of macronucleus chromosomes correspond to TAD-like structures of micronucleus chromosomes in Tetrahymena thermophila.

The somatic macronucleus (MAC) and germline micronucleus (MIC) of Tetrahymena thermophila differ in chromosome numbers, sizes, functions, transcriptional activities, and cohesin complex location. However, the higher-order chromatin organization in T. thermophila is still largely unknown. Here, we explored the higher-order chromatin organization in the two distinct nuclei of T. thermophila using the Hi-C and HiChIP methods. We found that the meiotic crescent MIC has a specific chromosome interaction pattern, with all the telomeres or centromeres on the five MIC chromosomes clustering together, respectively, which is also helpful to identify the midpoints of centromeres in the MIC. We revealed that the MAC chromosomes lack A/B compartments, topologically associating domains (TADs), and chromatin loops. The MIC chromosomes have TAD-like structures but not A/B compartments and chromatin loops. The boundaries of the TAD-like structures in the MIC are highly consistent with the chromatin breakage sequence (CBS) sites, suggesting that each TAD-like structure of the MIC chromosomes develops into one MAC chromosome during MAC development, which provides a mechanism of the formation of MAC chromosomes during conjugation. Overall, we demonstrated the distinct higher-order chromatin organization in the two nuclei of the T. thermophila and suggest that the higher-order chromatin structures may play important roles during the development of the MAC chromosomes.

TOPORS, a tumor suppressor protein, contributes to the maintenance of higher-order chromatin architecture

In the nucleus, chromosomes are hierarchically folded into active (A) and inactive (B) compartments composed of topologically associating domains (TADs). Genomic regions interact with nuclear lamina, termed lamina-associated domains (LADs). However, the molecular mechanisms underlying these 3D chromatin architectures remain incompletely understood. Here, we investigated the role of a potential tumor suppressor, TOP1 Binding Arginine/Serine Rich Protein (TOPORS), in genome organization. In mouse hepatocytes, chromatin interactions between A and B compartments increase and compartmentalization strength is reduced significantly upon Topors knockdown. Correspondingly, strength of TAD boundaries located at A/B borders is weakened. In the absence of TOPORS, chromatin-lamina interactions decrease and the coverage of LADs reduces from 53.31% to 46.52%. Interestingly, these changes in 3D genome are associated with PML nuclear bodies and PML-associated domains (PADs). Moreover, chromatin accessibility is altered predominantly at intergenic regions upon Topors knockdown, including a subset of enhancers. These alterations of chromatin are concordant with transcriptome changes, which are associated with carcinogenesis. Collectively, our findings demonstrate that TOPORS functions as a regulator in chromatin structure, providing novel insight into the architectural roles of tumor suppressors in higher-order genome organization.

Abstract BS1-2: Genome regulation by long noncoding RNA genes

Abstract The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation. I will discuss genome-scale strategies to discover and characterize lncRNAs, including RNA chemical modifications and RNA structures. LncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators, and target these enzymatic activities to appropriate locations in the genome. Long noncoding RNAs can function as modular scaffolds to specify higher order organization in RNP complexes and in chromatin states. A new emerging theme is that DNA elements that mediate lncRNA expression can also have powerful effects in controlling chromatin neighborhoods, which alter oncogene expression and activity. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs in human diseases. Citation Format: HY Chang. Genome regulation by long noncoding RNA genes [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr BS1-2.

Abstract P4-04-03: High-throughput epigenetic screening in hormone receptor positive (HR+) breast cancer cells

Abstract Background & Objective: Substantial progress has been made in treating HR+ metastatic breast cancer patients with CDK4/6, mTOR and PIK3CA inhibitors (all FDA approved), but there are no current available therapies to directly restore sensitivity to endocrine therapy once patients’ cells develop epigenetic suppression of ER pathways. CCCTC-binding factor (CTCF), which regulates higher-order chromatin organization and estrogen reception (ER) transcription, is altered by hemizygous deletion or mutation in more than 50% of HR+ luminal type breast tumors. The goal of this project is to characterize an epigenetic signature in CTCF-altered breast cancers to identify novel epigenetic targets for therapeutics development. Targeting CTCF influenced pathways with or without its chaperones may restore sensitivity to endocrine therapy in patients with HR+ breast cancer. Methods: We identified patient-derived cell lines from the Cancer Cell Line Encyclopedia (CCLE) with known CTCF gene copy number. Selected cell lines were tested for proliferation with a custom siRNA library targeting 1,300 genes involved in epigenetic regulation while in the presence of different concentrations of estradiol, tamoxifen and fulvestrant. We confirmed presence of CTCF protein by western blot and mRNA expression by real-time PCR. Proliferation of the lines in vitro was quantified by the MTT assay. Further functional annotation and pathway enrichment analysis of the identified genes were performed by using the g:ProfileR package. Results: We have screened more than 60 breast cancer cell lines and focused on six HR+ luminal (3 each “a” and “b” type) lines with different levels of CTCF copy number; a normal breast epithelial line was used as control. We observed that cell growth responded to anti-estrogen therapy differently based on the copy number of CTCF. Cells with high copy number of CTCF are resistant to endocrine therapy, the sensitivity was restored by silencing CTCF using siRNA. We then selected two cell lines with low and high copy number of CTCF for further high-throughput screening with the epigenetic siRNA library. A total of 67 and 35 candidate sensitizer target genes, and 56 and 44 candidate enhancer target genes were prioritized in cells with low and high copy number of CTCF, respectively. Functional annotation and pathway enrichment analysis of the identified genes showed that cells with low copy number of CTCF had apoptosis related hits, such as BCL family and caspase genes; however, cells with high copy number of CTCF were found to relate to other epigenetic regulators such as DNMTs and CHD. This suggests that CTCF might form complexes with other epigenetic proteins to form abnormal chromatin configuration with abnormal regulation of ER pathway as consequence. Conclusions: To our knowledge, this is the first epigenetic library screen of HR+ luminal breast cancer cells with responses categorized by differential CTCF copy number status. We have confirmed CTCF as an important protein in predicting response to endocrine therapy and identified possible interactions between CTCF, DNMT and CHD. We conclude that development of therapeutics against targets identified in our screen to reverse endocrine resistance may be achievable for treatment of luminal breast cancers that undertake epigenetic deregulation. Keywords: Breast cancer; Hormone receptor positive; epigenetic screening Citation Format: Fengting Yan, Olga Nikolova, VK Gadi. High-throughput epigenetic screening in hormone receptor positive (HR+) breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-03.

Radial Organization in the Mammalian Nucleus.

In eukaryotic cells, most of the genetic material is contained within a highly specialized organelle—the nucleus. A large body of evidence indicates that, within the nucleus, chromatinized DNA is spatially organized at multiple length scales. The higher-order organization of chromatin is crucial for proper execution of multiple genome functions, including DNA replication and transcription. Here, we review our current knowledge on the spatial organization of chromatin in the nucleus of mammalian cells, focusing in particular on how chromatin is radially arranged with respect to the nuclear lamina. We then discuss the possible mechanisms by which the radial organization of chromatin in the cell nucleus is established. Lastly, we propose a unifying model of nuclear spatial organization, and suggest novel approaches to test it.

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

Plant Chromatin Catches the Sun.

Plants use solar radiation as energy source for photosynthesis. They also take advantage of the information provided by the varying properties of sunlight, such as wavelength, orientation, and periodicity, to trigger physiological and developmental adaptations to a changing environment. After more than a century of research efforts in plant photobiology, multiple light signaling pathways converging onto chromatin-based mechanisms have now been identified, which in some instances play critical roles in plant phenotypic plasticity. In addition to locus-specific changes linked to transcription regulation, light signals impact higher-order chromatin organization. Here, we summarize current knowledge on how light can affect the global composition and the spatial distribution of chromatin domains. We introduce emerging questions on the functional links between light signaling and the epigenome, and further discuss how different chromatin regulatory layers may interconnect during plant adaptive responses to light.

Probing 3D genome by CRISPR/Cas9.

CRISPR/Cas9 system has significant advantages in gene editing strategy, offering cost-effective and efficient means to modify and edit the genomes of animals, plants, and microorganisms. Three-dimensional (3D) genome is an emerging and interdisciplinary field catapulted by combined technological breakthroughs of chromosome conformation capture with next-generation sequencing and live imaging with super-resolution microscopy. An important aspect of 3D genomics is to model structural variations and label specific genomic fragments to investigate the effects of manipulation of genomic elements on gene expression regulation, cell development and differentiation, and spatial location of chromosomal regions. Therefore, CRISPR/Cas9 system and its derivative technologies of DNA-fragment editing are excellent toolboxes for investigating dynamics and functions of the higher-order chromatin organization and three-dimensional genome structure. In this review, we describe the opportunities and challenges of CRISPR as well as its derivative technologies in 3D genome research, thereby providing some critical references and future research directions in the field.

Distinct roles for prominin-1 and photoreceptor cadherin in outer segment disc morphogenesis in CRISPR-altered X. laevis.

Mutations in prominin-1 (prom1) and photoreceptor cadherin (cdhr1) are associated with inherited retinal degenerative disorders but their functions remain unknown. Here, we used CRISPR-Cas9 to generate prom1-null, cdhr1-null, and prom1pluscdhr1 double-null Xenopuslaevis and then documented the effects of these mutations on photoreceptor structure and function. Prom1-null mutations resulted in severely dysmorphic photoreceptors comprising overgrown and disorganized disc membranes. Cone outer segments were more severely affected than rods and had an impaired electroretinogram response. Cdhr1-null photoreceptors did not appear grossly dysmorphic, but ultrastructural analysis revealed that some disc membranes were overgrown or oriented vertically within the plasma membrane. Double-null mutants did not differ significantly from prom1-null mutants. Our results indicate that neither prom1 nor cdhr1 are necessary for outer segment disc membrane evagination or the fusion event that controls disc sealing. Rather, they are necessary for the higher-order organization of the outer segment. Prom1 may align and reinforce interactions between nascent disc leading edges, a function more critical in cones for structural support. Cdhr1 may secure discs in a horizontal orientation prior to fusion and regulate cone lamellae size.This article has an associated First Person interview with the first author of the paper.