Interferon regulatory factor 4 modulates epigenetic silencing and cancer-critical pathways in melanoma cells.

Interferon regulatory factor 4 (IRF4) was initially identified as a key controller in lymphocyte differentiation and function, and subsequently as a dependency factor and therapy target in lymphocyte-derived cancers. In melanocytes, IRF4 takes part in pigmentation. Although genetic studies have implicated IRF4 in melanoma, how IRF4 functions in melanoma cells has remained largely elusive. Here, we confirmed prevalent IRF4 expression in melanoma and showed that high expression is linked to dependency in cells and mortality in patients. Analysis of genes activated by IRF4 uncovered, as a novel target category, epigenetic silencing factors involved in DNA methylation (DNMT1, DNMT3B, UHRF1) and histone H3K27 methylation (EZH2). Consequently, we show that IRF4 controls the expression of tumour suppressor genes known to be silenced by these epigenetic modifications, for instance cyclin-dependent kinase inhibitors CDKN1A and CDKN1B, the PI3-AKT pathway regulator PTEN, and primary cilium components. Furthermore, IRF4 modulates activity of key downstream oncogenic pathways, such as WNT/ß-catenin and AKT, impacting cell proliferation and survival. Accordingly, IRF4 modifies the effectiveness of pertinent epigenetic drugs on melanoma cells, a finding that encourages further studies towards therapeutic targeting of IRF4 in melanoma.

Navigating centriolar satellites: the role of PCM1 in cellular and organismal processes.

Centriolar satellites are ubiquitous membrane-less organelles that play critical roles in numerous cellular and organismal processes. They were initially discovered through electron microscopy as cytoplasmic granules surrounding centrosomes in vertebrate cells. These structures remained enigmatic until the identification of pericentriolar material 1 protein (PCM1) as their molecular marker, which has enabled their in-depth characterization. Recently, centriolar satellites have come into the spotlight due to their links to developmental and neurodegenerative disorders. This review presents a comprehensive summary of the major advances in centriolar satellite biology, with a focus on studies that investigated their biology associated with the essential scaffolding protein PCM1. We begin by exploring the molecular, cellular, and biochemical properties of centriolar satellites, laying the groundwork for a deeper understanding of their functions and mechanisms at both cellular and organismal levels. We then examine the implications of their dysregulation in various diseases, particularly highlighting their emerging roles in neurodegenerative and developmental disorders, as revealed by organismal models of PCM1. We conclude by discussing the current state of knowledge and posing questions about the adaptable nature of these organelles, thereby setting the stage for future research.

Development of a mouse embryonic stem cell model for investigating the functions of the linker histone H1-4.

The linker histone H1 C-terminal domain (CTD) plays a pivotal role in chromatin condensation. De novo frameshift mutations within the CTD coding region of H1.4 have recently been reported to be associated with Rahman syndrome, a neurological disease that causes intellectual disability and overgrowth. To investigate the mechanisms and pathogenesis of Rahman syndrome, we developed a cellular model using murine embryonic stem cells (mESCs) and CRISPR/Cas9 genome engineering. Our engineered mES cells facilitate detailed investigations, such as H1-4 dynamics, immunoprecipitation, and nuclear localization; in addition, we tagged the mutant H1-4 with a photoactivatable GFP (PA-GFP) and an HA tag to facilitate pulldown assays. We anticipate that these engineered cells could also be used for the development of a mouse model to study the in vivo role of the H1-4 protein.

The multifaceted roles of microtubule-associated proteins in the primary cilium and ciliopathies.

The primary cilium is a conserved microtubule-based organelle that is critical for transducing developmental, sensory and homeostatic signaling pathways. It comprises an axoneme with nine parallel doublet microtubules extending from the basal body, surrounded by the ciliary membrane. The axoneme exhibits remarkable stability, serving as the skeleton of the cilium in order to maintain its shape and provide tracks to ciliary trafficking complexes. Although ciliary trafficking and signaling have been exhaustively characterized over the years, less is known about the unique structural and functional complexities of the axoneme. Recent work has yielded new insights into the mechanisms by which the axoneme is built with its proper length and architecture, particularly regarding the activity of microtubule-associated proteins (MAPs). In this Review, we first summarize current knowledge about the architecture, composition and specialized compartments of the primary cilium. Next, we discuss the mechanistic underpinnings of how a functional cilium is assembled, maintained and disassembled through the regulation of its axonemal microtubules. We conclude by examining the diverse localizations and functions of ciliary MAPs for the pathobiology of ciliary diseases.

CCDC15 localizes to the centriole inner scaffold and controls centriole length and integrity.

Centrioles are microtubule-based organelles responsible for forming centrosomes and cilia, which serve as microtubule-organizing, signaling, and motility centers. Biogenesis and maintenance of centrioles with proper number, size, and architecture are vital for their functions during development and physiology. While centriole number control has been well-studied, less is understood about their maintenance as stable structures with conserved size and architecture during cell division and ciliary motility. Here, we identified CCDC15 as a centriole protein that colocalizes with and interacts with the inner scaffold, a crucial centriolar subcompartment for centriole size control and integrity. Using ultrastructure expansion microscopy, we found that CCDC15 depletion affects centriole length and integrity, leading to defective cilium formation, maintenance, and response to Hedgehog signaling. Moreover, loss-of-function experiments showed CCDC15's role in recruiting both the inner scaffold protein POC1B and the distal SFI1/Centrin-2 complex to centrioles. Our findings reveal players and mechanisms of centriole architectural integrity and insights into diseases linked to centriolar defects.

CCDC66 regulates primary cilium length and signaling via interactions with transition zone and axonemal proteins.

The primary cilium is a microtubule-based organelle that serves as a hub for many signaling pathways. It functions as part of the centrosome or cilium complex, which also contains the basal body and the centriolar satellites. Little is known about the mechanisms by which the microtubule-based ciliary axoneme is assembled with a proper length and structure, particularly in terms of the activity of microtubule-associated proteins (MAPs) and the crosstalk between the different compartments of the centrosome or cilium complex. Here, we analyzed CCDC66, a MAP implicated in cilium biogenesis and ciliopathies. Live-cell imaging revealed that CCDC66 compartmentalizes between centrosomes, centriolar satellites, and the ciliary axoneme and tip during cilium biogenesis. CCDC66 depletion in human cells causes defects in cilium assembly, length and morphology. Notably, CCDC66 interacts with the ciliopathy-linked MAPs CEP104 and CSPP1, and regulates axonemal length and Hedgehog pathway activation. Moreover, CCDC66 is required for the basal body recruitment of transition zone proteins and intraflagellar transport B (IFT-B) machinery. Overall, our results establish CCDC66 as a multifaceted regulator of the primary cilium and provide insight into how ciliary MAPs and subcompartments cooperate to ensure assembly of functional cilia.

An LED-Based structured illumination microscope using a digital micromirror device and GPU accelerated image reconstruction.

When combined with computational approaches, fluorescence imaging becomes one of the most powerful tools in biomedical research. It is possible to achieve resolution figures beyond the diffraction limit, and improve the performance and flexibility of high-resolution imaging systems with techniques such as structured illumination microscopy (SIM) reconstruction. In this study, the hardware and software implementation of an LED-based super-resolution imaging system using SIM employing GPU accelerated parallel image reconstruction is presented. The sample is illuminated with two-dimensional sinusoidal patterns with various orientations and lateral phase shifts generated using a digital micromirror device (DMD). SIM reconstruction is carried out in frequency space using parallel CUDA kernel functions. Furthermore, a general purpose toolbox for the parallel image reconstruction algorithm and an infrastructure that allows all users to perform parallel operations on images without developing any CUDA kernel code is presented. The developed image reconstruction algorithm was run separately on a CPU and a GPU. Two different SIM reconstruction algorithms have been developed for the CPU as mono-thread CPU algorithm and multi-thread OpenMP CPU algorithm. SIM reconstruction of 1024 × 1024 px images was achieved in 1.49 s using GPU computation, indicating an enhancement by ∼28 and ∼20 in computation time when compared with mono-thread CPU computation and multi-thread OpenMP CPU computation, respectively.

The ciliopathy protein CCDC66 controls mitotic progression and cytokinesis by promoting microtubule nucleation and organization.

Precise spatiotemporal control of microtubule nucleation and organization is critical for faithful segregation of cytoplasmic and genetic material during cell division and signaling via the primary cilium in quiescent cells. Microtubule-associated proteins (MAPs) govern assembly, maintenance, and remodeling of diverse microtubule arrays. While a set of conserved MAPs are only active during cell division, an emerging group of MAPs acts as dual regulators in dividing and nondividing cells. Here, we elucidated the nonciliary functions and molecular mechanism of action of the ciliopathy-linked protein CCDC66, which we previously characterized as a regulator of ciliogenesis in quiescent cells. We showed that CCDC66 dynamically localizes to the centrosomes, the bipolar spindle, the spindle midzone, the central spindle, and the midbody in dividing cells and interacts with the core machinery of centrosome maturation and MAPs involved in cell division. Loss-of-function experiments revealed its functions during mitotic progression and cytokinesis. Specifically, CCDC66 depletion resulted in defective spindle assembly and orientation, kinetochore fiber stability, chromosome alignment in metaphase as well as central spindle and midbody assembly and organization in anaphase and cytokinesis. Notably, CCDC66 regulates mitotic microtubule nucleation via noncentrosomal and centrosomal pathways via recruitment of gamma-tubulin to the centrosomes and the spindle. Additionally, CCDC66 bundles microtubules in vitro and in cells by its C-terminal microtubule-binding domain. Phenotypic rescue experiments showed that the microtubule and centrosome-associated pools of CCDC66 individually or cooperatively mediate its mitotic and cytokinetic functions. Collectively, our findings identify CCDC66 as a multifaceted regulator of the nucleation and organization of the diverse mitotic and cytokinetic microtubule arrays and provide new insight into nonciliary defects that underlie ciliopathies.

ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium content regulation.

Centrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their dysfunction causes cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of stable microtubules that maintain their length at a steady-state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified the Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that regulates microtubule organization and stability. Consistently, we observed an increase in tubulin polymerization and microtubule stability, as well as disrupted microtubule organization in ENKD1 overexpression. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results advance our understanding of the functional and regulatory relationship between MAPs and the primary cilium.

Microtubule-associated proteins and emerging links to primary cilium structure, assembly, maintenance, and disassembly.

The primary cilium is a microtubule-based structure that protrudes from the cell surface in diverse eukaryotic organisms. It functions as a key signaling center that decodes a variety of mechanical and chemical stimuli and plays fundamental roles in development and homeostasis. Accordingly, structural and functional defects of the primary cilium have profound effects on the physiology of multiple organ systems including kidney, retina, and central nervous system. At the core of the primary cilium is the microtubule-based axoneme, which supports the cilium shape and acts as the scaffold for bidirectional transport of cargoes into and out of cilium. Advances in imaging, proteomics, and structural biology have revealed new insights into the ultrastructural organization and composition of the primary cilium, the mechanisms that underlie its biogenesis and functions, and the pathologies that result from their deregulation termed ciliopathies. In this viewpoint, we first discuss the recent studies that identified the three-dimensional native architecture of the ciliary axoneme and revealed that it is considerably different from the well-known '9 + 0' paradigm. Moving forward, we explore emerging themes in the assembly and maintenance of the axoneme, with a focus on how microtubule-associated proteins regulate its structure, length, and stability. This far more complex picture of the primary cilium structure and composition, as well as the recent technological advances, open up new avenues for future research.

Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium.

Centriolar satellites are dynamic, membraneless granules composed of over 200 proteins. They store, modify, and traffic centrosome and primary cilium proteins, and help to regulate both the biogenesis and some functions of centrosomes and cilium. In most cell types, satellites cluster around the perinuclear centrosome, but their integrity and cellular distribution are dynamically remodeled in response to different stimuli, such as cell cycle cues. Dissecting the specific and temporal functions and mechanisms of satellites and how these are influenced by their cellular positioning and dynamics has been challenging using genetic approaches, particularly in ciliated and proliferating cells. To address this, we developed a chemical-based trafficking assay to rapidly and efficiently redistribute satellites to either the cell periphery or center, and fuse them into stable clusters in a temporally controlled way. Induced satellite clustering at either the periphery or center resulted in antagonistic changes in the pericentrosomal levels of a subset of proteins, revealing a direct and selective role for their positioning in protein targeting and sequestration. Systematic analysis of the interactome of peripheral satellite clusters revealed enrichment of proteins implicated in cilium biogenesis and mitosis. Importantly, induction of peripheral satellite targeting in ciliated cells revealed a function for satellites not just for efficient cilium assembly but also in the maintenance of steady-state cilia and in cilia disassembly by regulating the structural integrity of the ciliary axoneme. Finally, perturbing satellite distribution and dynamics inhibited their mitotic dissolution, and mitotic progression was perturbed only in cells with centrosomal satellite clustering. Collectively, our results for the first time showed a direct link between satellite functions and their pericentrosomal clustering, suggested new mechanisms underlying satellite functions during cilium assembly, and provided a new tool for probing temporal satellite functions in different contexts.

A Proximity Mapping Journey into the Biology of the Mammalian Centrosome/Cilium Complex.

The mammalian centrosome/cilium complex is composed of the centrosome, the primary cilium and the centriolar satellites, which together regulate cell polarity, signaling, proliferation and motility in cells and thereby development and homeostasis in organisms. Accordingly, deregulation of its structure and functions is implicated in various human diseases including cancer, developmental disorders and neurodegenerative diseases. To better understand these disease connections, the molecular underpinnings of the assembly, maintenance and dynamic adaptations of the centrosome/cilium complex need to be uncovered with exquisite detail. Application of proximity-based labeling methods to the centrosome/cilium complex generated spatial and temporal interaction maps for its components and provided key insights into these questions. In this review, we first describe the structure and cell cycle-linked regulation of the centrosome/cilium complex. Next, we explain the inherent biochemical and temporal limitations in probing the structure and function of the centrosome/cilium complex and describe how proximity-based labeling approaches have addressed them. Finally, we explore current insights into the knowledge we gained from the proximity mapping studies as it pertains to centrosome and cilium biogenesis and systematic characterization of the centrosome, cilium and centriolar satellite interactomes.

CCDC57 Cooperates with Microtubules and Microcephaly Protein CEP63 and Regulates Centriole Duplication and Mitotic Progression.

Centrosomes function in key cellular processes ranging from cell division to cellular signaling. Their dysfunction is linked to cancer and developmental disorders. Here, we identify CCDC57 as a pleiotropic regulator of centriole duplication, mitosis, and ciliogenesis. Combining proximity mapping with superresolution imaging, we show that CCDC57 localizes to the proximal end of centrioles and interacts with the microcephaly protein CEP63, centriolar satellite proteins, and microtubules. Loss of CCDC57 causes defects in centriole duplication and results in a failure to localize CEP63 and CEP152 to the centrosome. Additionally, CCDC57 depletion perturbs mitotic progression both in wild-type and centriole-less cells. Importantly, its centrosome-targeting region is required for its interaction with CEP63 and functions during centriole duplication and cilium assembly, whereas the microtubule-targeting region is required for its mitotic functions. Together, our results identify CCDC57 as a critical interface between centrosome and microtubule-mediated cellular processes that are deregulated in microcephaly.

Unraveling the mysteries of centriolar satellites: time to rewrite the textbooks about the centrosome/cilium complex.

Centriolar satellites are membraneless granules that localize and move around centrosomes and cilia. Once referred to as structures with no obvious function, research in the past decade has identified satellites as key regulators of a wide range of cellular and organismal processes. Importantly, these studies have revealed a substantial overlap between functions, proteomes, and disease links of satellites with centrosomes and cilia. Therefore, satellites are now accepted as the "third component" of the vertebrate centrosome/cilium complex, which profoundly changes the way we think about the assembly, maintenance, and remodeling of the complex at the cellular and organismal levels. In this perspective, we first provide an overview of the cellular and structural complexities of centriolar satellites. We then describe the progress in the identification of the satellite interactome, which have paved the way to a molecular understanding of their mechanism of action and assembly mechanisms. After exploring current insights into their functions as recently described by loss-of-function studies and comparative evolutionary approaches, we discuss major unanswered questions regarding their functional and compositional diversity and their functions outside centrosomes and cilia.

Proximity mapping of the microtubule plus-end tracking protein SLAIN2 using the BioID approach.

The centrosome is the main microtubule-organizing center of animal cells, which plays key roles in critical cellular processes ranging from cell division to cellular signaling. Accordingly, defects in the structure and function of centrosomes cause various human diseases such as cancer and primary microcephaly. To elucidate the molecular defects underlying these diseases, the biogenesis and functions of the centrosomes have to be fully understood. An essential step towards addressing these questions is the identification and functional dissection of the full repertoire of centrosome proteins. Here, we used high-resolution imaging and showed that the microtubule plus-end tracking protein SLAIN2 localizes to the pericentriolar material at the proximal end of centrioles. To gain insight into its cellular functions and mechanisms, we applied in vivo proximity-dependent biotin identification to SLAIN2 and generated its proximity interaction map. Gene ontology analysis of the SLAIN2 interactome revealed extensive interactions with centriole duplication, ciliogenesis, and microtubule-associated proteins, including previously characterized and uncharacterized interactions. Collectively, our results define SLAIN2 as a component of pericentriolar material and provide an important resource for future studies aimed at elucidating SLAIN2 functions at the centrosome.

Oncogenic K-Ras4B Dimerization Enhances Downstream Mitogen-activated Protein Kinase Signaling.

Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) ß-interface, mapped to Switch I and effector-binding regions, (ii) α-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the α- and ß-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. α-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While ß-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked K-Ras4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the α-interface in K-Ras4B homodimerization and the ß-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both α- and ß-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling.

Centrosomal and ciliary targeting of CCDC66 requires cooperative action of centriolar satellites, microtubules and molecular motors.

Mammalian centrosomes and cilia play key roles in many cellular processes and their deregulation is linked to cancer and ciliopathies. Spatiotemporal regulation of their biogenesis and function in response to physiological stimuli requires timely protein targeting. This can occur by different pathways, including microtubule-dependent active transport and via centriolar satellites, which are key regulators of cilia assembly and signaling. How satellites mediate their functions and their relationship with other targeting pathways is currently unclear. To address this, we studied retinal degeneration gene product CCDC66, which localizes to centrosomes, cilia, satellites and microtubules and functions in ciliogenesis. FRAP experiments showed that its centrosomal pool was dynamic and the ciliary pool associated with the ciliary axoneme and was stable. Centrosomal CCDC66 abundance and dynamics required microtubule-dependent active transport and tethering, and was inhibited by sequestration at satellites. Systematic quantitation of satellite dynamics identified only a small fraction to display microtubule-based bimodal motility, consistent with trafficking function. Majority displayed diffusive motility with unimodal persistence, supporting sequestration function. Together, our findings reveal new mechanisms of communication between membrane-less compartments.

Ecofriendly and Efficient Luminescent Solar Concentrators Based on Fluorescent Proteins.

In recent years, luminescent solar concentrators (LSCs) have received renewed attention as a versatile platform for large-area, high-efficiency, and low-cost solar energy harvesting. So far, artificial or engineered optical materials, such as rare-earth ions, organic dyes, and colloidal quantum dots (QDs) have been incorporated into LSCs. Incorporation of nontoxic materials into efficient device architectures is critical for environmental sustainability and clean energy production. Here, we demonstrated LSCs based on fluorescent proteins, which are biologically produced, ecofriendly, and edible luminescent biomaterials along with exceptional optical properties. We synthesized mScarlet fluorescent proteins in Escherichia coli expression system, which is the brightest protein with a quantum yield of 61% in red spectral region that matches well with the spectral response of silicon solar cells. Moreover, we integrated fluorescent proteins in an aqueous medium into solar concentrators, which preserved their quantum efficiency in LSCs and separated luminescence and wave-guiding regions due to refractive index contrast for efficient energy harvesting. Solar concentrators based on mScarlet fluorescent proteins achieved an external LSC efficiency of 2.58%, and the integration at high concentrations increased their efficiency approaching to 5%, which may facilitate their use as "luminescent solar curtains" for in-house applications. The liquid-state integration of proteins paves a way toward efficient and "green" solar energy harvesting.

The ciliopathy gene product Cep290 is required for primary cilium formation and microtubule network organization.

The mammalian centrosome/cilium complex is composed of the centrosome, the primary cilium, and the centriolar satellites, which together function in key cellular processes including signaling. Defective assembly, maintenance, and function of the centrosome/ cilium complex cause the human genetic diseases known as ciliopathies, which are characterized by a multitude of developmental syndromes including retinal degeneration and kidney cysts. The molecular mechanisms underlying pathogenesis in ciliopathies remain poorly understood, which requires structural and functional characterization of the mutated ciliopathy proteins at the cellular level. To this end, we elucidated the function and regulation of Cep290, which is the most frequently mutated gene in ciliopathies and importantly its functions remain poorly understood. First, we generated Cep290-null cells using the CRISPR/Cas9 genome editing approach. Using functional assays, we showed that Cep290-null cells do not ciliate and that they have defects in centriolar satellites dynamics and interphase microtubule organization. The centriolar satellites were tightly clustered around the centrosome in Cep290-null cells, and the interphase microtubule network lost its radial organization. Our results provide phenotypic insight into the disease mechanisms of Cep290 ciliopathy mutations and also the tools for studying genotype/phenotype relationships in ciliopathies.