Sub-ciliary localization of CEP290 and effects of its loss in mouse photoreceptors during development.

The most common genetic cause of the childhood blindness disease Leber congenital amaurosis is mutation of the ciliopathy gene CEP290. Despite extensive study, the photoreceptor-specific roles of CEP290 remain unclear. Using advanced microscopy techniques, we investigated the sub-ciliary localization of CEP290 and its role in mouse photoreceptors during development.

P23H rhodopsin accumulation causes transient disruptions to synaptic protein levels in rod photoreceptors in a model of retinitis pigmentosa.

Rod photoreceptor neurons in the retina detect scotopic light through the visual pigment rhodopsin (Rho) in their outer segment (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health. However, given the importance of protein trafficking to the OS, little is known about the trafficking of rod synaptic proteins.

Ciliopathy-associated protein, CEP290, is required for ciliary necklace and outer segment membrane formation in retinal photoreceptors.

The most common genetic cause of the childhood blinding disease Leber Congenital Amaurosis is mutation of the ciliopathy gene . Though studied extensively, the photoreceptor-specific roles of CEP290 remain unclear. Using advanced microscopy techniques, we investigated the sub-ciliary localization of CEP290 and its role in mouse photoreceptors during development.

Rabin8 phosphorylated by NDR2, the canine early retinal degeneration gene product, directs rhodopsin Golgi-to-cilia trafficking.

The Rab11-Rabin8-Rab8 ciliogenesis complex regulates the expansion of cilia-derived light-sensing organelles, the rod outer segments, via post-Golgi rhodopsin transport carriers (RTCs). Rabin8 (also known as RAB3IP), an effector of Rab11 proteins and a nucleotide exchange factor (GEF) for Rab8 proteins, is phosphorylated at S272 by NDR2 kinase (also known as STK38L), the canine early retinal degeneration (erd) gene product linked to the human ciliopathy Leber congenital amaurosis (LCA). Here, we define the step at which NDR2 phosphorylates Rabin8 and regulates Rab11-to-Rab8 succession in Xenopus laevis transgenic rod photoreceptors expressing human GFP-Rabin8 and its mutants.

P23H rhodopsin aggregation in the ER causes synaptic protein imbalance in rod photoreceptors.

Rod photoreceptor neurons in the retina detect scotopic light through the visual pigment rhodopsin (Rho) in their outer segments (OS). Efficient Rho trafficking to the OS through the inner rod compartments is critical for long-term rod health. Given the importance of protein trafficking to the OS, less is known about the trafficking of rod synaptic proteins.

Excessive tubulin glutamylation leads to progressive cone-rod dystrophy and loss of outer segment integrity.

Mutations in Cytosolic Carboxypeptidase-like Protein 5 (CCP5) are associated with vision loss in humans. To decipher the mechanisms behind CCP5-associated blindness, we generated a novel mouse model lacking CCP5. In this model, we found that increased tubulin glutamylation led to progressive cone-rod dystrophy, with cones showing a more pronounced and earlier functional loss than rod photoreceptors.

Expansion Microscopy of Mouse Photoreceptor Cilia.

The small size of ciliary structures that underlies photoreceptor function and inherited ciliopathies requires imaging techniques adapted to visualizing them at the highest possible resolution. In addition to powerful super-resolution imaging modalities, emerging approaches to sample preparation, including expansion microscopy (ExM), can provide a robust route to imaging specific molecules at the nanoscale level in the retina. We describe a protocol for applying ExM to whole retinas in order to achieve nanoscale fluorescence imaging of ciliary markers, including tubulin, CEP290, centrin, and CEP164.

Mapping rhodopsin trafficking in rod photoreceptors with quantitative super-resolution microscopy.

Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient long-term phototransduction. Rhodopsin, the visual pigment in rod photoreceptors, is densely packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in the mammalian rod inner segment remain undefined.

Rapid 3D-STORM imaging of diverse molecular targets in tissue.

Fine-scale molecular architecture is critical for nervous system and other biological functions. Methods to visualize these nanoscale structures would benefit from enhanced accessibility, throughput, and tissue compatibility. Here, we report RAIN-STORM, a rapid and scalable nanoscopic imaging optimization approach that improves three-dimensional visualization for subcellular targets in tissue at depth.

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knock-in mouse model of retinitis pigmentosa.

The P23H mutation in rhodopsin (Rho), the rod visual pigment, is the most common allele associated with autosomal-dominant retinitis pigmentosa (adRP). The fate of misfolded mutant Rho in rod photoreceptors has yet to be elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP).

Super-resolution microscopy reveals photoreceptor-specific subciliary location and function of ciliopathy-associated protein CEP290.

Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multiorgan ciliopathies or as retina-specific diseases. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used super-resolution fluorescence microscopy and electron microscopy to localize CEP290 in the CC and in the primary cilia of cultured cells with subdiffraction resolution and to determine effects of CEP290 deficiency in 3 mutant models.

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.

Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy.

Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins.

Single-Atom Fluorescence Switch: A General Approach toward Visible-Light-Activated Dyes for Biological Imaging.

Photoactivatable fluorophores afford powerful molecular tools to improve the spatial and temporal resolution of subcellular structures and dynamics. By performing a single sulfur-for-oxygen atom replacement within common fluorophores, we have developed a facile and general strategy to obtain photoactivatable fluorogenic dyes across a broad spectral range. Thiocarbonyl substitution within fluorophores results in significant loss of fluorescence via a photoinduced electron transfer-quenching mechanism as suggested by theoretical calculations.

A Large Endoplasmic Reticulum-Resident Pool of TRPM1 in Retinal ON-Bipolar Cells.

The chemical signal of light onset, a decrease in glutamate release from rod and cone photoreceptors, is processed by a postsynaptic G protein signaling cascade in ON-bipolar cells (BPCs). The metabotropic glutamate receptor mGluR6, along with other cascade elements, is localized synaptically at the BPC dendritic tips. The effector ion channel protein transient receptor potential melastatin-1 (TRPM1), in contrast, is located not only at the dendritic tips but also in BPC bodies and axons.

SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium.

Photoreceptor-specific ciliopathies often affect a structure that is considered functionally homologous to the ciliary transition zone (TZ) called the connecting cilium (CC). However, it is unclear how mutations in certain ciliary genes disrupt the photoreceptor CC without impacting the primary cilia systemically. By applying stochastic optical reconstruction microscopy technology in different genetic models, we show that the CC can be partitioned into two regions: the proximal CC (PCC), which is homologous to the TZ of primary cilia, and the distal CC (DCC), a photoreceptor-specific extension of the ciliary TZ.

Structural and molecular bases of rod photoreceptor morphogenesis and disease.

The rod cell has an extraordinarily specialized structure that allows it to carry out its unique function of detecting individual photons of light. Both the structural features of the rod and the metabolic processes required for highly amplified light detection seem to have rendered the rod especially sensitive to structural and metabolic defects, so that a large number of gene defects are primarily associated with rod cell death and give rise to blinding retinal dystrophies. The structures of the rod, especially those of the sensory cilium known as the outer segment, have been the subject of structural, biochemical, and genetic analysis for many years, but the molecular bases for rod morphogenesis and for cell death in rod dystrophies are still poorly understood.

EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure.

The two cortical hemispheres of the mammalian forebrain are interconnected by major white matter tracts, including the corpus callosum (CC) and the posterior branch of the anterior commissure (ACp), that bridge the telencephalic midline. We show here that the intracellular signaling domains of the EphB1 and EphB2 receptors are critical for formation of both the ACp and CC. We observe partial and complete agenesis of the corpus callosum, as well as highly penetrant ACp misprojection phenotypes in truncated EphB1/2 mice that lack intracellular signaling domains.

EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding.

In early brain development, ascending thalamocortical axons (TCAs) navigate through the ventral telencephalon (VTel) to reach their target regions in the young cerebral cortex. Descending, deep-layer cortical axons subsequently target appropriate thalamic and subcortical target regions. However, precisely how and when corticothalamic axons (CTAs) identify their appropriate, reciprocal thalamic targets remains unclear.

Signaling mechanisms of axon guidance and early synaptogenesis.

The development of the vertebrate nervous system, including the brain and spinal cord, progresses in a step-wise fashion that involves the function of thousands of genes. The birth of new neurons (also known as neurogenesis) and their subsequent migration to appropriate locations within the developing brain mark the earliest stages of CNS development. Subsequently, these newborn neurons extend axons and dendrites to make stereotyped synaptic connections within the developing brain, which is a complex process involving cell intrinsic mechanisms that respond to specific extracellular signals.

EphB2 receptor forward signaling controls cortical growth cone collapse via Nck and Pak.

EphB receptors and their ephrinB ligands transduce bidirectional signals that mediate contact-dependent axon guidance primarily by promoting growth cone repulsion. However, how EphB receptor-mediated forward signaling induces axonal repulsion remains poorly understood. Here, we identify Nck and Pak proteins as essential forward signaling components of EphB2-dependent growth cone collapse in cortical neurons.

A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development.

EphB receptor tyrosine kinases control multiple steps in nervous system development. However, it remains unclear whether EphBs regulate these different developmental processes directly or indirectly. In addition, given that EphBs signal through multiple mechanisms, it has been challenging to define which signaling functions of EphBs regulate particular developmental events.