Dr. Robichaux's Publications
Rapid 3D-STORM imaging of diverse molecular targets in tissue
Nicholas E Albrecht, Danye Jiang, Viktor Akhanov, Robert Hobson, Colenso M Speer, Michael A Robichaux, Melanie A Samuel
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 improved three-dimensional visualization for subcellular targets in tissue at depth. RAIN-STORM uses conventional tissue samples and readily available reagents and is suitable for commercial instrumentation. To illustrate the efficacy of RAIN-STORM, we utilized the retina. We show that RAIN-STORM imaging is versatile and provide 3D nanoscopic data for over 20 synpase, neuron, glia, and vasculature targets. Sample preparation is also rapid, with a 1-day turnaround from tissue to image, and parameters are suitable for multiple tissue sources. Finally, we show that this method can be applied to clinical samples to reveal nanoscale features of human cells and synapse. RAIN-STORM thus paves the way for high-throughput studies of nanoscopic targets in tissue.
Subcellular localization of mutant P23H rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa
Michael A. Robichaux, Vy Nguyen, Fung Chan, lavanya Kailasam, Feng He, John H. Wilson, Theodore G. Wensel
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 not yet been elucidated. We generated a new mouse model, in which the P23H-Rho mutant allele is fused to the fluorescent protein Tag-RFP-T (P23HhRhoRFP). In heterozygotes, outer segments formed, and WT rhodopsin was properly localized, but mutant P23H-Rho protein was mislocalized in the inner segments. Heterozygotes exhibited slowly progressing retinal degeneration. Mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases. mRNA levels for both the mutant human rhodopsin allele and the WT mouse rhodopsin levels were reduced, but protein levels revealed selective degradation of the mutant protein. The results suggest the mutant rods undergo an adaptative process that prolongs survival despite unfolded protein accumulation in the ER. The P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.
Superresolution microscopy reveals photoreceptor-specific subciliary location and function of ciolopathy-associated protein, Cep290
Valencia L. Potter, Abigail R. Moye, Michael A. Robichaux, and Theodore G. Wensel
Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multi-organ ciliopathies or as retina-specific disease. The precise location and the functional roles of CEP290 within cilia and, specifically, the connecting cilia (CC) of photoreceptors, remain unclear. We used superresolution fluorescence microscopy and electron microscopy (TEM) to localiza CEP290 in the CC and in primary cilia of cultured cells with sub-diffraction resolution, and to determine effects of CEP290 deficiency in three mutant models. Radially, CEP290 localizes in close proximity to the microtubule doublets in the region between the doublets and the ciliary membrane. Longitudinally, it is distributed throughout the length of the CC whereas it is confined to the very base of primary cilia in hRPE-1 cells. We found Y-shaped links, ciliary sub-structures between microtubules and membrane, throughout the length of the CC. Severe CEP290 deficiencies in mouse models did not prevent assembly of cilia or cause obvious mislocalization of ciliary components in early stages of degeneration. There were fewer cilia and no normal outer segments in the mutants, but the Y-shaped links were clearly present. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.
Structure and dynamics of photoreceptor sensory cilia
Theodore G Wensel, Valenicia L Potter, Abigail Moye, Zhixian Zhang, Michael A Robichuax
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.
Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy
Robichaux MA, Potter VL, Zhang Z, He F, Liu J, Schmid MF, Wensel TG
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. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet–Biedl syndrome.
A General Approach Toward Visible-Light-Activated Dyes for Biological Imaging
Tang J, Robichaux MA, Wu KL, Pei J, Nguyen NT, Zhou Y, Wensel TG, Xiao H.
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. Significantly, upon exposure to air and visible light residing in their absorption regime (365–630 nm), thio-caged fluorophores can be efficiently desulfurized to their oxo derivatives, thus restoring strong emission of the fluorophores. The effective photoactivation makes thio-caged fluorophores promising candidates for super-resolution imaging, which was realized by photoactivated localization microscopy (PALM) with low-power activation light under physiological conditions in the absence of cytotoxic additives (e.g., thiols, oxygen scavengers), a feature superior to traditional PALM probes. The versatility of this thio-caging strategy was further demonstrated by multicolor super-resolution imaging of lipid droplets and proteins of interest.
A Large Endoplasmic Reticulum-Resident Pool of TRPM1 in retinal ON-Bipolar Cells
Agosto MA, Anastassov IA, Robichaux MA, Wensel TG.
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. Little is known about the intracellular localization of TRPM1, or its trafficking route to the dendritic tip plasma membrane. Recombinant TRPM1 expressed in mammalian cells colocalized with endoplasmic reticulum (ER) markers, with little or none detected at the plasma membrane. In mouse retina, somatic TRPM1 was similarly intracellular, and not at the plasma membrane. Labeling of ER membranes by expression of a fluorescent marker showed that in BPCs the ER extends into axons and dendrites, but not dendritic tips. In cell bodies, TRPM1 colocalized with the ER, and not with the Golgi apparatus. Fluorescence protease protection (FPP) assays with TRPM1-GFP fusions in heterologous cells revealed that the N and C termini are both accessible to the cytoplasm, consistent with the transmembrane domain topology of related TRP channels. These results indicate that the majority of TRPM1 is present in the ER, from which it can potentially be transported to the dendritic tips as needed for ON light responses. The excess of ER-resident TRPM1 relative to the amount needed at the dendritic tips suggests a potential new function for TRPM1 in the ER
SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium
Dharmat R, Eblimit A, Robichaux MA, Zhang Z, Nguyen TT, Jung SY, He F, Jain A, Li Y, Qin J, Overbeek P, Roepman R, Mardon G, Wensel TG, Chen R.
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. This specialized distal zone of the CC in photoreceptors is maintained by SPATA7, which interacts with other photoreceptor-specific ciliary proteins such as RPGR and RPGRIP1. The absence of Spata7 results in the mislocalization of DCC proteins without affecting the PCC protein complexes. This collapse results in destabilization of the axonemal microtubules, which consequently results in photoreceptor degeneration. These data provide a novel mechanism to explain how genetic disruption of ubiquitously present ciliary proteins exerts tissue-specific ciliopathy phenotypes.
Structural and molecular bases of rod photoreceptor morphogenesis and disease
Wensel TG, Zhang Z, Anastassov IA, Gilliam JC, He F, Schmid MF, Robichaux MA.
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. Recent developments in imaging technology, such as cryo-electron tomography and super-resolution fluorescence microscopy, in gene sequencing technology, and in gene editing technology are rapidly leading to new breakthroughs in our understanding of these questions. A summary is presented of our current understanding of selected aspects of these questions, highlighting areas of uncertainty and contention as well as recent discoveries that provide new insights. Examples of structural data from emerging imaging technologies are presented.
EphB1 and EphB2 intracellular domains regulate the formation of the corpus callosum and anterior commissure
Robichaux MA, Chenaux G, Ho HY, Soskis MJ, Greenberg ME, Henkemeyer M, Cower CW.
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. Consistent with the roles for these receptors in formation of the CC and ACp, we detect expression of these receptors in multiple brain regions associated with the formation of these forebrain structures. Taken together, our findings suggest that a combination of forward and reverse EphB1/2 receptor‐mediated signaling contribute to ACp and CC axon guidance.
EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding
Robichaux MA, Chenaux G, Ho HY, Soskis MJ, Dravis C, Kwan KY, Sestan N, Greenberg ME, Henkemeyer M, Cowan CW.
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. We show here that EphB1 and EphB2 receptors control proper navigation of a subset of TCA and CTA projections through the VTel. We show in vivo that EphB receptor forward signaling and the ephrinB1 ligand are required during the early navigation of L1-CAM+ thalamic fibers in the VTel, and that the misguided thalamic fibers in EphB1/2 KO mice appear to interact with cortical subregion-specific axon populations during reciprocal cortical axon guidance. As such, our findings suggest that descending cortical axons identify specific TCA subpopulations in the dorsal VTel to coordinate reciprocal cortical–thalamic connectivity in the early developing brain.
Signaling mechanisms of axon guidance and early synaptogenesis
Robichaux MA, Cowan CW.
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. The extension and navigation of the axon to its appropriate target region in the brain and body is dependent upon many cell surface proteins that detect extracellular cues and transduce signals to the inside of the cell. In turn, intracellular signaling mechanisms orchestrate axon structural reorganization and appropriate turning toward or away from a guidance cue. Once the target region is reached, chemical synapses are formed between the axon and target cell, and again, this appears to involve cell surface proteins signaling to the inside of the neuron to stabilize and mature a synapse. Here, we describe some of the key convergent and, in some cases, divergent molecular pathways that regulate axon guidance and synaptogenesis in early brain development. Mutations in genes involved in early brain wiring and synapse formation and pruning increase the risk for developing autism, further highlighting the relevance of brain development factors in the pathophysiology of neurodevelopmental disorders.
EphB2 receptor forward signaling controls cortical growth cone collapse via Nck and Pak
Srivastava N, Robichaux MA, Chenaux G, Henkemeyer M, Cowan CW.
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. We show that kinase-active EphB2 binds to Pak and promotes growth cone repulsion via Pak kinase activity, Pak–Nck binding, RhoA signaling and endocytosis. However, Pak's function in this context appears to be independent of Rac/Cdc42-GTP, consistent with the absence of Rac-GTP production after ephrinB treatment of cortical neurons. Taken together, our findings suggest that ephrinB-activated EphB2 receptors recruit a novel Nck/Pak signaling complex to mediate repulsive cortical growth cone guidance, which may be relevant for EphB forward signaling-dependent axon guidance in vivo.
A chemical genetic approach reveals distinct EphB signaling mechanisms during brain development
Soskis MJ, Ho HY, Bloodgood BL, Robichaux MA, Malik AN, Ataman B, Rubin AA, Zieg J, Zhang C, Shokat KM, Sharma N, Cowan CW, Greenberg ME.
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. To address these issues, we engineered triple knock-in mice in which the kinase activity of three neuronally expressed EphBs can be rapidly, reversibly and specifically blocked. We found that the tyrosine kinase activity of EphBs was required for axon guidance in vivo. In contrast, EphB-mediated synaptogenesis occurred normally when the kinase activity of EphBs was inhibited, suggesting that EphBs mediate synapse development by an EphB tyrosine kinase–independent mechanism. Taken together, our data indicate that EphBs control axon guidance and synaptogenesis by distinct mechanisms and provide a new mouse model for dissecting EphB function in development and disease.