Biology subjects

Wensel, T. G.

Publications and source records attributed to Wensel, T. G..

9 recordsLinked to original sources

bioRxiv2026

Differential and compensatory roles for type I phosphatidylinositol-4-phosphate-5-kinase isoforms in retinal function and health.

Phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) plays important roles in development, signaling, intracellular trafficking and regulation throughout the nervous system. Using selective and combined gene ablation strategies, we have determined the roles of this lipid and the kinase isoforms of the PIP5KI family primarily responsible for its synthesis in mouse retina. In rod cells, PI(4,5)P2 localizes predominantly to the plasma membrane of inner and outer segments and is enriched in membranes near the synaptic termini. Disruption of the gene encoding the {gamma} PIP5KI isoform, Pip5k1c, throughout the developing retina, using Cre expression driven by a Six3 transcription factor-dependent promoter, yields dramatic, but not complete, loss of the protein, with no apparent effects on morphology or function through the first 3-4 months after birth. Slowly progressing photoreceptor degeneration is observed at later ages. Complete loss of the {gamma} isoform in rods, driven by the rhodopsin promoter-based iCre75 transgene, leads to no obvious developmental defects, but results in an earlier-onset rod degeneration. Germ-line ablation of neither the Pip5k1a nor the Pip5k1b gene leads to any observable morphological defects. Homozygous Pip5k1a ablation leads to functional defects in photoreceptors as revealed by reduced a-wave and b-wave amplitudes in the electroretinograms. On the background of rod-specific Pip5k1c ablation, Pip5k1a deficiency greatly accelerates retinal degeneration. These results reveal a complex interplay among PIP5KI isoforms in ensuring proper photoreceptor function and health, with apparent partial redundancy in fulfilling their critical functions. They underscore the important role of PI(4,5)P2 in neuronal signaling and homeostasis. Significance StatementPhosphatidylinositol(4,5)P2, PI(4,5)P2, plays essential roles in nervous system development and function, but its roles in retina have been unknown. This study combines biochemistry, mouse genetics, light- and electron microscopy to reveal both specific and redundant functions for PIP2 formed by different kinase isoforms in the mammalian retina. It has implications for retinal function, disease and therapy, and for the broader field of phosphoinositide regulation.

neuroscience↗

bioRxiv2025

Computational design of allosteric pathways reprograms ligand-selective GPCR signaling

G-protein-coupled receptors (GPCRs) constitute the largest family of signaling receptors and drug targets. However, understanding how variations in receptor sequence, ligand chemical structure, and binding impact signaling functions remains a challenge, hindering drug discovery. Here, we developed a computational protein structure and dynamics approach to infer and design GPCR responses to multiple ligands. We created 32 dopamine D1 and D2 receptor variants with widely reprogrammed agonist-induced signal transductions. Subtle natural and designed receptor sequence variations, predicted to alter specific structural and dynamic mechanisms of ligand responses, profoundly impacted ligand potency and efficacy in agreement with our calculations. Our study provides a rational blueprint for computing the effect of sequence polymorphisms on ligand-selective protein signaling and paves the way for advancements in pharmacogenomics, drug selectivity, and the design of signaling receptors from first principles.

biochemistry↗

bioRxiv2025

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 CEP290. 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. CEP290 was found throughout the connecting cilium between the microtubules and membrane, with nine-fold symmetry. In the absence of CEP290 ciliogenesis occurs, but the connecting cilium membrane is aberrant, and sub-structures, such as the ciliary necklace and Y-links, are defective or absent throughout the mid to distal connecting cilium. Transition zone proteins AHI1 and NPHP1 were abnormally restricted to the proximal connecting cilium in the absence of CEP290, while others like NPHP8 and CEP89 were unaffected. Although outer segment disc formation is inhibited in CEP290 mutant retina, we observed large numbers of extracellular vesicles. These results suggest roles for CEP290 in ciliary membrane structure, outer segment disc formation and photoreceptor-specific spatial distribution of a subset of transition zone proteins, which collectively lead to failure of outer segment formation and photoreceptor degeneration.

cell biology↗

bioRxiv2023

Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography

Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the "Y-shaped links" and the "ciliary necklace". We have used cryo-electron tomography with subtomogram averaging and conventional TEM to elucidate the structures associated with the basal region of the "connecting cilia" of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules, identified genetics, proteomics, and superresolution fluorescence, can be placed in our emerging model of photoreceptor sensory cilia. SummaryCryo-electron tomography and subtomogram averaging reveal new structural features at the base of the light sensing cilia of retinal rods. These include the basal body, the Y-links between axoneme and membrane, and the ciliary necklace of the transition zone.

cell biology↗

bioRxiv2022

Ciliary tip actin dynamics regulate the cadenceof photoreceptor disc formation

As signalling organelles, primary cilia regulate their membrane G protein-coupled receptor (GPCR) content by ectocytosis, a process requiring localised actin dynamics at their tip to alter membrane shape.(1, 2) Mammalian photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia (CC), in which photosensitive GPCRs like rhodopsin are concentrated. In an extraordinary feat of biology, outer segment discs are shed and remade daily.(3) Defects in this process, due to genetic mutations, cause retinitis pigmentosa (RP), an untreatable, blinding disease. The mechanism by which photoreceptor cilia generate outer segments is therefore fundamental for vision yet poorly understood. Here, we show the membrane deformation required for outer segment disc genesis is driven by dynamic changes in the actin cytoskeleton in a process akin to ectocytosis. Further, we show RPGR, a leading causal RP gene, regulates activity of actin binding proteins crucial to this process. Disc genesis is compromised in Rpgr mouse models, slowing the actin dynamics required for timely disc formation, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Manipulation of actin dynamics partially rescues the phenotype, suggesting this pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.

cell biology↗

bioRxiv2021

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa

The P23H mutation in rhodopsin (Rho), the visual pigment protein in rod photoreceptor neurons, is the most common genetic cause of autosomal dominant retinitis pigmentosa (adRP), a retinal disease that causes blindness. Despite multiple studies in animal models, the subcellular details of the fate of misfolded mutant Rho in rod photoreceptors have not been completely defined. We generated a new mouse model of adRP, 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 there, but mutant P23H-Rho protein was specifically mislocalized in the inner segments of rods. Despite this cellular phenotype, the P23HhRhoRFP heterozygous mice exhibited only slowly progressing retinal degeneration; in ERG recordings, scotopic a-wave amplitudes were reduced by 24% and 26% at 30 days and 90 days respectively, and the corresponding scotopic b-waves by 18% and 24%. Outer nuclear layer thickness was still 80% of WT at 90 days, but at 364 days had declined to 40% of WT. Transmission electron microscopy revealed greatly expanded membrane lamellae in the inner segment, and by fluorescence imaging, we determined that the mislocalized P23HhRhoRFP was contained in greatly expanded endoplasmic reticulum (ER) membranes. TUNEL staining revealed a slow pace of cell death involving chromosomal endonucleolytic degradation. Quantification of mRNA for markers of ER stress and the unfolded protein response revealed little or no increases in levels of messages encoding the proteins BiP, CHOP, ATF6, XBP1, PERK, Eif2 and Derlin-1, but a decreased level of total Rhodopsin (mouse + human) mRNA levels. The decline in the rate of cell death after an initial burst suggests that P23HhRhoRFP mutant rods undergo an adaptative process that prolongs survival despite gross P23HhRhoRFP protein accumulation in the ER. Because of its slowly progressing nature, and easy visualization of the mutant protein, the P23H-Rho-RFP mouse may represent a useful tool for the future study of the pathology and treatment of P23H-Rho and adRP.

neuroscience↗

bioRxiv2020

Superresolution microscopy reveals photoreceptor-specific subciliary location and function of Cep290

Mutations in the cilium-associated protein CEP290 cause retinal degeneration as part of multi-organ syndromic 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 superresolution fluorescence microscopy and electron microscopy (TEM) to localize CEP290 in the CC and in primary cilia of cultured cells with sub-diffraction resolution, and to determine effects of CEP290 deficiency. Radially, CEP290 co-localizes with the microtubule doublets and extends beyond them. Longitudinally, it is distributed throughout the length of the CC but is strictly confined to the very base of primary cilia in hRPE-1 cells. We found Y-shaped links, the ciliary sub-structures between microtubules and membrane, at the base of the transition zone in primary cilia of epithelial cells and 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. They did not lead to loss of the Y-shaped links but caused changes in their structures. These results point to photoreceptor-specific functions of CEP290 essential for CC maturation and stability following the earliest stages of ciliogenesis.

cell biology↗

bioRxiv2020

Multiple phosphatidylinositol(3)phosphate roles in retinal pigment epithelium membrane recycling

The low-abundance lipid phosphatidylinositol-3-phosphate (PI(3)P) is important for membrane dynamics in autophagy, endosome processing, and phagocytosis. In retinal pigmented epithelial cells (RPE) all three pathways are important, but phagocytosis of disk membranes shed from adjacent photoreceptors is especially important for ensuring health of photoreceptors and preventing retinal degeneration. By eliminating Vps34, the kinase responsible for synthesizing PI(3)P in RPE, we have found that PI(3)P plays distinct roles in each pathway. In phagocytosis it is not required for disk engulfment or phagosome transport but is essential for recruitment and lipidation of LC3. In contrast, initiation of autophagy and LC3 recruitment to autophagosomes does not require PI(3)P, which can be bypassed by an alternative mechanism of ATG16L recruitment that does not require PI(3)P, Rab11a, or WIPI2. In all three pathways, PI(3)P is essential for fusion with lysosomes; autophagosomes, phagosomes, and Rab7-positive late endosomes, as well as enlarged lysosomes, accumulate in large numbers in the absence of Vps34, leading to cell death.

cell biology↗

bioRxiv2017

Defining the Layers of a Sensory Cilium with STORM and Cryo-Electron Nanoscopies

Primary cilia are cylindrical organelles extending from the surface of most animal cells that have been implicated in a host of signaling and sensory functions. Genetic defects in their component molecules, known as \"ciliopathies\" give rise to devastating symptoms, ranging from defective development, to kidney disease, to progressive blindness. The detailed structures of these organelles and the true functions of proteins encoded by ciliopathy genes are poorly understood because of the small size of cilia and the limitations of conventional microscopic techniques. We describe the combination of cryo-electron tomography, enhanced by sub-tomogram averaging, with super-resolution stochastic reconstruction microscopy (STORM) to define substructures and subdomains within the light-sensing rod sensory cilium of the mammalian retina. Longitudinal and radial domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of these compartments, including Ca2+-binding protein centrin-2 in the lumen of the axoneme, acetylated tubulin forming the axoneme, the glycocalyx extending outward from the surface of the plasma membrane, and molecular residents of the space between axoneme and ciliary membrane, including Arl13B, intraflagellar transport proteins, BBS5, and syntaxin-3. Within this framework we document that deficiencies in the ciliopathy proteins BBS2, BBS7 and BBS9 lead to inappropriate accumulation of proteins in rod outer segments while largely preserving their sub-domain localization within the connecting cilium region, but alter the distribution of syntaxin-3 clusters.

cell biology↗