Rhodopsin is a cilia-specific GPCR essential for vision. Rhodopsin mislocalization is associated with blinding diseases called retinal ciliopathies. The mechanism by which rhodopsin mislocalizes in rod photoreceptor neurons is not well understood. Therefore, we investigated the roles of trafficking signals in rhodopsin mislocalization. Rhodopsin and its truncation mutants were fused to a photoconvertible fluorescent protein, Dendra2, and expressed in Xenopus laevis rod photoreceptors. Photoconversion of Dendra2 causes a color change from green to red, enabling visualization of the dynamic events associated with rhodopsin trafficking and renewal. We found that rhodopsin mislocalization is a facilitated process for which a signal located within 322-326 aa (CCGKN) is essential. An additional signal within 327-336 aa further facilitated the mislocalization. This collective mistrafficking signal confers toxicity to rhodopsin and causes mislocalization when the VXPX cilia-targeting motif is absent. We also determined that the VXPX motif neutralizes this mistrafficking signal, enhances ciliary targeting at least 10-fold, and accelerates trafficking of post-Golgi vesicular structures. In the absence of the VXPX motif, mislocalized rhodopsin is actively cleared through secretion of vesicles into the extracellular milieu. Therefore, this study unveiled the multiple roles of trafficking signals in rhodopsin localization and renewal.

It is unclear how unconventional secretion interplays with conventional secretion for the normal maintenance and renewal of membrane structures. The photoreceptor sensory cilium is recognized for fast membrane renewal, for which rhodopsin and peripherin/rds (P/rds) play critical roles. Here, we provide evidence that P/rds is targeted to the cilia by an unconventional secretion pathway. When expressed in ciliated hTERT-RPE1 human cell line, P/rd is localized to cilia. Cilium trafficking of P/rds was sustained even when the Golgi functions, including trans-Golgi-mediated conventional secretion, were inhibited by the small molecules brefeldin A, 30N12, and monensin. The unconventional cilia targeting of P/rds is dependent on COPII-mediated exit from the ER, but appears to be independent of GRASP55-mediated secretion. The regions in the C-terminal tail of P/rds are essential for this unconventional trafficking. In the absence of the region required for cilia targeting, P/rds was prohibited from entering the secretory pathways and was retained in the Golgi apparatus. A region essential for this Golgi retention was also found in the C-terminal tail of P/rds and supported the cilia targeting of P/rds mediated by unconventional secretion. In ciliated cells, including bovine and Xenopus laevis rod photoreceptors, P/rds was robustly sensitive to endoglycosidase H, which is consistent with its bypassing the medial Golgi and traversing the unconventional secretory pathway. Because rhodopsin is known to traffic through conventional secretion, this study of P/rds suggests that both conventional secretion and unconventional secretion need to cooperate for the renewal of the photoreceptor sensory cilium.

Understanding the pressure dependence of the nonlinear behavior of ultrasonically excited phospholipid-stabilized nanobubbles (NBs) is important for optimizing ultrasound exposure parameters for implementations of contrast enhanced ultrasound, critical to molecular imaging. The viscoelastic properties of the shell can be controlled by the introduction of membrane additives, such as propylene glycol as a membrane softener or glycerol as a membrane stiffener. We report on the production of high-yield NBs with narrow dispersity and different shell properties. Through precise control over size and shell structure, we show how these shell components interact with the phospholipid membrane, change their structure, affect their viscoelastic properties, and consequently change their acoustic response. A two-photon microscopy technique through a polarity-sensitive fluorescent dye, C-laurdan, was utilized to gain insights on the effect of membrane additives to the membrane structure. We report how the shell stiffness of NBs affects the pressure threshold ( P t ) for the sudden amplification in the scattered acoustic signal from NBs. For narrow size NBs with 200 nm mean size, we find P t to be between 123 and 245 kPa for the NBs with the most flexible membrane as assessed using C-Laurdan, 465–588 kPa for the NBs with intermediate stiffness, and 588–710 kPa for the NBs with stiff membranes. Numerical simulations of the NB dynamics are in good agreement with the experimental observations, confirming the dependence of acoustic response to shell properties, thereby substantiating further the development in engineering the shell of ultrasound contrast agents. The viscoelastic-dependent threshold behavior can be utilized for significantly and selectively enhancing the diagnostic and therapeutic ultrasound applications of potent narrow size NBs.

Usher syndrome type III (USH3) characterized by progressive deafness, variable balance disorder, and blindness is caused by destabilizing mutations in the gene encoding the clarin-1 protein (CLRN1). Here we report a novel strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1N48K that involved a cell-based high-throughput screening of small molecules capable of stabilizing CLRN1N48K, a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure activity relationships. This resulted in the identification of BF844. To test the efficacy of BF844, a mouse model was developed that mimicked the progressive hearing loss of USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the human CLRN1N48K mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

Mutations that are Clrn1-/- biallelic cause visual defects when placed under A/J background. The absence of apparent rod degeneration suggests that the observed phenotype is due to functional defects, and not due to loss of rods. Biallelic Clrn1N48K/N48K mutations did not cause discernible visual defects, suggesting that Clrn1- allele is more severely dysfunctional than ClrnN48K allele.

Photoreceptor outer segments (OSs) are essential for our visual perception, and take either rod or cone forms. The cell biological basis for the formation of rods is well established, however, the mechanism of cone formation is ill characterized. While Xenopus rods are called rods, they exhibit cone shaped OSs during the early process of development. To visualize the dynamic reorganization of disk membranes, opsin and peripherin/rds were fused to a fluorescent protein Dendra2 and expressed in early developing rod photoreceptors, in which OSs are still cone shaped. Dendra2 is a fluorescent protein which can be converted from green to red irreversibly, and thus allows spatiotemporal labeling of proteins. Using a photoconversion technique, we found that disk membranes are assembled at the base of cone shaped OSs. After incorporation into disks, however, Opsin-Dendra2 was also trafficked from old to new disk membranes, consistent with the hypothesis that retrograde trafficking of membrane components contributes to the larger disk membrane observed toward the base of the cone shaped OS. Such retrograde trafficking is cargo specific and was not observed for peripherin/rds-Dendra2. The trafficking is unlikely mediated by diffusion, since the disk membranes have a closed configuration, as evidenced by CNGA1 labeling of the plasma membrane. Consistent with retrograde trafficking, the axoneme, which potentially mediates retrograde intraflagellar trafficking, runs through the entire axis of OSs. This study provides an insight into the role of membrane reorganization in developing photoreceptor OSs, and proves that retrograde trafficking of membrane cargoes can occur there.