PNAS Liu Chunqiao's team reveals a new mechanism for protein transport in the outer segment of photoreceptors

Visual information is an important means for humans and animals to survive, and 80-90% of external information comes from vision
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The acquisition of visual information begins with the capture of photons by photoreceptors
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The membrane disc of the photoreceptor outer segment (OS) captures photons through opsin proteins and initiates the light signaling cascade
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Membrane disc is renewed about 10% every day, which requires the synthesis of a large amount of lipids and proteins, but it does not have the ability to synthesize proteins and lipids
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The protein elements of the light signaling pathway need to be continuously transported to the OS through the synthesis of narrow connecting cilium (CC) in the inner segment (IS) to maintain their renewal
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The failure of protein transport in the outer segment will lead to its accumulation in the inner segment, causing stress necrosis of photoreceptor cells and triggering retinal degenerative diseases such as the common blinding Retinitis pigmentosa (RP)
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According to incomplete statistics, the worldwide incidence of hereditary RP is about 1:4000~5000, affecting nearly 300,000 people in China.
Many mutant genes of RP are related to protein transport in the outer segment of photoreceptor cilia
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Although there have been many functional studies on the protein trafficking of cilia in the outer segment of photoreceptors, most of these studies have focused on the microtubules of cilia and their associated structural proteins, dyneins (motors) and the vesicular trafficking complex (IFT)
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However, the transport function of ciliary membrane proteins in the outer segment of photoreceptors is unclear
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Recently, Chunqiao Liu from Zhongshan Ophthalmology Center of Sun Yat-sen University jointly published a research paper Tmem138 is localized to the connecting cilium essential for rhodopsin localization and outer segment biogenesis in PNAS, Proceedings of the National Academy of Sciences
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This study reveals for the first time that the neuroretinal photoreceptor ciliary membrane protein complex plays an important role in the trafficking of Rhodopsin and other outer segment proteins, thus providing a necessary complement to the pathological molecular mechanism of RP
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This study also provides an important reference for the molecular mechanism of cerebral edema in the central nervous system caused by ciliary lesions
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The research group has been focusing on the development and genetic diseases of the neural retina
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Previous studies on the Wnt/PCP signaling pathway that regulates ciliary development and cell polarity trafficking (Guo et al, 2018, IOVS; 2019, EXER; 2020, IOVS; 2020, Development) found several transmembrane proteins regulated by it
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One mutation in Tmem138 causes Joubert syndrome with retinal dystrophy
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In order to study the molecular mechanism of retinal diseases caused by Tmem138 mutation, the authors first created a Tmem138 knockout mouse model, which successfully mimicked the phenotype of human Joubert syndrome, including ventriculomegaly, spermatogenesis disorders and retinal degeneration (Fig.
1)
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Germline deletion of Tmem138 resulted in failure of photoreceptor OS by immunofluorescence, transmission and scanning electron microscopy observations
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Figure 1: Failure of OS development in retinal degenerative diseases due to deletion of Tmem138 may be due to defects in any or all of cilia biogenesis, protein trafficking, or assembly of the OS membrane disc
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The authors first observed the formation of OS in mutant mice by electron microscopy and confirmed that the cilia structure was normal; immunofluorescence showed that the localization of IFT subunits and their dynein (IFT88, Kif3A)-dependent microtubule transport components did not change significantly; membrane disc assembly proteins (Rds/Prominin-1/Prcad) was relatively normal in neonatal OS
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The above experiments generally rule out the possibility of initial developmental defects of ciliary structure and outer segment membrane disc
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Next, the authors used multiple immunofluorescence molecular markers to distinguish different regions of the outer segment of the cilia, and confirmed the localization of Tmem138 at the proximal end of the CC by knockout mice (Fig.
2A-C), and further observed mutations.
Loss of the localization domain of distal Ahi1 in the mouse CC suggests that Tmem138 is closely related to the ciliated cytoplasmic protein Ahi1
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Consistent with this, there is published evidence that AHI1 mutations in humans also cause Joubert syndrome and fail to develop the outer photoreceptor segment
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These results suggest that the Tmem138 membrane protein is likely to be involved in the trafficking of ectoproteins together with Ahi1
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After examining elements of a range of phototransduction signaling pathways, the authors found that Rhodopsin was the first to aberrantly localize, coinciding with the onset of outer segment development
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Figure 2: Tmem138 localizes to CC and mediates Rhodopsin transport mechanism.
Next, the authors found that Tmem138 could directly interact with Ahi1 and Rhodopsin through co-immunoprecipitation and pull down experiments of photoreceptor outer segment protein extracts
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At the same time, it was found that another membrane protein Tmem231 can interact with Tmem138 and Rhodopsin in pairs, and the ciliary membrane localization of Tmem231 in Tmem138 mutant mice is abnormal
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Taking all the evidence together, the authors propose that the Tmem138/Tmem231/Ahi1 junction ciliary complex is critical for transciliary trafficking of Rhodopsin and other outer segment proteins (Fig.
2D)
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This study is the only paper to describe the transport process of resident CC membrane proteins in photoreceptor proteins
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Zhongshan Ophthalmology Center of Sun Yat-sen University and State Key Laboratory of Ophthalmology are the first units
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Postdoctoral fellow Guo Dianlei and Dr.
Ru Jiali are the first authors of this paper.
Professor Wei Yanhong, School of Public Health, Sun Yat-sen University, and Professor Liu Xialin, Zhongshan Ophthalmology Center participated in the research.
Researcher Liu Chunqiao and Professor Liu Yizhi are the corresponding authors of this paper
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