Jen-Zen Chuang

The cell biology of vision

Humans possess the remarkable ability to perceive color, shape, and motion, and to differentiate between light intensities varied by over nine orders of magnitude. Phototransduction—the process in which absorbed photons are converted into electrical responses—is the first stage of visual processing, and occurs in the outer segment, the light-sensing organelle of the photoreceptor cell. Studies of genes linked to human inherited blindness have been crucial to understanding the biogenesis of the outer segment and membrane-trafficking of photoreceptors.

SARA-Regulated Vesicular Targeting Underlies Formation of the Light-Sensing Organelle in Mammalian Rods

The light-sensing organelle of the vertebrate rod photoreceptor, the outer segment (OS), is a modified cilium containing approximately 1,000 stacked disc membranes that are densely packed with visual pigment rhodopsin. The mammalian OS is renewed every ten days; new discs are assembled at the base of the OS by a poorly understood mechanism. Our results suggest that discs are formed and matured in a process that involves specific phospholipid-directed vesicular membrane targeting. Rhodopsin-laden vesicles in the OS axonemal cytoplasm fuse with nascent discs that are highly specialized with abundant phosphatidylinositol 3-phosphate (PI3P). This membrane coupling is regulated by the FYVE domain-containing protein, SARA, through its direct interaction with PI3P, rhodopsin, and SNARE protein syntaxin 3. Our model, in contrast to the previously proposed evagination model, suggests that the vesicular delivery of rhodopsin in the OS concentrates rhodopsin into discs, and this process directly participates in disc biogenesis.

G protein βγ subunit interaction with the dynein light‐chain component Tctex‐1 regulates neurite outgrowth

Tctex‐1, a light‐chain component of the cytoplasmic dynein motor complex, can function independently of dynein to regulate multiple steps in neuronal development. However, how dynein‐associated and dynein‐free pools of Tctex‐1 are maintained in the cell is not known. Tctex‐1 was recently identified as a Gβγ‐binding protein and shown to be identical to the receptor‐independent activator of G protein signaling AGS2. We propose a novel role for the interaction of Gβγ with Tctex‐1 in neurite outgrowth. Ectopic expression of either Tctex‐1 or Gβγ promotes neurite outgrowth whereas interfering with their function inhibits neuritogenesis. Using embryonic mouse brain extracts, we demonstrate an endogenous Gβγ–Tctex‐1 complex and show that Gβγ co‐segregates with dynein‐free fractions of Tctex‐1. Furthermore, Gβ competes with the dynein intermediate chain for binding to Tctex‐1, regulating assembly of Tctex‐1 into the dynein motor complex. We propose that Tctex‐1 is a novel effector of Gβγ, and that Gβγ–Tctex‐1 complex plays a key role in the dynein‐independent function of Tctex‐1 in regulating neurite outgrowth in primary hippocampal neurons, most likely by modulating actin and microtubule dynamics.

Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations.

SARA, a FYVE domain protein, affects Rab5-mediated endocytosis.

Rab5, a member of the small GTPase family of proteins, is primarily localized on early endosomes and has been proposed to participate in the regulation of early endosome trafficking. It has been reported that phosphatidylinositol 3-kinases and FYVE domain proteins, such as EEA1, can be recruited onto early endosomes and act as Rab5 effectors. SARA (Smad anchor for receptor activation), also a FYVE domain protein, was initially isolated as a participant in signal transduction from the transforming growth factorβ receptor. Overexpressed SARA has been found on EEA1-positive early endosomes. In this report, we show that endogenous SARA is present on early endosomes and overexpression of SARA causes endosomal enlargement. Functionally, SARA overexpression significantly delays the recycling of transferrin. The transferrin receptor distributed on the cell surfaces was also greatly reduced in cells overexpressing SARA. However, the internalization rate of transferrin is not affected by SARA overexpression. The morphological and functional alterations caused by SARA overexpression resemble those caused by overexpression of Rab5:GTP mutant Rab5Q79L. Finally, all SARA-mediated phenotypic changes can be counteracted by overexpression Rab5:GDP mutant Rab5S34N. These results collectively suggested that SARA plays an important functional role downstream of Rab5-regulated endosomal trafficking.

IGF-1 Activates a Cilium-Localized Noncanonical Gβγ Signaling Pathway that Regulates Cell-Cycle Progression

Primary cilia undergo cell-cycle-dependent assembly and disassembly. Emerging data suggest that ciliary resorption is a checkpoint for S phase reentry and that the activation of phospho(T94)Tctex-1 couples these two events. However, the environmental cues and molecular mechanisms that trigger these processes remain unknown. Here, we show that insulin-like growth-1 (IGF-1) accelerates G1-S progression by causing cilia to resorb. The mitogenic signals of IGF-1 are predominantly transduced through IGF-1 receptor (IGF-1R) on the cilia of fibroblasts and epithelial cells. At the base of the cilium, phosphorylated IGF-1R activates an AGS3-regulated Gβγ signaling pathway that subsequently recruits phospho(T94)Tctex-1 to the transition zone. Perturbing any component of this pathway in cortical progenitors induces premature neuronal differentiation at the expense of proliferation. These data suggest that during corticogenesis, a cilium-transduced, noncanonical IGF-1R-Gβγ-phospho(T94)Tctex-1 signaling pathway promotes the proliferation of neural progenitors through modulation of ciliary resorption and G1 length.

Regulatory Dissociation of Tctex‐1 Light Chain from Dynein Complex Is Essential for the Apical Delivery of Rhodopsin

Post‐Golgi to apical surface delivery in polarized epithelial cells requires the cytoplasmic dynein motor complex. However, the nature of dynein–cargo interactions and their underlying regulation are largely unknown. Previous studies have shown that the apical surface targeting of rhodopsin requires the dynein light chain, Tctex‐1, which binds directly to both dynein intermediate chain (IC) and rhodopsin. In this report, we show that the S82E mutant of Tctex‐1, which mimics Tctex‐1 phosphorylated at serine 82, has a reduced affinity for dynein IC but not for rhodopsin. Velocity sedimentation experiments further suggest that S82E is not incorporated into the dynein complex. The dominant‐negative effect of S82E causes rhodopsin mislocalization in polarized Madin‐Darby canine kidney (MDCK) cells. The S82A mutant, which mimics dephosphorylated Tctex‐1, can be incorporated into dynein complex but is impaired in its release. Expression of S82A also causes disruption of the apical localization of rhodopsin in MDCK cells. Taken together, these results suggest that the dynein complex disassembles to release cargo due to the specific phosphorylation of Tctex‐1 at the S82 residue and that this process is critical for the apical delivery of membrane cargoes.

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates

Significance The establishment and maintenance of epithelial polarity relies on the tight regulation of vesicular trafficking, which ensures that apical and basolateral membrane proteins reach their designated plasma membrane domains. Here, we report the direct visualization of biosynthetic trans-endosomal trafficking of apically targeted rhodopsin in polarized epithelial cells. Our work provides novel insights into the crosstalk between biosynthetic and endocytic pathways. We demonstrate that the small GTPase Rab11a regulates sorting at apical recycling endosomes (AREs) and is also implicated in carrier vesicle docking at the apical plasma membrane. We further unveil a surprising role for dynamin-2 in the release of apical carriers from AREs. Our data indicate that trans-endosomal trafficking is indispensable for accurate and high-fidelity apical delivery. Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.

Chloride intracellular channel 4 is critical for the epithelial morphogenesis of RPE cells and retinal attachment

A plasmid-based transfection method was used to cell-autonomously silence chloride intracellular channel 4 (CLIC4) in RPE in situ. These results show CLIC4 is critical for epithelial morphogenesis and retinal attachment. Novel candidate targets for retinal detachment therapy have also been identified.

Evidence for the Involvement of Lfc and Tctex-1 in Axon Formation

RhoA and Rac play key and opposite roles during neuronal polarization. We now show that Lfc, a guanosine nucleotide exchange factor (GEF), localizes to the Golgi apparatus and growth cones of developing neurons and negatively regulates neurite sprouting and axon formation through a Rho signaling pathway. Tctex-1, a dynein light chain implicated in axon outgrowth by modulating actin dynamics and Rac activity, colocalizes and physically interacts with Lfc, thus inhibiting its GEF activity, decreasing Rho-GTP levels, and functionally antagonizing Lfc during neurite formation.