Showing papers by "Tiansen Li published in 2005"

Rootletin Interacts with C-Nap1 and May Function as a Physical Linker between the Pair of Centrioles/Basal Bodies in Cells

Abstract: Rootletin, a major structural component of the ciliary rootlet, is located at the basal bodies and centrosomes in ciliated and nonciliated cells, respectively. Here we investigated its potential role in the linkage of basal bodies/centrioles and the mechanism involved in such linkages. We show that rootletin interacts with C-Nap1, a protein restricted at the ends of centrioles and functioning in centrosome cohesion in interphase cells. Their interaction in vivo is supported by their colocalization at the basal bodies/centrioles and coordinated association with the centrioles during the cell cycle. Ultrastructural examinations demonstrate that rootletin fibers connect the basal bodies in ciliated cells and are present both at the ends of and in between the pair of centrioles in nonciliated cells. The latter finding stands in contrast with C-Nap1, which is present only at the ends of the centrioles. Transient expression of C-Nap1 fragments dissociated rootletin fibers from the centrioles, resulting in centrosome separation in interphase. Overexpression of rootletin in cells caused multinucleation, micronucleation, and irregularity of nuclear shape and size, indicative of defects in chromosome separation. These data suggest that rootletin may function as a physical linker between the pair of basal bodies/centrioles by binding to C-Nap1.

The Ciliary Rootlet Maintains Long-Term Stability of Sensory Cilia

Abstract: The striated ciliary rootlet is a prominent cytoskeleton originating from basal bodies of ciliated cells. Although a familiar structure in cell biology, its function has remained unresolved. In this study, we carried out targeted disruption in mice of the gene for rootletin, a component of the rootlet. In the mutant, ciliated cells are devoid of rootlets. Phototransduction and ciliary beating in sensory and motile cilia initially exhibit no apparent functional deficits. However, photoreceptors degenerate over time, and mutant lungs appear prone to pathological changes consistent with insufficient mucociliary clearance. Further analyses revealed a striking fragility at the ciliary base in photoreceptors lacking rootlets. In vitro assays suggest that the rootlet is among the least dynamic of all cytoskeletons and interacts with actin filaments. Thus, a primary function of the rootlet is to provide structural support for the cilium. Inasmuch as photoreceptors elaborate an exceptionally enlarged sensory cilium, they are especially dependent on the rootlet for structural integrity and long-term survival.

Gene replacement therapy rescues photoreceptor degeneration in a murine model of Leber congenital amaurosis lacking RPGRIP.

Abstract: Purpose Retinitis pigmentosa GTPase regulator (RPGR) is a photoreceptor protein anchored in the connecting cilia by an RPGR-interacting protein (RPGRIP). Loss of RPGRIP causes Leber congenital amaurosis (LCA), a severe form of photoreceptor degeneration. The current study was an investigation of whether somatic gene replacement could rescue degenerating photoreceptors in a murine model of LCA due to a defect in RPGRIP. Methods An RPGRIP expression cassette, driven by a mouse opsin promoter, was packaged into recombinant adeno-associated virus (AAV). The AAV vector was delivered into the right eyes of RPGRIP(-/-) mice by a single subretinal injection into the superior hemisphere. The left eyes received a saline injection as a control. Full-field electroretinograms (ERGs) were recorded from both eyes at 2, 3, 4, and 5 months after injection. After the final follow-up, retinas were analyzed by immunostaining or by light and electron microscopy. Results Delivery of the AAV vector led to RPGRIP expression and restoration of normal RPGR localization at the connecting cilia. Photoreceptor preservation was evident by a thicker cell layer and well-developed outer segments in the treated eyes. Rescue was more pronounced in the superior hemisphere coincident with the site of delivery. Functional preservation was demonstrated by ERG. Conclusions AAV-mediated RPGRIP gene replacement preserves photoreceptor structure and function in a mouse model of LCA, despite ongoing cell loss at the time of intervention. These results indicate that gene replacement therapy may be effective in patients with LCA due to a defect in RPGRIP and suggest that further preclinical development of gene therapy for this disorder is warranted.

A single, abbreviated RPGR-ORF15 variant reconstitutes RPGR function in vivo.

Abstract: Purpose The retinitis pigmentosa GTPase regulator (RPGR) is essential for the maintenance of photoreceptor viability. RPGR is expressed as constitutive and ORF15 variants because of alternative splicing. This study was designed to examine whether the retina-specific ORF15 variant alone could substantially substitute for RPGR function. A further objective was to test whether the highly repetitive purine-rich region of ORF15 could be abbreviated without ablating the function, so as to accommodate RPGR replacement genes in adenoassociated virus (AAV) vectors. Methods A cDNA representing RPGR-ORF15 but shortened by 654 bp in the repetitive region was placed under the control of a chicken beta-actin (CBA) hybrid promoter. The resultant construct was transfected into mouse embryonic stem cells. Clones expressing the transgene were selected and injected into mouse blastocysts. Transgenic chimeras were crossed with RPGR knockout (KO) mice. Mice expressing the transgene but null for endogenous RPGR (Tg/KO) were studied from 1 month to 18 months of age by light and electron microscopy, immunofluorescence, and electroretinography (ERG). The results were compared with those of wild-type (WT) and RPGR-null control mice. Results Transgenic RPGR-ORF15 was found in the connecting cilia of rod and cone photoreceptors, at approximately 20% of the WT level. Photoreceptor morphology, cone opsin localization, expression of GFAP (a marker for retinal degeneration) and ERGs were consistent with the transgene exerting substantial rescue of retinal degeneration due to loss of endogenous RPGR. Conclusions RPGR-ORF15 is the functionally significant variant in photoreceptors. The length of its repetitive region can be reduced while preserving its function. The current findings should facilitate the design of gene replacement therapy for RPGR-null mutations.

Genetic replacement of cyclin D1 function in mouse development by cyclin D2.

Abstract: The progression of mammalian cells through the G1 phase of the cell cycle is driven by the D-type and E-type cyclins (43). These cyclins bind, activate, and provide substrate specificity for their associated cyclin-dependent kinases (CDKs). In contrast to other cyclins, which are induced periodically during cell cycle progression, the expression of D cyclins is controlled largely by the extracellular environment. For this reason, D cyclins are regarded as links between the external mitogenic milieu and the core cell cycle machinery (39, 45). Three D-type cyclins, D1, D2 and D3, have been enumerated in mammalian cells (21, 33, 34, 37, 38, 57). These three proteins are encoded by separate genes located on different chromosomes, but they show significant amino acid similarity, suggesting that they arose from a common primordial ancestor gene (19, 58). On average, D cyclins show 50 to 60% identity throughout the entire coding sequence and 75 to 78% identity within the most conserved cyclin box domain (19, 58). All three D cyclins associate with CDK4 or CDK6, yielding six different combinations of cyclin D-CDK holoenzymes (2, 10, 20, 31, 32, 36). An important issue is whether each of the D cyclins performs unique, possibly cell type-specific functions or the three proteins represent tissue-specific isoforms with virtually identical functions. At a biochemical level, all three D cyclins were shown to physically associate with CDK4 and CDK6 and to drive phosphorylation of the retinoblastoma protein, pRB, and pRB-related “pocket” proteins p107 and p130 (3, 28, 32, 36, 54, 56). The phosphorylation of these pocket proteins may represent the major function for cyclin D-CDK complexes in cell cycle progression, as shown by the observations that cells lacking pRB or p107 and p130 no longer require D cyclins for proliferation (1, 4, 16, 23, 29, 35, 40, 53). However, biochemical differences between the three D cyclins were noted. Thus, cyclins D2 and D3 can form active complexes with CDK2, while cyclin D1 was reported to lack this ability (10, 17). Moreover, in addition to their well-established CDK-dependent functions, D cyclins were shown to interact with tissue-specific transcription factors, such as estrogen receptor, androgen receptor, thyroid receptor, and retinoic acid receptor alpha, C/EBP binding protein β, DMP1, and others (8, 27). In some cases, this interaction was uniquely ascribed to a particular D-type cyclin (9, 60). To address the functions of the D-type cyclins in development, we and others generated mice lacking cyclin D1, D2, or D3 and characterized their phenotypes (12, 46-48). We found that mice lacking individual D cyclins were viable and displayed narrow, tissue-specific abnormalities. For instance, cyclin D1-deficient mice showed underdeveloped, hypoplastic retinas and presented a developmental neurological abnormality. Moreover, cyclin D1-deficient females displayed a normal mammary epithelial tree at the end of sexual maturation, but they failed to undergo full lobuloalveolar development during pregnancy (12, 48). Importantly, all these compartments developed normally in cyclin D2- or D3-deficient animals (46, 47), revealing a unique requirement for cyclin D1 in vivo in selected tissues. In the present study, we asked whether the requirement for cyclin D1 function in these compartments was caused by tissue-specific pattern of D cyclin expression or alternatively reflected the presence of specialized tissue-specific functions for cyclin D1. To address this question by genetic means, we generated a knock-in strain of mice expressing cyclin D2 in place of cyclin D1. We next asked whether cyclin D2 could drive the normal development of cyclin D1-dependent tissues.