Essential role of the chaperonin CCT in rod outer segment biogenesis

Abstract

Purpose: While some evidence suggests an essential role for the chaperonin containing t-complex protein 1 (CCT) in ciliogenesis, this function remains poorly understood mechanistically. We used transgenic mice, previously generated in our lab, and characterized by a genetically-induced suppression of CCT in rod photoreceptors as well as a malformation of the rod sensory cilia, the outer segments, to gain new insights into this underlying molecular mechanism.

Methods: The CCT activity in rod photoreceptors of mice was suppressed by overexpressing the chaperonin inhibitor, phosducin-like protein short, and the ensuing changes of cellular morphology were analyzed by light and electron microscopy. Protein expression levels were studied by fluorescent microscopy and Western blotting.

Results: Suppressing the chaperonin made the photoreceptors incompetent to build their outer segments. Specifically, the CCT-deficient rods appeared unable to expand the outer segment plasma membrane, and accommodate growth of this compartment. Seeking the molecular mechanisms underlying such a shortcoming, we found that the affected rods could not express normal levels of Bardet-Biedl Syndrome (BBS) proteins 2, 5, and 7 and, owing to that deficiency, were unable to assemble the BBSome, a multisubunit complex responsible for ciliary trafficking. A similar effect in response to the chaperonin suppression was also observed in cultured ciliated cells.

Conclusions: Our data provide new evidence indicating the essential role of the chaperonin CCT in the biogenesis of vertebrate photoreceptor sensory cilia, and suggest that it may be due to the direct participation of the chaperonin in the posttranslational processing of selected BBS proteins and assembly of the BBSome.

Keywords: BBSome; Bardet-Biedl Syndrome; chaperonin CCT; outer segments; rods.

Figure 1

Suppressing the CCT activity in mouse photoreceptors by phosducin-like protein short. (A) Whole-retina extract from 8-day-old mice were analyzed by pull down with anti-FLAG agarose. A representative Western blot shows the amounts of captured ^Δ1-83PhLP-FLAG, as visualized with antibody against FLAG, in transgene-positive (^Δ1-83PhLP-FLAG^±) and control (^Δ1-83PhLP-FLAG^−/−) mice from the lines 1 to 3. Graph: protein bands in each experiment were quantified and their values expressed as percent of the highest value found in line 3. Bars are SEM with n = 3, and P < 0.05 as determined by paired t-test. (B) Paraffin-embedded retina cross-sections were stained with hematoxylin and eosin to visualize its cellular composition at the age P10 and P21. (C) Nuclei count across the outer nuclear layer (ONL) as a function of mouse age. Bars are SEM, n = 6. OS, outer segments; IS, inner segments.

Figure 2

Suppressing the CCT activity disrupts development of rod outer segments. Retinas from wild-type (left panel) and ^Δ1-83PhLP-FLAG^± (right panel) littermates were analyzed by TEM at P10. OLM, outer limiting membrane.

Figure 3

Formation of membrane disks in healthy and affected rods. Ultrastructure of rod photoreceptor connecting cilium (*) and OS membrane disks (md) in wild-type and ^Δ1-83PhLP-FLAG^± littermates at P10.

Figure 4

Targeting of proteins to the OS disks. Subcellular localization of rhodopsin (red), peripherin/rds (green), and PDE6-α (magenta) as determined by immunofluorescence microscopy in rod photoreceptors at P10. Cell nuclei are visualized with DAPI.

Figure 5

Reduced levels of BBS proteins in the CCT-deficient rods. (A) A representative Western blot showing the levels of indicated BBS proteins in the whole-retina extracts of wild-type and ^Δ1–83PhLP-FLAG^± littermates at P10. (B) The fluorescence value of each specific band of a ^Δ1–83PhLP-FLAG^± mouse was normalized to that in a wild-type mouse, SEM, n = 4, P < 0.01 as determined by paired t-test (**).

Figure 6

An incomplete BBSome complex is assembled in the CCT-deficient rods. Whole-eye extracts from ^Δ1–83PhLP-FLAG^−/− and ^Δ1–83PhLP-FLAG^± littermates at P10 were fractionated by ultracentrifugation on a 10% to 40% sucrose gradient calibrated using sedimentation (S) coefficient standards. Collected fractions were analyzed by Western blotting using specific antibodies against the indicated BBS proteins. A mature BBSome contains BBS1, -2, -4, -5, -7, -8, and -9 and migrates as a 14S complex (▴); an incomplete BBSome contains BBS1, -4, -8, -9, and migrates as a 9S complex (Δ).

Figure 7

Bardet-Biedl syndrome protein 4 becomes mislocalized in ciliated cells in response to the CCT suppression. (A) Transfection protocol for transient expression of ^Δ1–83PhLP-FLAG and phosducin in RPE1-hTERT cells. (B) Representative Western blotting showing the expression of the indicated proteins. (C) RPE1-hTERT cells stably expressing GFP-BBS4 fusion protein were cotransfected with empty- and tdTomato vectors. GFP-BBS4 was immuno-enhanced with antibody against GFP (green), cell nuclei were visualized with DAPI (blue), transfected cells are distinguished from nontransfected cells by the presence of tdTomato fluorescence (red). (D) A number of cells with GFP-BBS4 was determined and expressed as a fraction of all transfected (red) cells. Such fraction in the cells expressing ^Δ1–83PhLP-FLAG and phosducin was normalized to that found in the control cells treated with empty vector. Error bars are SEM, n = 700, P = 0.008, as determined by paired t-test (***).

Figure 8

Characterization of visual function, photoreceptor morphology, and protein expression in 21-day-old mice from transgenic line 1. (A) Representative ERG responses to 0.00025, 0.025, and 0.1577 cd s⁻¹ m² flashes (−40, −20, and −12 dB). (B) The a-wave amplitude as a function of flash intensity in ^Δ1–83PhLP-FLAG^± (white circles) and wild-type (black circles); SEM, n = 6, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) as determined by t-test. (C) Paraffin-embedded retina cross-sections were stained with hematoxylin and eosin to visualize their cellular composition. (D) Subcellular localization of rhodopsin (red) and peripherin/rds (green) as determined by immunofluorescence microscopy. Cell nuclei are stained with DAPI. (E) Levels of the indicated proteins determined by Western blotting of whole retina extracts of ^Δ1–83PhLP-FLAG^± mice were normalized to the corresponding values of wild-type mice. Error bars represent SEM, n = 3 (rhodopsin), n = 6 (peripherin/rds), n = 4 (BBS), P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***) as determined by paired t-test.