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First published online May 10, 2006
doi: 10.1242/10.1242/jcs.02940

Journal of Cell Science 119, 2138-2144 (2006)
Published by The Company of Biologists 2006
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Knock-in of {alpha}3 connexin prevents severe cataracts caused by an {alpha}8 point mutation

Chun-hong Xia1, Debra Cheung1, Adam M. DeRosa2, Bo Chang3, Woo-Kuen Lo4, Thomas W. White2,5 and Xiaohua Gong1,*

1 School of Optometry and Vision Science Program, University of California at Berkeley, Berkeley, CA, USA
2 Program in Genetics, State University of New York, Stony Brook, NY, USA
3 The Jackson Laboratory, Bar Harbor, ME, USA
4 Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta, GA, USA
5 Department of Physiology and Biophysics, State University of New York, Stony Brook, NY, USA


Figure 1
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  		Fig. 1. Knocked-in {alpha}3 connexin suppresses the severe cataract caused by the {alpha}8-G22R mutation. Comparison of three compound mutant lenses of mice aged 3 weeks. (A) The {alpha}3(-/-) {alpha}8(KI{alpha}3/-) lens is transparent. (B) The {alpha}3(-/-) {alpha}8(G22R/-) lens has severe cataract with posterior rupture. (C) The {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) lens is intact with only a very mild nuclear cataract.

 

Figure 2
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  		Fig. 2. Histology reveals normal fiber morphology in the lens of {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) mice. (A) The {alpha}3(-/-) {alpha}8(G22R/-) lens of a 2-week-old mouse has severely degenerated inner fibers and posterior rupture of the lens capsule. (B) The {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) lens section of a 2-week-old mouse shows relatively normal inner lens fibers without any obvious cell degeneration. The defect in the center fibers could either be caused by sectioning artifacts or the mild nuclear cataract.

 

Figure 3
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  		Fig. 3. Immunohistochemical staining reveals that the knocked-in {alpha}3 connexin restores targeting of {alpha}8-G22R mutant subunits to gap junctions in the lens fiber cells. (A,B) {alpha}8 connexin staining (red) in cross sections of (A) {alpha}3(-/-) {alpha}8(G22R/-) and (B) {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) lenses of 3-week-old mice. (C,D) Merged images of {alpha}8 connexin (red) and F-actin (FITC-phalloidin staining, green) in cross sections of (C) {alpha}3(-/-) {alpha}8(G22R/-) and (D) {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) lenses. Boxed regions in C and D are magnified in C' and D', respectively, showing that very few punctate fluorescent spots appear in the peripheral fiber cells of the {alpha}3(-/-) {alpha}8(G22R/-) lens, whereas typical punctate fluorescent signals of gap junctions predominantly localize in the long sides of the fiber cells of the {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) lens. Bar, 20 µm.

 

Figure 4
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  		Fig. 4. Western blot data show that the {alpha}8-G22R connexin protein level is greatly increased in the lenses of {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) mice. Membrane-enriched lens protein samples were prepared from different mice aged 3 weeks. Lane 1, wild type {alpha}3(+/+) {alpha}8(+/+); lane 2, {alpha}3(-/-) {alpha}8(KI{alpha}3/-); lane 3, {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3); lane 4, {alpha}3(-/-) {alpha}8(G22R/-). Equal amounts of lens samples were loaded onto the gel and probed with (A) anti-{alpha}3 connexin antibody or (B) anti-{alpha}8 connexin antibody. Lane 1 is the wild-type control showing the endogenous {alpha}3 and {alpha}8 connexins. All three mutant mice contain no endogenous wild-type {alpha}3 and {alpha}8 alleles, thus the bands in lanes 2 and 3 in A are knocked-in {alpha}3 connexin. The band in lane 3 in B is the {alpha}8-G22R mutant. The molecular mass markers are listed on the left and give values in kDa.

 

Figure 5
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  		Fig. 5. (A-C') TEM photographs show gap junctions (arrows) in (A,A') {alpha}3(-/-) {alpha}8(KI{alpha}3/-) and (C,C') {alpha}3(-/-) {alpha}8(G22R/KI{alpha}3) lens fiber cells, but not in (B,B') {alpha}3(-/-) {alpha}8(G22R/-) lens fiber cells. A',B' and C' are magnified images of the regions indicated by arrows or arrowheads in A,B and C, respectively. Bars, 200 nm (A-C) and 20 nm (A'-C').

 

Figure 6
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  		Fig. 6. Functional expression of {alpha}3 and {alpha}8-G22R connexins in Xenopus oocytes. (A) Western blot analysis of Xenopus oocytes shows similar levels of {alpha}8-G22R and {alpha}3 proteins when expressed alone or co-injected. Equal amounts of oocyte membrane fractions were loaded into each lane, and the blots were probed with a polyclonal anti-{alpha}8 connexin (left), or anti-{alpha}3 connexin (right) antibody. Similar levels of {alpha}8-G22R and {alpha}3 protein synthesis were seen in individually and co-injected oocytes. Quantitation of band intensity by densitometry confirmed that levels of connexin expression were not statistically different (P>0.05). Densitometry values are the mean of three independent experiments. (B) {alpha}8-G22R connexin forms functional heteromeric channels with {alpha}3 connexin. Junctional conductance values were measured between oocyte pairs injected with wild-type {alpha}3, {alpha}8-G22R, co-injected {alpha}8-G22R and {alpha}3, or water. Oocyte pairs containing wild-type {alpha}3 alone (n=20), or co-injected {alpha}8-G22R and {alpha}3 (n=57) exhibited conductance values >100-fold higher than the pairs injected with water for the background value (n=40). However, conductance of co-injected {alpha}3 and {alpha}8-G22R was reduced ~75% compared with {alpha}3 alone (P<0.05). Homotypic {alpha}8-G22R (n=55) cell pairs failed to produce levels of coupling that were significantly higher than background (P>0.05). Similarly, heterotypic {alpha}8-G22R/{alpha}3 cell pairs, formed by pairing one oocyte expressing {alpha}3 with another injected with {alpha}8-G22R, were poorly coupled (n=16).

 

Figure 7
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  		Fig. 7. Voltage-gating properties of homotypic {alpha}3, and heteromeric {alpha}8-G22R and {alpha}3 channels. (A) The decay in junctional current (Ij) induced by transjunctional voltage (Vj) was plotted as a function of time for channels comprised of {alpha}3 (left), and {alpha}8-G22R plus {alpha}3 (right). Vj was stepped in 20-mV increments to ±120 mV. At all potentials >±20 mV, heteromeric {alpha}8-G22R and {alpha}3 channels showed a more rapid current decay of greater magnitude. (B) Analysis of channel kinetics. Representative initial current decays for gap junctions comprised of homotypic {alpha}3 connexin (left), and heteromeric {alpha}8-G22R with {alpha}3 connexins (right) after application of +80 mV transjunctional voltage. Current traces were fit to a mono-exponential decay to determine the time constant, {tau}. Heteromeric {alpha}8-G22R and {alpha}3 channels closed significantly faster than homotypic {alpha}3 channels (P<0.05). {tau} values are the mean ± s.e. of four independent experiments. (C) Comparison of equilibrium conductance. Steady-state conductance was measured when current decay reached equilibrium, normalized to the values at ±20 mV and plotted as a function of Vj. The steady-state reduction in conductance for heteromeric {alpha}8-G22R and {alpha}3 channels was greater than the reduction for homotypic {alpha}3 channels at Vj values. Smooth lines are fits to the Boltzmann equation whose parameters are given in Table 2. Consistent with the formation of heteromeric channels, co-expression of {alpha}8-G22R with wild-type {alpha}3 results in significantly altered gating properties.

 

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© The Company of Biologists Ltd 2006