Limits...
Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

Caceres A, Shang F, Wawrousek E, Liu Q, Avidan O, Cvekl A, Yang Y, Haririnia A, Storaska A, Fushman D, Kuszak J, Dudek E, Smith D, Taylor A - PLoS ONE (2010)

Bottom Line: Of the 7 lysines (K) least is known about effects of modification of K6.Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA.These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.

Methodology and principal findings: We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

Conclusions and significance: K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

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Related in: MedlinePlus

K6 on Ub is essential for proper lens formation and clarity.Slit-lamp photographs of P90 mouse lenses. Lenses expressing high levels of K6W-Ub (A) show severe cataracts whereas lenses from animals expressing low levels of K6W-Ub (C) are clear, comparable to wild type (B). (D, E, F) Head-on photographs of P30 lenses. Lenses expressing high levels of K6W-Ub are cloudy and opaque. Note that the print behind the lens in panel E cannot be seen as it is in panels D and F. Lens from animals expressing low levels of K6W-Ub are clear comparable to Wt. (G, H) Light micrographs from E18.5 days Wt and K6W-Ub lenses. Lenses expressing high levels of K6W-Ub were ∼2/3 the size of Wt lenses. (I) P30 lenses from high or low K6W-Ub-expressing animals show different levels of K6W-Ub-containing conjugates. Lenses from Wt and transgenic animals were lysed and expression of transgene was determined by western blotting using anti-RGS(His)4. (J–M) Fluorescent micrographs of E18.5 K6W-Ub-expressing lens show attenuated proliferation compared to Wt. (J, K) BrdU (red) incorporation assay was used to detect S-phase cells in mouse lenses. K6W-Ub-expressing lenses show limited incorporation of BrdU compared to Wt. (L, M) Phospho-H3 (green), also shows that K6W-Ub-expressing lenses have decreased proliferation compared to Wt lenses. Immunohistochemistry was used to detect incorporation of BrdU and expression of phospho-H3, using anti-BrdU and anti-phospho-H3 antibodies respectively. DAPI was used to stain nuclei.
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pone-0013331-g002: K6 on Ub is essential for proper lens formation and clarity.Slit-lamp photographs of P90 mouse lenses. Lenses expressing high levels of K6W-Ub (A) show severe cataracts whereas lenses from animals expressing low levels of K6W-Ub (C) are clear, comparable to wild type (B). (D, E, F) Head-on photographs of P30 lenses. Lenses expressing high levels of K6W-Ub are cloudy and opaque. Note that the print behind the lens in panel E cannot be seen as it is in panels D and F. Lens from animals expressing low levels of K6W-Ub are clear comparable to Wt. (G, H) Light micrographs from E18.5 days Wt and K6W-Ub lenses. Lenses expressing high levels of K6W-Ub were ∼2/3 the size of Wt lenses. (I) P30 lenses from high or low K6W-Ub-expressing animals show different levels of K6W-Ub-containing conjugates. Lenses from Wt and transgenic animals were lysed and expression of transgene was determined by western blotting using anti-RGS(His)4. (J–M) Fluorescent micrographs of E18.5 K6W-Ub-expressing lens show attenuated proliferation compared to Wt. (J, K) BrdU (red) incorporation assay was used to detect S-phase cells in mouse lenses. K6W-Ub-expressing lenses show limited incorporation of BrdU compared to Wt. (L, M) Phospho-H3 (green), also shows that K6W-Ub-expressing lenses have decreased proliferation compared to Wt lenses. Immunohistochemistry was used to detect incorporation of BrdU and expression of phospho-H3, using anti-BrdU and anti-phospho-H3 antibodies respectively. DAPI was used to stain nuclei.

Mentions: It is not possible to target proteasome inhibitors to the lens without affecting most other organs. To overcome this barrier multiple transgenic K6W-Ub lines were generated in which the Ub variant was expressed at high and low levels in the lens. The lens was chosen because a) metabolism in lens epithelia is comparable to that of cells in many other tissues, b) to avoid embryonic lethal effects that might affect critical organs, c) aberrations in the proteome might lead to readily observed opacity, and d) because patterns of cell proliferation and differentiation are well documented and readily observed. Mice which expressed higher levels of K6W-Ub showed severe opacities or cataract and had higher levels of high mass Ub conjugates (Figure 2B, E, I), whereas lenses from animals which expressed lower levels of K6W-Ub (Figure 2C, F) or overexpressed Wt-Ub were clear (Supplementary Figure S1B) comparable to non- transgenic animals (Figure 2A, D). They also accumulated lower levels of high mass conjugates (Figure 2I).


Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

Caceres A, Shang F, Wawrousek E, Liu Q, Avidan O, Cvekl A, Yang Y, Haririnia A, Storaska A, Fushman D, Kuszak J, Dudek E, Smith D, Taylor A - PLoS ONE (2010)

K6 on Ub is essential for proper lens formation and clarity.Slit-lamp photographs of P90 mouse lenses. Lenses expressing high levels of K6W-Ub (A) show severe cataracts whereas lenses from animals expressing low levels of K6W-Ub (C) are clear, comparable to wild type (B). (D, E, F) Head-on photographs of P30 lenses. Lenses expressing high levels of K6W-Ub are cloudy and opaque. Note that the print behind the lens in panel E cannot be seen as it is in panels D and F. Lens from animals expressing low levels of K6W-Ub are clear comparable to Wt. (G, H) Light micrographs from E18.5 days Wt and K6W-Ub lenses. Lenses expressing high levels of K6W-Ub were ∼2/3 the size of Wt lenses. (I) P30 lenses from high or low K6W-Ub-expressing animals show different levels of K6W-Ub-containing conjugates. Lenses from Wt and transgenic animals were lysed and expression of transgene was determined by western blotting using anti-RGS(His)4. (J–M) Fluorescent micrographs of E18.5 K6W-Ub-expressing lens show attenuated proliferation compared to Wt. (J, K) BrdU (red) incorporation assay was used to detect S-phase cells in mouse lenses. K6W-Ub-expressing lenses show limited incorporation of BrdU compared to Wt. (L, M) Phospho-H3 (green), also shows that K6W-Ub-expressing lenses have decreased proliferation compared to Wt lenses. Immunohistochemistry was used to detect incorporation of BrdU and expression of phospho-H3, using anti-BrdU and anti-phospho-H3 antibodies respectively. DAPI was used to stain nuclei.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958118&req=5

pone-0013331-g002: K6 on Ub is essential for proper lens formation and clarity.Slit-lamp photographs of P90 mouse lenses. Lenses expressing high levels of K6W-Ub (A) show severe cataracts whereas lenses from animals expressing low levels of K6W-Ub (C) are clear, comparable to wild type (B). (D, E, F) Head-on photographs of P30 lenses. Lenses expressing high levels of K6W-Ub are cloudy and opaque. Note that the print behind the lens in panel E cannot be seen as it is in panels D and F. Lens from animals expressing low levels of K6W-Ub are clear comparable to Wt. (G, H) Light micrographs from E18.5 days Wt and K6W-Ub lenses. Lenses expressing high levels of K6W-Ub were ∼2/3 the size of Wt lenses. (I) P30 lenses from high or low K6W-Ub-expressing animals show different levels of K6W-Ub-containing conjugates. Lenses from Wt and transgenic animals were lysed and expression of transgene was determined by western blotting using anti-RGS(His)4. (J–M) Fluorescent micrographs of E18.5 K6W-Ub-expressing lens show attenuated proliferation compared to Wt. (J, K) BrdU (red) incorporation assay was used to detect S-phase cells in mouse lenses. K6W-Ub-expressing lenses show limited incorporation of BrdU compared to Wt. (L, M) Phospho-H3 (green), also shows that K6W-Ub-expressing lenses have decreased proliferation compared to Wt lenses. Immunohistochemistry was used to detect incorporation of BrdU and expression of phospho-H3, using anti-BrdU and anti-phospho-H3 antibodies respectively. DAPI was used to stain nuclei.
Mentions: It is not possible to target proteasome inhibitors to the lens without affecting most other organs. To overcome this barrier multiple transgenic K6W-Ub lines were generated in which the Ub variant was expressed at high and low levels in the lens. The lens was chosen because a) metabolism in lens epithelia is comparable to that of cells in many other tissues, b) to avoid embryonic lethal effects that might affect critical organs, c) aberrations in the proteome might lead to readily observed opacity, and d) because patterns of cell proliferation and differentiation are well documented and readily observed. Mice which expressed higher levels of K6W-Ub showed severe opacities or cataract and had higher levels of high mass Ub conjugates (Figure 2B, E, I), whereas lenses from animals which expressed lower levels of K6W-Ub (Figure 2C, F) or overexpressed Wt-Ub were clear (Supplementary Figure S1B) comparable to non- transgenic animals (Figure 2A, D). They also accumulated lower levels of high mass conjugates (Figure 2I).

Bottom Line: Of the 7 lysines (K) least is known about effects of modification of K6.Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA.These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.

Methodology and principal findings: We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

Conclusions and significance: K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

Show MeSH
Related in: MedlinePlus