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Ubiquitination is involved in secondary growth, not initial formation of polyglutamine protein aggregates in C. elegans.

Skibinski GA, Boyd L - BMC Cell Biol. (2012)

Bottom Line: Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation.However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate.The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Alabama in Huntsville, Huntsville AL 35899, USA. boydl@uah.edu

ABSTRACT

Background: Protein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The specific UBC used during ubiquitination can determine the type of polyubiquitin chain linkage, which in turn plays an important role in determining the fate of the ubiquitinated protein. Thus, UBCs may serve an important role in the cellular response to misfolded proteins and the fate of protein aggregates.

Results: The Q82 strain of C. elegans harbors a transgene encoding an aggregation prone tract of 82 glutamine residues fused to green fluorescent protein (Q82::GFP) that is expressed in the body wall muscle. When measured with time-lapse microscopy in young larvae, the initial formation of individual Q82::GFP aggregates occurs in approximately 58 minutes. This process is largely unaffected by a mutation in the C. elegans E1 ubiquitin activating enzyme. RNAi of ubc-22, a nematode homolog of E2-25K, resulted in higher pre-aggregation levels of Q82::GFP and a faster initial aggregation rate relative to control. Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation. However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate. This result is consistent with previous findings that aggregates in young adult worms are smaller after ubc-13 RNAi. mCherry::ubiquitin becomes localized to Q82::GFP aggregates during the fourth larval (L4) stage of life, a time point long after most aggregates have formed. FLIP and FRAP analysis indicate that mCherry::ubiquitin is considerably more mobile than Q82::GFP within aggregates.

Conclusions: These data indicate that initial formation of Q82::GFP aggregates in C. elegans is not directly dependent on ubiquitination, but is more likely a spontaneous process driven by biophysical properties in the cytosol such as the concentration of the aggregating species. The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.

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FRAP analysis of mCherry::ubiquitin and Q82::GFP in polyglutamine aggregates reveals differential mobility of ubiquitin and polyglutamine proteins within aggregates. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FRAP using a confocal laser scanning microscope system. Fluorescence intensity is indicated by a heat map of either Q82::GFP (A) or mCherry::ubiquitin (B). Red or yellow rectangles indicate regions to where bleaching was directed. White rectangles indicate regions that were used to control for acquisition photobleaching. Measurements of fluorescence recovery were taken every 0.1 s for 3 minutes. Quantitative analysis (C) of fluorescence recovery in bleached regions indicates higher overall mobility of mCherry::ubiquitin fusion protein when compared to Q82::GFP protein. Data plotted are the mean ± SEM.
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Figure 6: FRAP analysis of mCherry::ubiquitin and Q82::GFP in polyglutamine aggregates reveals differential mobility of ubiquitin and polyglutamine proteins within aggregates. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FRAP using a confocal laser scanning microscope system. Fluorescence intensity is indicated by a heat map of either Q82::GFP (A) or mCherry::ubiquitin (B). Red or yellow rectangles indicate regions to where bleaching was directed. White rectangles indicate regions that were used to control for acquisition photobleaching. Measurements of fluorescence recovery were taken every 0.1 s for 3 minutes. Quantitative analysis (C) of fluorescence recovery in bleached regions indicates higher overall mobility of mCherry::ubiquitin fusion protein when compared to Q82::GFP protein. Data plotted are the mean ± SEM.

Mentions: Ubiquitin within aggregates may be attached to the primary aggregating protein, attached to other proteins that coaggregate, or associated as a free monomer. The mobility of the protein may provide insights into its conjugation state. FRAP and FLIP experiments were performed in order to examine the mobility of mCherry::ubiquitin within the aggregates of Q82::GFP. In the FRAP experiments, mCherry or GFP was bleached within a region of the Q82::GFP aggregates in adult worms and recovery was observed (Figure 6A, B). Q82::GFP showed a slight recovery (Figure 6C) with a mobile fraction of 23.3 ± 9.2. The mCherry::ubiquitin fusion protein showed a higher degree of recovery, with a mobile fraction of 70.8 ± 17.0. This result indicates that while the polyglutamine protein, Q82::GFP, is highly immobile within aggregates, ubiquitin shows a greater rate of diffusion. The slow, continued increase in mCherry recovery after the initial rapid recovery may indicate the continued accumulation into aggregates of mCherry::ubiquitin or substrates to which it is attached.


Ubiquitination is involved in secondary growth, not initial formation of polyglutamine protein aggregates in C. elegans.

Skibinski GA, Boyd L - BMC Cell Biol. (2012)

FRAP analysis of mCherry::ubiquitin and Q82::GFP in polyglutamine aggregates reveals differential mobility of ubiquitin and polyglutamine proteins within aggregates. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FRAP using a confocal laser scanning microscope system. Fluorescence intensity is indicated by a heat map of either Q82::GFP (A) or mCherry::ubiquitin (B). Red or yellow rectangles indicate regions to where bleaching was directed. White rectangles indicate regions that were used to control for acquisition photobleaching. Measurements of fluorescence recovery were taken every 0.1 s for 3 minutes. Quantitative analysis (C) of fluorescence recovery in bleached regions indicates higher overall mobility of mCherry::ubiquitin fusion protein when compared to Q82::GFP protein. Data plotted are the mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368771&req=5

Figure 6: FRAP analysis of mCherry::ubiquitin and Q82::GFP in polyglutamine aggregates reveals differential mobility of ubiquitin and polyglutamine proteins within aggregates. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FRAP using a confocal laser scanning microscope system. Fluorescence intensity is indicated by a heat map of either Q82::GFP (A) or mCherry::ubiquitin (B). Red or yellow rectangles indicate regions to where bleaching was directed. White rectangles indicate regions that were used to control for acquisition photobleaching. Measurements of fluorescence recovery were taken every 0.1 s for 3 minutes. Quantitative analysis (C) of fluorescence recovery in bleached regions indicates higher overall mobility of mCherry::ubiquitin fusion protein when compared to Q82::GFP protein. Data plotted are the mean ± SEM.
Mentions: Ubiquitin within aggregates may be attached to the primary aggregating protein, attached to other proteins that coaggregate, or associated as a free monomer. The mobility of the protein may provide insights into its conjugation state. FRAP and FLIP experiments were performed in order to examine the mobility of mCherry::ubiquitin within the aggregates of Q82::GFP. In the FRAP experiments, mCherry or GFP was bleached within a region of the Q82::GFP aggregates in adult worms and recovery was observed (Figure 6A, B). Q82::GFP showed a slight recovery (Figure 6C) with a mobile fraction of 23.3 ± 9.2. The mCherry::ubiquitin fusion protein showed a higher degree of recovery, with a mobile fraction of 70.8 ± 17.0. This result indicates that while the polyglutamine protein, Q82::GFP, is highly immobile within aggregates, ubiquitin shows a greater rate of diffusion. The slow, continued increase in mCherry recovery after the initial rapid recovery may indicate the continued accumulation into aggregates of mCherry::ubiquitin or substrates to which it is attached.

Bottom Line: Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation.However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate.The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Alabama in Huntsville, Huntsville AL 35899, USA. boydl@uah.edu

ABSTRACT

Background: Protein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The specific UBC used during ubiquitination can determine the type of polyubiquitin chain linkage, which in turn plays an important role in determining the fate of the ubiquitinated protein. Thus, UBCs may serve an important role in the cellular response to misfolded proteins and the fate of protein aggregates.

Results: The Q82 strain of C. elegans harbors a transgene encoding an aggregation prone tract of 82 glutamine residues fused to green fluorescent protein (Q82::GFP) that is expressed in the body wall muscle. When measured with time-lapse microscopy in young larvae, the initial formation of individual Q82::GFP aggregates occurs in approximately 58 minutes. This process is largely unaffected by a mutation in the C. elegans E1 ubiquitin activating enzyme. RNAi of ubc-22, a nematode homolog of E2-25K, resulted in higher pre-aggregation levels of Q82::GFP and a faster initial aggregation rate relative to control. Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation. However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate. This result is consistent with previous findings that aggregates in young adult worms are smaller after ubc-13 RNAi. mCherry::ubiquitin becomes localized to Q82::GFP aggregates during the fourth larval (L4) stage of life, a time point long after most aggregates have formed. FLIP and FRAP analysis indicate that mCherry::ubiquitin is considerably more mobile than Q82::GFP within aggregates.

Conclusions: These data indicate that initial formation of Q82::GFP aggregates in C. elegans is not directly dependent on ubiquitination, but is more likely a spontaneous process driven by biophysical properties in the cytosol such as the concentration of the aggregating species. The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.

Show MeSH
Related in: MedlinePlus