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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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Aggregation profile of Q82::GFP protein in C. elegans. C. elegans larvae expressing a Q82:GFP transgene in a wild type (black diamonds) or in a uba-1 mutant (red squares) were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. The sum of the pixel intensity in a square region of the image sequence in which an aggregate formed was measured over time. Plots of individual formation events were aligned along the time axis so that the frame at which the aggregation rate is highest occurs at 60 minutes. For each individual aggregate formation, the fraction of the peak intensity was calculated at each time point. This chart shows the mean (± SEM) fractional aggregate intensity at each time point for a total of 58 aggregate formations in 5 different time-lapse experiments for the Q82 strain in the wild type background and 30 aggregate formations in 3 experiments for the Q82 in the uba-1(ts) background.
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Figure 1: Aggregation profile of Q82::GFP protein in C. elegans. C. elegans larvae expressing a Q82:GFP transgene in a wild type (black diamonds) or in a uba-1 mutant (red squares) were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. The sum of the pixel intensity in a square region of the image sequence in which an aggregate formed was measured over time. Plots of individual formation events were aligned along the time axis so that the frame at which the aggregation rate is highest occurs at 60 minutes. For each individual aggregate formation, the fraction of the peak intensity was calculated at each time point. This chart shows the mean (± SEM) fractional aggregate intensity at each time point for a total of 58 aggregate formations in 5 different time-lapse experiments for the Q82 strain in the wild type background and 30 aggregate formations in 3 experiments for the Q82 in the uba-1(ts) background.

Mentions: The transgenic Q82 strain of C. elegans expresses a fusion protein that consists of 82 glutamine residues fused to GFP, under control of the unc-54 promoter for expression in the body wall muscle cells. Aggregates form throughout the animal's development to adulthood [47]. We have employed this model to examine the early formation of aggregates, and the role of ubiquitination in this process. L1- and L2-stage worms readily form distinct puncta of Q82::GFP that appear concurrently with the disappearance of diffuse, putatively soluble fluorescent material (Additional files 1 and 2 Figure: Videos 1 and 2). This process of initial aggregate formation occurs rapidly, with the time for aggregation to occur (defined as the time taken for total fluorescence to increase from 10% to 90% of maximum) being 58.1 ± 21.5 minutes. Initial formation is represented by a sigmoidal curve (Figure 1) when plotted as a function of time. This is followed by secondary growth, in which fluorescence increases at a slower rate. Aggregates forming during time-lapse observation are similar in size to those that were formed prior to microscopic observation. They consists of largely immobile Q82::GFP, as they do not show fluorescence recovery after photobleaching (G.S., data not shown).


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

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

Aggregation profile of Q82::GFP protein in C. elegans. C. elegans larvae expressing a Q82:GFP transgene in a wild type (black diamonds) or in a uba-1 mutant (red squares) were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. The sum of the pixel intensity in a square region of the image sequence in which an aggregate formed was measured over time. Plots of individual formation events were aligned along the time axis so that the frame at which the aggregation rate is highest occurs at 60 minutes. For each individual aggregate formation, the fraction of the peak intensity was calculated at each time point. This chart shows the mean (± SEM) fractional aggregate intensity at each time point for a total of 58 aggregate formations in 5 different time-lapse experiments for the Q82 strain in the wild type background and 30 aggregate formations in 3 experiments for the Q82 in the uba-1(ts) background.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Aggregation profile of Q82::GFP protein in C. elegans. C. elegans larvae expressing a Q82:GFP transgene in a wild type (black diamonds) or in a uba-1 mutant (red squares) were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. The sum of the pixel intensity in a square region of the image sequence in which an aggregate formed was measured over time. Plots of individual formation events were aligned along the time axis so that the frame at which the aggregation rate is highest occurs at 60 minutes. For each individual aggregate formation, the fraction of the peak intensity was calculated at each time point. This chart shows the mean (± SEM) fractional aggregate intensity at each time point for a total of 58 aggregate formations in 5 different time-lapse experiments for the Q82 strain in the wild type background and 30 aggregate formations in 3 experiments for the Q82 in the uba-1(ts) background.
Mentions: The transgenic Q82 strain of C. elegans expresses a fusion protein that consists of 82 glutamine residues fused to GFP, under control of the unc-54 promoter for expression in the body wall muscle cells. Aggregates form throughout the animal's development to adulthood [47]. We have employed this model to examine the early formation of aggregates, and the role of ubiquitination in this process. L1- and L2-stage worms readily form distinct puncta of Q82::GFP that appear concurrently with the disappearance of diffuse, putatively soluble fluorescent material (Additional files 1 and 2 Figure: Videos 1 and 2). This process of initial aggregate formation occurs rapidly, with the time for aggregation to occur (defined as the time taken for total fluorescence to increase from 10% to 90% of maximum) being 58.1 ± 21.5 minutes. Initial formation is represented by a sigmoidal curve (Figure 1) when plotted as a function of time. This is followed by secondary growth, in which fluorescence increases at a slower rate. Aggregates forming during time-lapse observation are similar in size to those that were formed prior to microscopic observation. They consists of largely immobile Q82::GFP, as they do not show fluorescence recovery after photobleaching (G.S., data not shown).

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