Limits...
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.

Show MeSH

Related in: MedlinePlus

RNAi knockdown of UBCs affects the cellular level and aggregation rate of Q82::GFP fusion protein. C. elegans expressing a Q82:GFP transgene were fed bacterial clones expressing dsRNA against the indicated genes or the empty pL4440 vector as a control, beginning at the L2 stage. The progeny of these worms were imaged at a rate of 1 frame per minute. 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. This chart shows the mean (± SEM) aggregate intensity at each time point for each RNAi treatment. At least 50 aggregates were analyzed for each RNAi treatment. See Table 1 for calculation of aggregation rates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3368771&req=5

Figure 2: RNAi knockdown of UBCs affects the cellular level and aggregation rate of Q82::GFP fusion protein. C. elegans expressing a Q82:GFP transgene were fed bacterial clones expressing dsRNA against the indicated genes or the empty pL4440 vector as a control, beginning at the L2 stage. The progeny of these worms were imaged at a rate of 1 frame per minute. 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. This chart shows the mean (± SEM) aggregate intensity at each time point for each RNAi treatment. At least 50 aggregates were analyzed for each RNAi treatment. See Table 1 for calculation of aggregation rates.

Mentions: We have previously shown that RNAi knockdown of specific UBCs can affect the size and number of aggregates in worms at an age of 48 hours [17]. Specifically, the most dramatic phenotypes were seen with ubc-1, -2, -13, -22, and uev-1. Therefore, these UBCs were chosen for further analysis. For RNAi-mediated knockdown, the identical RNAi feeding strains and protocols were used that have been shown to reduce RNA to barely detectable levels [17]. Time-lapse microscopy was used to observe initial formation of aggregates in UBC knock down worms. RNAi treatment was started in L2 worms, and the L1 progeny of those worms were used for time-lapse imaging. Fluorescence measurements of time-lapse image series were made by recording the total intensity of all pixels over time in a square region framing the boundaries of the final aggregate. After performing multiple experiments, aggregation data were pooled by treatment group, aligned for the coincidence of aggregate formation, averaged at each time point, and plotted to form composite curves (Figure 2). Notably, RNAi of ubc-22, and, to a lesser extent, uev-1, resulted in an increase in initial levels of Q82::GFP, and an increase in the rate of initial aggregate formation. To compare the effects of the RNAi treatments on the rate of aggregation, a linear regression was performed on composite curves at the period of initial rapid aggregation (minutes 58-62) These data are shown in Table 1. The table shows that almost all UBCs had an effect on initial fluorescence levels. This may be due to a general disruption in the UPS by disruption of key components in that pathway. ubc-13 was the only UBC to show no significant effect on initial fluorescence level. In general, higher initial levels of Q82::GFP are associated with higher rates of aggregation. This correlation suggests that once an aggregate is initially seeded, its formation is largely diffusion-limited and dependent on concentration, rather than active cellular pathways.


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

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

RNAi knockdown of UBCs affects the cellular level and aggregation rate of Q82::GFP fusion protein. C. elegans expressing a Q82:GFP transgene were fed bacterial clones expressing dsRNA against the indicated genes or the empty pL4440 vector as a control, beginning at the L2 stage. The progeny of these worms were imaged at a rate of 1 frame per minute. 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. This chart shows the mean (± SEM) aggregate intensity at each time point for each RNAi treatment. At least 50 aggregates were analyzed for each RNAi treatment. See Table 1 for calculation of aggregation rates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: RNAi knockdown of UBCs affects the cellular level and aggregation rate of Q82::GFP fusion protein. C. elegans expressing a Q82:GFP transgene were fed bacterial clones expressing dsRNA against the indicated genes or the empty pL4440 vector as a control, beginning at the L2 stage. The progeny of these worms were imaged at a rate of 1 frame per minute. 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. This chart shows the mean (± SEM) aggregate intensity at each time point for each RNAi treatment. At least 50 aggregates were analyzed for each RNAi treatment. See Table 1 for calculation of aggregation rates.
Mentions: We have previously shown that RNAi knockdown of specific UBCs can affect the size and number of aggregates in worms at an age of 48 hours [17]. Specifically, the most dramatic phenotypes were seen with ubc-1, -2, -13, -22, and uev-1. Therefore, these UBCs were chosen for further analysis. For RNAi-mediated knockdown, the identical RNAi feeding strains and protocols were used that have been shown to reduce RNA to barely detectable levels [17]. Time-lapse microscopy was used to observe initial formation of aggregates in UBC knock down worms. RNAi treatment was started in L2 worms, and the L1 progeny of those worms were used for time-lapse imaging. Fluorescence measurements of time-lapse image series were made by recording the total intensity of all pixels over time in a square region framing the boundaries of the final aggregate. After performing multiple experiments, aggregation data were pooled by treatment group, aligned for the coincidence of aggregate formation, averaged at each time point, and plotted to form composite curves (Figure 2). Notably, RNAi of ubc-22, and, to a lesser extent, uev-1, resulted in an increase in initial levels of Q82::GFP, and an increase in the rate of initial aggregate formation. To compare the effects of the RNAi treatments on the rate of aggregation, a linear regression was performed on composite curves at the period of initial rapid aggregation (minutes 58-62) These data are shown in Table 1. The table shows that almost all UBCs had an effect on initial fluorescence levels. This may be due to a general disruption in the UPS by disruption of key components in that pathway. ubc-13 was the only UBC to show no significant effect on initial fluorescence level. In general, higher initial levels of Q82::GFP are associated with higher rates of aggregation. This correlation suggests that once an aggregate is initially seeded, its formation is largely diffusion-limited and dependent on concentration, rather than active cellular pathways.

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