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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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RNAi knockdown of UBCs alters the secondary growth of aggregates. 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, belonging to the same population as the worms analyzed in Figure 2, were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. Aggregates that had formed prior to mounting on the slide were located using the automatic object tracking feature of Image Pro Plus 6.1, which uses an intensity threshold to define the borders of the aggregates. The sum pixel intensity within each automatically defined region was measured over time, omitting objects that formed during the observation period. The Y axis represents the mean pixel intensity for all aggregates, plotted by gene. At least 50 aggregates were analyzed for each RNAi treatment. See Table 2 for calculation of aggregation rates.
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Figure 3: RNAi knockdown of UBCs alters the secondary growth of aggregates. 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, belonging to the same population as the worms analyzed in Figure 2, were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. Aggregates that had formed prior to mounting on the slide were located using the automatic object tracking feature of Image Pro Plus 6.1, which uses an intensity threshold to define the borders of the aggregates. The sum pixel intensity within each automatically defined region was measured over time, omitting objects that formed during the observation period. The Y axis represents the mean pixel intensity for all aggregates, plotted by gene. At least 50 aggregates were analyzed for each RNAi treatment. See Table 2 for calculation of aggregation rates.

Mentions: In the C. elegans Q82 model, aggregates in adult worms are much larger than those seen in young larvae. The data Figures 1 and 2 show that initially aggregates form quickly, but that after initial formation, aggregates continue to grow at a slower rate. Since RNAi of UBCs affects aggregate size, we wanted to investigate whether the UBCs might affect this secondary growth phase. Thus, from the same population of worms in which we observed aggregate formation, we recorded the growth of aggregates that had formed prior to observation under the microscope (Figure 3). Table 2 summarizes the growth rates of these aggregates, as determined by a linear regression performed on the data from Figure 3. In this case initial fluorescence level indicates the level of fluorescence in the existing aggregate at the time of observation during the L1 larval period. Initial fluorescence of aggregates was higher in worms subjected to RNAi of ubc-22, consistent with our previous findings [17]. In addition, ubc-22 and ubc-1 RNAi aggregates grew at rates faster than the control (Table 2). Aggregates in worms treated with ubc-13 and uev-1 RNAi showed smaller aggregates, consistent with results from our previous study [17]. In addition, secondary growth was markedly reduced upon RNAi of ubc-13 (Table 2). Since homologs of ubc-13 and uev-1 are known to dimerize and to catalyze the formation of K63-linked polyubiquitin chains [9,19], it is possible that the reduced secondary growth rate may be related to reduced levels of K63 ubiquitination.


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 alters the secondary growth of aggregates. 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, belonging to the same population as the worms analyzed in Figure 2, were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. Aggregates that had formed prior to mounting on the slide were located using the automatic object tracking feature of Image Pro Plus 6.1, which uses an intensity threshold to define the borders of the aggregates. The sum pixel intensity within each automatically defined region was measured over time, omitting objects that formed during the observation period. The Y axis represents the mean pixel intensity for all aggregates, plotted by gene. At least 50 aggregates were analyzed for each RNAi treatment. See Table 2 for calculation of aggregation rates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: RNAi knockdown of UBCs alters the secondary growth of aggregates. 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, belonging to the same population as the worms analyzed in Figure 2, were imaged using a microscope with a 10X objective lens at a rate of 1 frame per minute. Aggregates that had formed prior to mounting on the slide were located using the automatic object tracking feature of Image Pro Plus 6.1, which uses an intensity threshold to define the borders of the aggregates. The sum pixel intensity within each automatically defined region was measured over time, omitting objects that formed during the observation period. The Y axis represents the mean pixel intensity for all aggregates, plotted by gene. At least 50 aggregates were analyzed for each RNAi treatment. See Table 2 for calculation of aggregation rates.
Mentions: In the C. elegans Q82 model, aggregates in adult worms are much larger than those seen in young larvae. The data Figures 1 and 2 show that initially aggregates form quickly, but that after initial formation, aggregates continue to grow at a slower rate. Since RNAi of UBCs affects aggregate size, we wanted to investigate whether the UBCs might affect this secondary growth phase. Thus, from the same population of worms in which we observed aggregate formation, we recorded the growth of aggregates that had formed prior to observation under the microscope (Figure 3). Table 2 summarizes the growth rates of these aggregates, as determined by a linear regression performed on the data from Figure 3. In this case initial fluorescence level indicates the level of fluorescence in the existing aggregate at the time of observation during the L1 larval period. Initial fluorescence of aggregates was higher in worms subjected to RNAi of ubc-22, consistent with our previous findings [17]. In addition, ubc-22 and ubc-1 RNAi aggregates grew at rates faster than the control (Table 2). Aggregates in worms treated with ubc-13 and uev-1 RNAi showed smaller aggregates, consistent with results from our previous study [17]. In addition, secondary growth was markedly reduced upon RNAi of ubc-13 (Table 2). Since homologs of ubc-13 and uev-1 are known to dimerize and to catalyze the formation of K63-linked polyubiquitin chains [9,19], it is possible that the reduced secondary growth rate may be related to reduced levels of K63 ubiquitination.

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