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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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FLIP analysis of mCherry::ubiquitin and Q82::GFP. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FLIP analysis of the mCherry::ubiquitin protein to study mobility of ubiquitin. Fluorescence intensity is indicated by a heat map of mCherry::ubiquitin prior to bleaching and at various times after commencement of repeated bleach pulses. Red squares indicate regions where bleach pulses were directed, black and white squares indicate regions that were quantitatively analyzed for fluorescence loss, and yellow squares indicate regions in non-bleached cells that were used to control for acquisition photobleaching. Separate experiments were carried out in which bleaching was directed to the cytoplasm (A) or the aggregate of Q82::GFP (B). A quantitative analysis (C) was carried out to analyze fluorescence loss over time. Results indicate no loss of mCherry fluorescence in aggregates when bleaching was directed to either a separate region within the aggregate itself (blue diamonds) or an area in the cytoplasm. The loss of fluorescence in the cytoplasm when a region within the cytoplasm was bleached indicates the effectiveness of the bleaching protocol (red squares), while the limited loss of fluorescence in the cytoplasm when a region within the aggregate was bleached indicates the limited access of mCherry::ubiquitin to the Q82::GFP aggregates.
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Figure 7: FLIP analysis of mCherry::ubiquitin and Q82::GFP. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FLIP analysis of the mCherry::ubiquitin protein to study mobility of ubiquitin. Fluorescence intensity is indicated by a heat map of mCherry::ubiquitin prior to bleaching and at various times after commencement of repeated bleach pulses. Red squares indicate regions where bleach pulses were directed, black and white squares indicate regions that were quantitatively analyzed for fluorescence loss, and yellow squares indicate regions in non-bleached cells that were used to control for acquisition photobleaching. Separate experiments were carried out in which bleaching was directed to the cytoplasm (A) or the aggregate of Q82::GFP (B). A quantitative analysis (C) was carried out to analyze fluorescence loss over time. Results indicate no loss of mCherry fluorescence in aggregates when bleaching was directed to either a separate region within the aggregate itself (blue diamonds) or an area in the cytoplasm. The loss of fluorescence in the cytoplasm when a region within the cytoplasm was bleached indicates the effectiveness of the bleaching protocol (red squares), while the limited loss of fluorescence in the cytoplasm when a region within the aggregate was bleached indicates the limited access of mCherry::ubiquitin to the Q82::GFP aggregates.

Mentions: To further investigate the mobility of mCherry::ubiquitin within Q82::GFP aggregates, fluorescence loss in photobleaching (FLIP) was used. mCherry was continuously bleached in a region either within the Q82::GFP aggregate or in the cytoplasm of a cell expressing the two fusion proteins. Loss of fluorescence in either a separate region within the aggregate or in the cytoplasm was monitored to examine mobility of the fluorescence material (Figure 7A, B). Directing bleach pulses to either the cytoplasm or the aggregate itself did not result in loss of fluorescence within the aggregate, indicating mCherry::ubiquitin is sequestered within aggregates. Bleaching within the cytoplasm reduced cytoplasmic mCherry fluorescence, indicating the effectiveness of the bleaching protocol and the mobility of mCherry::ubiquitin within the cytoplasm (Figure 7C). These results support the notion that the mCherry::ubiquitin is sequestered within the Q82::GFP aggregates, but is not itself in an aggregated, immobile configuration.


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

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

FLIP analysis of mCherry::ubiquitin and Q82::GFP. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FLIP analysis of the mCherry::ubiquitin protein to study mobility of ubiquitin. Fluorescence intensity is indicated by a heat map of mCherry::ubiquitin prior to bleaching and at various times after commencement of repeated bleach pulses. Red squares indicate regions where bleach pulses were directed, black and white squares indicate regions that were quantitatively analyzed for fluorescence loss, and yellow squares indicate regions in non-bleached cells that were used to control for acquisition photobleaching. Separate experiments were carried out in which bleaching was directed to the cytoplasm (A) or the aggregate of Q82::GFP (B). A quantitative analysis (C) was carried out to analyze fluorescence loss over time. Results indicate no loss of mCherry fluorescence in aggregates when bleaching was directed to either a separate region within the aggregate itself (blue diamonds) or an area in the cytoplasm. The loss of fluorescence in the cytoplasm when a region within the cytoplasm was bleached indicates the effectiveness of the bleaching protocol (red squares), while the limited loss of fluorescence in the cytoplasm when a region within the aggregate was bleached indicates the limited access of mCherry::ubiquitin to the Q82::GFP aggregates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: FLIP analysis of mCherry::ubiquitin and Q82::GFP. Worms co-expressing an mCherry::ubiquitin fusion protein with Q82::GFP were subjected to FLIP analysis of the mCherry::ubiquitin protein to study mobility of ubiquitin. Fluorescence intensity is indicated by a heat map of mCherry::ubiquitin prior to bleaching and at various times after commencement of repeated bleach pulses. Red squares indicate regions where bleach pulses were directed, black and white squares indicate regions that were quantitatively analyzed for fluorescence loss, and yellow squares indicate regions in non-bleached cells that were used to control for acquisition photobleaching. Separate experiments were carried out in which bleaching was directed to the cytoplasm (A) or the aggregate of Q82::GFP (B). A quantitative analysis (C) was carried out to analyze fluorescence loss over time. Results indicate no loss of mCherry fluorescence in aggregates when bleaching was directed to either a separate region within the aggregate itself (blue diamonds) or an area in the cytoplasm. The loss of fluorescence in the cytoplasm when a region within the cytoplasm was bleached indicates the effectiveness of the bleaching protocol (red squares), while the limited loss of fluorescence in the cytoplasm when a region within the aggregate was bleached indicates the limited access of mCherry::ubiquitin to the Q82::GFP aggregates.
Mentions: To further investigate the mobility of mCherry::ubiquitin within Q82::GFP aggregates, fluorescence loss in photobleaching (FLIP) was used. mCherry was continuously bleached in a region either within the Q82::GFP aggregate or in the cytoplasm of a cell expressing the two fusion proteins. Loss of fluorescence in either a separate region within the aggregate or in the cytoplasm was monitored to examine mobility of the fluorescence material (Figure 7A, B). Directing bleach pulses to either the cytoplasm or the aggregate itself did not result in loss of fluorescence within the aggregate, indicating mCherry::ubiquitin is sequestered within aggregates. Bleaching within the cytoplasm reduced cytoplasmic mCherry fluorescence, indicating the effectiveness of the bleaching protocol and the mobility of mCherry::ubiquitin within the cytoplasm (Figure 7C). These results support the notion that the mCherry::ubiquitin is sequestered within the Q82::GFP aggregates, but is not itself in an aggregated, immobile configuration.

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