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

mCherry::ubiquitin colocalization to Q82::GFP aggregates. Worms co-expressing an mCherry::ubiquitin with Q82::GFP were exposed to RNAi of ubc-22, ubc-1, or pL4440 control. A typical control aggregate is shown in (A) with a 10.16-micron line (45 pixels) measuring a cross-section of fluorescence intensity. Data for this line profile is shown in (B). (C) Averaged line profile data for mCherry::ubiquitin fluorescence of Q82::GFP aggregates at multiple time points. Higher levels of colocalization of mCherry::ubiqutin occur at an age of 36 hours. Peaks represent fluorescence values in the aggregates while the peripheral points represent intracellular fluorescence. Values are the mean (± SEM) of all values at that distance coordinate for aggregates at the given time point. All values have been normalized with respect to exposure time and background levels of fluorescence. (D) Time course of mCherry::ubiquitin colocalization to Q82::GFP aggregates shows the spike in ubiquitin colocalization at 36 hours, which is diminished by RNAi of ubc-1 or ubc-22. Data shown are the mean (± SEM) ratio of mCherry fluorescence to GFP fluorescence from line profile measurements of composite images for multiple aggregates at each time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: mCherry::ubiquitin colocalization to Q82::GFP aggregates. Worms co-expressing an mCherry::ubiquitin with Q82::GFP were exposed to RNAi of ubc-22, ubc-1, or pL4440 control. A typical control aggregate is shown in (A) with a 10.16-micron line (45 pixels) measuring a cross-section of fluorescence intensity. Data for this line profile is shown in (B). (C) Averaged line profile data for mCherry::ubiquitin fluorescence of Q82::GFP aggregates at multiple time points. Higher levels of colocalization of mCherry::ubiqutin occur at an age of 36 hours. Peaks represent fluorescence values in the aggregates while the peripheral points represent intracellular fluorescence. Values are the mean (± SEM) of all values at that distance coordinate for aggregates at the given time point. All values have been normalized with respect to exposure time and background levels of fluorescence. (D) Time course of mCherry::ubiquitin colocalization to Q82::GFP aggregates shows the spike in ubiquitin colocalization at 36 hours, which is diminished by RNAi of ubc-1 or ubc-22. Data shown are the mean (± SEM) ratio of mCherry fluorescence to GFP fluorescence from line profile measurements of composite images for multiple aggregates at each time point.

Mentions: A strain expressing mCherry::ubiquitin with Q82::GFP in muscle cells (hereby referred to as Q82 + Ub) was used to assess the dynamics of ubiquitin localization to aggregates. A time course of mCherry::ubiquitin colocalization was performed, in which worms were imaged every 12 hours and line profile measurements of individual aggregates were taken to evaluate mCherry::ubiquitin colocalization to Q82::GFP aggregates. Our results show a spike in mCherry::ubiquitin colocalization in aggregates at 36 hours (Figure 5C, D). In the same experiment, groups exposed to RNAi of ubc-1 or ubc-22 show reduced colocalization of mCherry::ubiquitin to the polyglutamine aggregates (Figure 5D) at 36 hours.


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

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

mCherry::ubiquitin colocalization to Q82::GFP aggregates. Worms co-expressing an mCherry::ubiquitin with Q82::GFP were exposed to RNAi of ubc-22, ubc-1, or pL4440 control. A typical control aggregate is shown in (A) with a 10.16-micron line (45 pixels) measuring a cross-section of fluorescence intensity. Data for this line profile is shown in (B). (C) Averaged line profile data for mCherry::ubiquitin fluorescence of Q82::GFP aggregates at multiple time points. Higher levels of colocalization of mCherry::ubiqutin occur at an age of 36 hours. Peaks represent fluorescence values in the aggregates while the peripheral points represent intracellular fluorescence. Values are the mean (± SEM) of all values at that distance coordinate for aggregates at the given time point. All values have been normalized with respect to exposure time and background levels of fluorescence. (D) Time course of mCherry::ubiquitin colocalization to Q82::GFP aggregates shows the spike in ubiquitin colocalization at 36 hours, which is diminished by RNAi of ubc-1 or ubc-22. Data shown are the mean (± SEM) ratio of mCherry fluorescence to GFP fluorescence from line profile measurements of composite images for multiple aggregates at each time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: mCherry::ubiquitin colocalization to Q82::GFP aggregates. Worms co-expressing an mCherry::ubiquitin with Q82::GFP were exposed to RNAi of ubc-22, ubc-1, or pL4440 control. A typical control aggregate is shown in (A) with a 10.16-micron line (45 pixels) measuring a cross-section of fluorescence intensity. Data for this line profile is shown in (B). (C) Averaged line profile data for mCherry::ubiquitin fluorescence of Q82::GFP aggregates at multiple time points. Higher levels of colocalization of mCherry::ubiqutin occur at an age of 36 hours. Peaks represent fluorescence values in the aggregates while the peripheral points represent intracellular fluorescence. Values are the mean (± SEM) of all values at that distance coordinate for aggregates at the given time point. All values have been normalized with respect to exposure time and background levels of fluorescence. (D) Time course of mCherry::ubiquitin colocalization to Q82::GFP aggregates shows the spike in ubiquitin colocalization at 36 hours, which is diminished by RNAi of ubc-1 or ubc-22. Data shown are the mean (± SEM) ratio of mCherry fluorescence to GFP fluorescence from line profile measurements of composite images for multiple aggregates at each time point.
Mentions: A strain expressing mCherry::ubiquitin with Q82::GFP in muscle cells (hereby referred to as Q82 + Ub) was used to assess the dynamics of ubiquitin localization to aggregates. A time course of mCherry::ubiquitin colocalization was performed, in which worms were imaged every 12 hours and line profile measurements of individual aggregates were taken to evaluate mCherry::ubiquitin colocalization to Q82::GFP aggregates. Our results show a spike in mCherry::ubiquitin colocalization in aggregates at 36 hours (Figure 5C, D). In the same experiment, groups exposed to RNAi of ubc-1 or ubc-22 show reduced colocalization of mCherry::ubiquitin to the polyglutamine aggregates (Figure 5D) at 36 hours.

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