Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans

Abstract

Green fluorescent protein (GFP) is widely used as a molecular tool to assess protein expression and localization. In C. elegans, the signal from weakly expressed GFP fusion proteins is masked by autofluorescence emitted from the intestinal lysosome-related gut granules. For instance, the GFP fluorescence from SKN-1 transcription factor fused to GFP is barely visible with common GFP (FITC) filter setups. Furthermore, this intestinal autofluorescence increases upon heat stress, oxidative stress (sodium azide), and during aging, thereby masking GFP expression even from proximal tissues. Here, we describe a triple band GFP filter setup that separates the GFP signal from autofluorescence, displaying GFP in green and autofluorescence in yellow. In addition, yellow fluorescent protein (YFP) remains distinguishable from both the yellowish autofluorescence and GFP with this triple band filter setup. Although some GFP intensity might be lost with the triple band GFP filter setup, the advantage is that no modification of currently used transgenic GFP lines is needed and these GFP filters are easy to install. Hence, by using this triple band GFP filter setup, the investigators can easily distinguish autofluorescence from GFP and YFP in their favorite transgenic C. elegans lines.

Rapid Degradation ofCaenorhabditis elegansProteins at Single-Cell Resolution with a Synthetic Auxin

“…Although degradation kinetics were slower than those observed in NAA in NGM plates or M9 alone, we still observed approximately 62-74% reduction of AID::GFP within the first 30 min of NAA or K-NAA exposure, and nearly 80% depletion of whole animal AID::GFP within 1 hr ( Figure 4B-C). Our results may be underrepresenting the overall loss of AID::GFP as we did not account for gut autofluorescence in our quantification of fluorescence intensity in whole animals (Teuscher and Ewald 2018). Taking this into consideration, we also quantified depletion of AID::GFP in the pharynx, observing greater than 77% AID::GFP depletion within the first 30 min in larval pharynxes (Supplemental Material, Figure S1A).…”

The Matrisome during Aging and Longevity: A Systems-Level Approach toward Defining Matreotypes Promoting Healthy Aging

“…While single-cell RNA-sequencing is feasible, single-cell proteomics is more challenging. Alternatively, tagging ECM proteins with fluorescent proteins could reveal the matreotypes during aging in vivo and noninvasively [37]. Thus, these few examples already indicate that it might be challenging but feasible to quantify and interpret matreotypic changes during aging.…”

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

“…Although degradation kinetics were slower than those 353 observed in NAA solubilized in M9 alone (Figure 3B-D) or NGM plates containing NAA 354 or K-NAA (Figure S1B-C), we still observed approximately 60-70% reduction of 355 AID::GFP expression within the first 30 minutes of NAA or K-NAA exposure, and nearly 356 80% depletion of whole animal AID::GFP within 1 hour (Figure 3E-F). Our results may 357 be under-representing the overall loss of AID::GFP as we did not account for gut 358 autofluorescence in our quantification of fluorescence intensity in whole animals 359 (Teuscher and Ewald 2018). Nonetheless, our results demonstrate that AID-tagged 360 proteins can be depleted in a microfluidic platform which, when combined with long-term 361 high-resolution imaging, provides a powerful tool for studying post-embryonic C. elegans 362 development at cellular resolution.…”