Abstract
The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane and cytosolic proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity, while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.
Highlights
The study of the consequences of the loss of gene function is a central technique in biology
The degradation of Histone 2A (H2A)-EGFP was significantly slower displaying a half-life of about 2.5 h (Figure 2 and Table 2)
Embryos were incubated at 28.5 °C from one-cell-stage (0 hpf) until the indicated time. cxcr4bt26035 [38] homozygous mutant embryos were generated by inbreeding heterozygous adults, crossing homozygous adult with heterozygous adults or inbreeding homozygous adults. tg(cxcr4b:cxcr4b-EGFP-internal ribosomal entry site (IRES)-Kate2364 CaaX) [27, 28] embryos were generated by crossing heterozygous adults with wild-type adults 365 and sorted by GFP expression
Summary
The study of the consequences of the loss of gene function is a central technique in biology. While the expected fraction of embryos that should lose Cdh activity and display a phenotype is 6.25% (cdh1-/-; hsp70l:zGrad; cdh1:cdh1-sfGFP embryos, Figure 4 – figure supplement 1A), we believe that not all embryos had enough Cdh protein depleted to cause a cell-cell adhesion defect due to the variability in zGrad levels These observations suggest that transient expression of zGrad can efficiently degrade GFP-tagged proteins and, if GFP-tagged proteins are depleted sufficiently, cause loss of protein function and uncover phenotypes past the initial requirements of essential proteins. A pulse of zGrad expression can deplete Cxcr4b-EGFP to levels unable to sustain directed cell migration of the primordium This suggests that temporal zGrad induction from the heat shock promoter can be used to induce reversible protein loss of function scenarios. These observations indicate that expression of zGrad from a tissue309 specific promoter can result in efficient degradation of GFP-tagged proteins to levels low enough to perturb protein function and cause tissue-specific defects
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