Target gene responses differ when transcription factor levels are acutely decreased by nuclear export versus degradation.

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that result in either nuclear export or degradation of Dorsal when exposed to blue light. Nuclear export of Dorsal results in loss of expression for the high threshold, ventrally-expressed target gene snail (sna) but retention of the low threshold, laterally-expressed target gene short-gastrulation (sog). In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels than when Dorsal is exported from the nucleus. To elucidate how nuclear export results in loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found it reenters the nucleus even under conditions of blue-light when export is favored. The associated kinetics of being imported and exported continuously are likely responsible for loss of sna but, alternatively, can support sog. Collectively, our results show that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention.

Diffusion and distal linkages govern interchromosomal dynamics during meiotic prophase.

SignificanceEssential for sexual reproduction, meiosis is a specialized cell division required for the production of haploid gametes. Critical to this process are the pairing, recombination, and segregation of homologous chromosomes (homologs). While pairing and recombination are linked, it is not known how many linkages are sufficient to hold homologs in proximity. Here, we reveal that random diffusion and the placement of a small number of linkages are sufficient to establish the apparent "pairing" of homologs. We also show that colocalization between any two loci is more dynamic than anticipated. Our study provides observations of live interchromosomal dynamics during meiosis and illustrates the power of combining single-cell measurements with theoretical polymer modeling.

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster.

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.

Twist-dependent ratchet functioning downstream from Dorsal revealed using a light-inducible degron.

Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

A comparison of methods for estimating the lactate threshold.

The purpose of this study was to examine the accuracy of tests that may be used by distance runners to estimate the lactate threshold. Competitive distance runners/triathletes (N = 27) performed a criterion test that directly measured (blood lactate of 4.0 mmol.L(-1)) the lactate threshold. Subjects then performed 4 tests (VDOT, 3,200-m time trial, 30-minute time trial, Conconi) that estimate the threshold. Mean estimations of the running velocity at the lactate threshold from the 30-minute time trial (standard error of the estimate, SEE, 0.21 m.s(-1)) and VDOT (SEE 0.41 m.s(-1)) methods did not differ (P>0.05) from the criterion. In terms of heart rate, the 30-minute time trial estimation did not significantly differ (SEE 8.0 b.min(-1)) from criterion. These findings suggest that the 30-minute time-trial method should be considered by coaches and distance runners/triathletes as a method for estimating both the running velocity and heart rate at the lactate threshold.