Green Fluorescent Protein: Applications & Protocols (Methods in Molecular Biology Vol 183)
The resulting diploids were grown, induced with galactose, washed, and imaged as in Figure 1B. A Diploids 73—77, B diploids 66—70, C diploids —, and D diploid strains 80— Thus access to a tag placed at the N-terminus of either of these two septins may be limited, resulting in a weaker signal in this assay format. To verify that the tripartite split-GFP method can also faithfully report direct interaction of other cellular proteins with septins at the bud neck, we first examined Bni5 residues.
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Taken together, these data support the conclusion that the C-terminal end of Bni5 is anchored to Cdc11 and Shs1, whereas the N-terminal end of this highly elongated protein is more flexible and can sweep out a much larger conformational space. Representative cells with a medium-to-large bud and an intact septin collar before cytokinesis top or with a split septin collar diagnostic of cells in cytokinesis bottom. C Quantification, as in Figure 1C , of the data in B. E Quantification, as in Figure 1C , of the data in D.
Although the septin-binding region of Hsl1 has been delineated, the subunit specificity of Hsl1 binding to the septin collar, if any, has not yet been determined. Only cells with an intact septin collar were scored because, upon the onset of anaphase and formation of the split collar, Hsl1 is degraded Burton and Solomon, , C Quantification, as in Figure 1C , of the data shown in B, except that only budded cells with an intact septin collar i.
Dashed green line, best fit trend line for the Cdc3 and Cdc12 data points; dashed blue line, best-fit trend line for Cdc10 data points. This arrangement yielded no detectable signal Figure 5, B , left, and C , left , suggesting that, even when bound at the septin collar, the N-terminus of Hsl1 which contains its catalytic domain is very far from the C-terminus of every septin. Reassuringly, using this approach, there was a clear trend in the 60 diploids constructed for this set of analyses Figure 5D.
Hence it is clear that the septin-binding element in Hsl1 associates primarily with Cdc12 and Cdc3, a specificity not previously characterized. For the majority of these proteins, there is little or no information about whether they localize to the bud neck because they directly bind to a septin s there, or not.
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Hence, as a final test of the usefulness of the tripartite split-GFP assay, we examined three bud neck—associated proteins whose capacity to physically associate with specific septins has, to our knowledge, not yet been definitively characterized. The first such protein we examined was a poorly studied bud neck—localized factor, Nis1 residues , whose purported ability to physically associate with septin Shs1 is based mainly on a two-hybrid interaction reported in a single study Iwase and Toh-e, In our hands, Nis1-eGFP expressed under its endogenous promoter in a strain coexpressing CdcmCh does not colocalize with the septin collar.
In cells with small or large buds, Nis1 is found just adjacent to the division site in a small patch Figure 5A , top ; in cells undergoing cytokinesis, Nis1 is localized between the split septin rings Figure 5A , bottom.
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We carefully examined all cells, but especially dividing cells that were actively undergoing cytokinesis i. We conclude, therefore, that Nis1 is sequestered between the split septins but does not make direct contact with any septin there. Thus recruitment of Nis1 to this location is not mediated by its binding to any septin.
Top, GFP signal white triangles adjacent to the septin collar in budded cells before cytokinesis. Bottom, during cytokinesis, Nis1-eGFP localizes between the two rings generated by splitting of the septin collar. Although prior work provided genetic and biochemical evidence that Bud4 makes direct contact with septins, the subunit specificity of its interaction was not defined. Therefore, and as for Hsl1, we focused our analysis on this previously identified, putative septin-binding domain of Bud4. Indeed, in our hands, a Bud4 -eGFP construct strongly colocalized with the septin collar marked with CdcmCh although a detectable amount of this fragment was also found in the nucleus; Figure 7A.
F Quantification, as in Figure 1C , of the data in E. As for Bud4, prior genetic and biochemical findings indicated that Hof1 makes direct contact with septins at the bud neck, but its subunit selectivity was not clearly identified. In our hands, an eGFP-Hof1 construct was found mainly in the cytosol but did localize detectably, although only rather weakly, at the bud neck, congruent with CdcmCh Figure 7D. This method permitted successful interrogation of protein—protein associations in situ under near-native conditions at endogenous levels of expression.
Any proteins examined are present in their normal modification state and in their natural intracellular location and milieu when their ability to physically interact is assessed. Given their small size, the tags 20 or 21 residues have a minimal effect on the solubility and other behaviors of the tagged proteins, whose interaction was readily detectable even in diploid cells in which a wild-type WT copy of each tagged partner was also present Supplemental Figure S1.
Hence background fluorescence is minimal. This arrangement allows for temporal control.
This arrangement also allows for spatial control. We found, however, that the occurrence and intensity of the fluorescence signal observed between pairs of septins largely mirrors the known subunit order within the hetero-octamer. Thus our observations indicate that the tripartite split-GFP method mainly measures intimate short-range physical contacts. Nonetheless, by increasing linker length, we could begin to detect such readouts. For example, in the hetero-octamer, any given Cdc10 is adjacent to another Cdc10 and to Cdc3 and gives a very strong signal with either partner, and yet, at the longest linker lengths we used, interaction albeit much weaker of Cdc10 with Cdc12 the next subunit over from Cdc3 was detectable.
Thus the shorter the linker length, the more stringent are the distance requirements for interaction; conversely, the longer the linker length, the more promiscuous are the associations that will be detected, up to a point. Membrane association may partially occlude access to the N-termini of these septins.
Thus this method provides a certain degree of insight into how septin-binding proteins are oriented when they dock on the septin collar Figure 8. The linear hetero-octamer is depicted as it resides in paired filaments conjoined via formation of cross-filament coiled coils between the CTEs on Cdc3 and Cdc The C-terminus of Bni5 exhibited preferential interaction with the N terminal faces of Cdc11 and Shs1 thick red arrows , whereas the N- terminus of Bni5 was able to interact weakly with the C-termini of all five septins dashed red arrows.
The N-terminus of the septin-binding domain of Hsl1 had preferential interaction with the C-termini of Cdc12 and Cdc3, whereas the C-terminus of the same fragment did not have a detectable interaction with any subunit. The C-terminus of the septin-binding domain of Bud4 exhibited preferential interaction with the N-termini of Cdc11, Shs1, and, weakly, Cdc3 most likely due to the extremely long N-terminal extension on Cdc3 , whereas the N-terminus of the same fragment did not have a detectable interaction with any subunit. The C-terminus of the septin-binding domain of Hof1 had preferential interaction with the N-termini of Cdc10, Cdc12, and, weakly, Cdc3, whereas the N-terminus of the same fragment did not have detectable interaction with any subunit.
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To verify that the tripartite split-GFP method reports interaction of authentic septin-binding domains, we turned to the much larger residues multidomain, septin collar—binding protein Hsl1. Aside from confirming that this Hsl1 segment associates directly with the septin collar, the tripartite split-GFP method revealed that it docks via its N-terminal end to the C-terminal ends of Cdc12 and Cdc3. Thus this technique shed light on how a septin-binding domain within a larger septin-binding protein engages the septin collar.
For Bud4 and Hof1, prior deletion analysis and fragment scanning identified segments that appear both necessary and sufficient for their septin association. Thus synergistic action of both PM-binding motifs and septin-binding elements in certain bud neck—localized proteins presumably coordinates their function with processes that influence the lipid composition of the PM. Whether the subunit arrangement within septin hetero-octamers and in the higher-order structures built from them arose due to evolutionary selection for efficient assembly or as a means to dictate more effectively the positioning of associated septin-binding proteins or both has been an open question.
There is some evidence for the latter. Whether the subunit-specific position of any other septin-associated protein is crucial for its function has, to our knowledge, not been explored. Such information is important for understanding how these factors exert their physiological actions. By contrast, the tripartite split-GFP method allows for facile assessment of the potential interaction between any two proteins of interest.
The yeast strains used in this study are listed in Table 1. Standard molecular biology techniques and protocols were followed Sambrook and Russell, To generate the C-terminally tagged versions of the proteins examined, a common methodology was used that involves a PCR-based in vivo ligation and homologous recombination method in yeast Finnigan and Thorner, Each construct was confirmed via DNA sequencing.
Next the entire cassette was PCR amplified using the plasmid DNA as the template, treated with Dpn I, and introduced by DNA-mediated transformation into yeast to replace a chromosomal deletion of the gene of interest. To maintain viability for integration of the tagged version of any essential gene, the cells also harbored a URA3- based covering plasmid carrying a WT copy of the cognate gene, which was then removed by selection on medium containing 5-fluoro-orotic acid 5-FOA. To generate the N-terminally tagged versions of the proteins examined, a modified protocol was used.
Fourth, the entire cassette was PCR amplified and integrated into the genome as described. DNA isolated from each resulting integrant was amplified with a high-fidelity polymerase and sequenced to verify correct construction. During the construction of any strain that involved introduction of a tagged version of an essential septin gene, the cells harbored a URA3 -marked covering plasmid expressing the corresponding WT septin gene.
For the constructs involving Bud4 and Hof1, a modified diploid selection protocol was used. Diploids then were selected and propagated on SD-Ura-Leu medium. For imaging of strains not containing any plasmids e. Cells were harvested, washed with water, placed on a glass slide with a coverslip, and imaged within 5 min. The cultures were back-diluted to an A nm of 0.
sandrettari.tk All images were treated identically and rescaled together. For clarity, the periphery of yeast cells was labeled using white dotted lines from an overexposed image or a corresponding differential interference contrast image. Unless otherwise indicated, all images were taken for the same exposure time and using the same light source power level.
The results described are average values for each diploid strain derived from experiments conducted in triplicate. For quantification of the percentage of the cell population that displayed a detectable reconstituted eGFP signal at the bud neck, 25— cells in separate fields were scored and divided by the total number of cells in the same fields that exhibited a detectable mCherry signal i.
For quantification of the pixel intensity of the eGFP fluorescence at the bud neck, the box tool in ImageJ was used to carefully outline the bud neck in 25— cells that also clearly displayed a septin collar as judged by the mCherry signal , and the average pixel intensity with SEM was calculated after subtracting the average background fluorescence in any given image from each measurement taken in the same image.
The average background fluorescence was determined by using the box tool to assess the pixel intensity of an equivalent area of five randomly chosen regions of each image that did not contain any cells. Conversely, for quantification of Nis1 at the bud neck, only budded cells that displayed a clear split septin ring were included, as native Nis1 is not present at the bud neck until cytokinesis Figure 5A.
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E on July 6, C-terminal extension. This work was supported by a Postdoctoral Fellowship from the Adolph C. This article is distributed by The American Society for Cell Biology under license from the author s. Two months after publication it is available to the public under an Attribution—Noncommercial—Share Alike 3. Molecular Biology of the Cell Vol. This is the final version - click for previous version. Gregory C. Angela Duvalyan Search for more papers by this author. Elizabeth N.
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Jeremy Thorner Search for more papers by this author. Add to favorites Download Citations Track Citations. Abstract Various methods can provide a readout of the physical interaction between two biomolecules. Int Rev Cell Mol Biol , Saccharomyces cerevisiae septins: supramolecular organization of heterooligomers and the mechanism of filament assembly.
Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae. Mol Biol Cell 23 , Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin filament assembly and organization.