The rosetteless gene controls development in the choanoflagellate S. rosetta

1) Demonstrate antibody specificity […]

2) Homology of Rtls to metazoan proteins […]

We start by addressing the generation and specificity of the Rosetteless antibody. As requested, we have revised the text (described in detail below) to more clearly explain the nature of the Rosetteless antibody and the evidence for its specificity. In addition, we have added two new pieces of data: (1) a silver-stained gel documenting the purity of the recombinant Rosetteless used to compete with endogenous Rosetteless in tests of antibody specificity and (2) a Western showing that Rosetteless antibody recognizes the recombinant protein (Figure 5–figure supplement 1B).

First, we wish to clarify some information on the design and generation of the antibody. The Rosetteless antibody was raised against an epitope that we predicted should be present in all wild type and mutant Rtls isoforms (Figure 5A; orange bracket). Therefore, our measurements of protein abundance (Figure 5F) are expected to recognize both wild type and truncated versions of Rtls. To clarify this section of the Results, we have added the following text:

“To investigate endogenous Rtls protein in S. rosetta cells, we generated an antibody against residues 438–539, a region of the protein that is unique to Rtls and was expected to be present in all wild type and mutant Rtls isoforms (Figure 5A, Methods, Figure 5–figure supplement 1). Using this antibody, we found that total Rtls protein levels in mutant cells were ∼25% that of wild type cells (Figure 5F, Figure 5–figure supplement 1B-D).”

We have also added the following section on Rtls epitope design to the Methods and provide the BLAST results below:

“The epitope is unique to Rtls and bears no resemblance to other predicted polypeptide sequences in S. rosetta; when the amino acid sequence of the epitope was used to search the full catalog of predicted S. rosetta proteins (using blastp), no other protein hit the epitope with an e-value less than 20 (i.e. the other hits were not statistically significant)”.

Unlike conventional antibodies, the anti-Rtls antibody was generated as a genomic antibody, using a process that involves the introduction into rabbits of a DNA construct encoding the epitope, rather than the injection of purified protein ([Brown, 2011]; http://www.sdix.com/Products/Custom-Antibody-Services/Antibody-Development/Genomic-Antibody-Technology.aspx). This means that there was no possibility of inadvertently introducing additional bacterial or choanoflagellate epitopes during antibody generation. After the rabbit antiserum was harvested, the antibody was also affinity purified and analyzed by ELISA by SDIX.

We now turn to a discussion of the evidence for the specificity of the Rosetteless antibody:

One of the gold standard methods for establishing antibody specificity is to show that the signal is present in wild type samples, but absent or altered in mutant or knock out samples. In the case of Rosetteless, the staining patterns detected by immunofluorescence microscopy in mutant and wild type cells were strikingly different (Figure 6C), as were the levels of protein detected by dot blot (Figure 5F). To summarize the main text, Rtls mutants are missing both the bright staining at the center of rosettes (because the mutants do not produce rosettes) and the fainter, cell-membrane associated patches found in wild type single cells and chains. By dot blot, the anti-Rtls signal was reduced in mutant cells (Figure 5F, Figure 5–figure supplement 1C) even though the antibody should recognize both wild type and truncated Rtls isoforms. These differences between mutant and wild type detected by immunofluorescence microscopy and dot blot are most consistent with the interpretation that the majority of the antibody signal recognizes an epitope that is altered between mutant and wild type cells.

As an additional test of the specificity of the antibody, we used recombinant protein as a competitor during immunofluorescence and in dot blots. As requested by the reviewers, we have added documentation of the purity of the recombinant protein (by silver stain) and a Western blot of the recombinant protein as recognized by anti-Rtls to the manuscript (Figure 5–figure supplement 1B). The epitope has a predicted size of 38 kDa, and in our purifications it consistently appears at this size as a doublet, suggesting that some cleavage of the recombinant protein occurs in the bacteria. We indeed see this doublet as the predominant set of bands in the silver-stained recombinant protein sample and the only clear signal by Western blot.

In cells stained with the Rosetteless antibody, we see three main areas of immunofluorescence: (1) an extracellular meshwork in the center of rosettes, (2) in single cells and chains, a thin patch of membrane-associated staining that is most often found at the base of cells but sometimes seen in other parts of the cell, and (3) faint staining throughout the cell body in single cells and rosettes. If the Rosetteless antibody is pre-incubated with recombinant Rosetteless protein, the first two staining patterns (i.e. extracellular meshwork in center of rosettes and thin membrane-associated patches) disappear, presumably because those patterns result from antibody binding that specifically recognizes Rosetteless protein. The third pattern (i.e. faint staining throughout the cell body) persists following pre-incubation of the Rosetteless; we interpret this third pattern then to reflect either autofluorescence or non-specific staining. We are careful in the text to delineate that portion of the staining pattern that is specific to Rosetteless vs. that which reflects either autofluorescence or non-specific staining (in the legend for Figure 6–figure supplement 2): “In all cells following competition with the epitope there remained faint staining in the cell body; we thus infer that this is non-specific staining that does not reflect the distribution of Rtls protein in the cells”).

Finally, we agree with the reviewers that a Western of S. rosetta cell lysates would be a valuable contribution to our efforts to validate the Rosetteless antibody. Indeed, we have been trying to establish Rosetteless Westerns on S. rosetta cell lysates for the past 5 months. However, likely due to the nature of Rosetteless protein itself, our attempts at these Westerns have failed.

We find either that there is no signal or that the primary signal detected by the anti-Rtls antibody by Western blot is of a very high molecular weight protein that does not enter the gel. We believe that the explanation for this phenomenon lies in the predicted mucin-like domains of the Rtls protein; in similar S/T-rich regions, mucins are heavily glycosylated, making these proteins sticky and quite large [Harrop et al, 2011]. Indeed, because such ECM proteins may be decorated with a variable number of sugars and often undergo other forms of posttranslational processing (e.g. cleavage into active forms), a single band on a Western may not even be expected for this type of protein.

Still, we have made a number of attempts to modify our Western blotting protocol to accommodate heavily glycosylated proteins and solubilize the mucous-like S. rosetta lysate. These attempts include: 1) various cell lysis and solubilization methods; 2) syringing and other means of physical shearing; 3) pre-incubation of cell lysates with deglycosylases; 4) resuspension of lysate in 8M urea; and 5) the use of low-percentage acrylamide and acrylamide/agarose gels. However, in all attempts to date, we observe either a high molecular weight signal that does not enter the gel, or no signal at all.

Due to the challenges in working with fully glycosylated mucins, the use of dot blots for semi-quantitative analyses of mucin levels is not unusual [Harrop et al, 2011, Thomsson et al 2011]. Therefore, because Western analysis of Rosetteless in S. rosetta cell lysates has not yet been possible, we used dot blot analysis to measure the relative abundance of Rosetteless protein in wild type and mutant cells [Harrop et al, 2011, Thomsson et al 2011]. As the reviewers pointed out, dot blots are not ideal, as the high concentration of protein can lead to non-specific binding of antibodies and, because the proteins are not separated by size, it is not possible to see whether the source of the signal is from one protein or multiple proteins. Nonetheless, for proteins that do not properly enter into gels, it can provide a means by which to estimate their abundance. Similar to our procedure for validating the antibody by immunofluorescence, we used the pre-incubation of the recombinant epitope with anti-Rtls antibody to compete away the staining that specifically recognized the Rosetteless protein. In Figure 5–figure supplement 1B, we demonstrate that this pre-incubation results in the loss of the majority, but not all, of the signal by dot blot. We also showed the residual signal following competition in the main figure (Figure 5F), and explained in the figure legend for this panel that it was only a “semi-quantitative” method, thereby acknowledging the limitations with dot blots.

In summary, although we have been unable to perform a Western analysis on S. rosetta cell lysates, we have a number of independent observations and experiments that are all consistent with the interpretation that the anti-Rtls antibody displays a high degree of specificity for Rtls protein.

We now discuss the relationship between Rosetteless protein and CTLD-containing proteins in animals:

We have generated a new figure, Figure 5–figure supplement 2, to illustrate the diversity of CTLD-containing proteins in choanoflagellates. This figure shows the full domain architecture of all CTLD-containing proteins in the S. rosetta and M. brevicollis genomes, with similar animal CTLD domain architectures for comparison.

While Rtls may indeed be a multicellularity gene, we have not yet demonstrated this to be the case. We refrain from making this assertion because it is not currently possible to clearly assess the homology between Rtls and animal lectins. Specifically, the divergence time between animals and choanoflagellates means that orthology assignments often must be made on the basis of domain architecture and organization instead of amino acid alignments. This analysis cannot be convincingly carried out for Rtls, as its simple domain architecture is not sufficiently diagnostic. Finally, within animals, C-type lectins are a family of rapidly evolving genes that exhibit extensive duplications and rearrangements among taxa (e.g. [Sattler et al, 2012; Drickamer et al 1999]). It is thus not currently possible to assign clear orthology relationships among many of the animal C-type lectins, much less between animal C-type lectins and more evolutionarily distant CTLD-containing proteins found in choanoflagellates and other eukaryotes (e.g. [Wheeler et al, 2008]). We discuss the limitations in identifying homologous C-type lectins in the legend for Figure 5–figure supplement 2.