Determination of oligomeric states of proteins via dual-color colocalization with single molecule localization microscopy
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Evaluation Summary:
The authors present a method for measuring the oligomerisation state of tagged membrane proteins by PALM co-localisation that is new, interesting and potentially very useful for identifying oligomerization states of unknown proteins in native cells. While the authors develop a basic theory and apply the method to a set of candidate proteins with solid results, their implementation could be refined and improved, which would help to better delineate the full scope and the limitations of their method. An open-source software tool would help other researchers to adopt this analysis. The work is relevant for cell biologists, especially those studying membrane proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The oligomeric state of plasma membrane proteins is the result of the interactions between individual subunits and an important determinant of their function. Most approaches used to address this question rely on extracting these complexes from their native environment, which may disrupt weaker interactions. Therefore, microscopy techniques have been increasingly used in recent years to determine oligomeric states in situ. Classical light microscopy suffers from insufficient resolution, but super-resolution methods such as single molecule localization microscopy (SMLM) can circumvent this problem. When using SMLM to determine oligomeric states of proteins, subunits are labeled with fluorescent proteins that only emit light following activation or conversion at different wavelengths. Typically, individual molecules are counted based on a binomial distribution analysis of emission events detected within the same diffraction-limited volume. This strategy requires low background noise, a high recall rate for the fluorescent tag and intensive post-imaging data processing. To overcome these limitations, we developed a new method based on SMLM to determine the oligomeric state of plasma membrane proteins. Our dual-color colocalization (DCC) approach allows for accurate in situ counting even with low efficiencies of fluorescent protein detection. In addition, it is robust in the presence of background signals and does not require temporal clustering of localizations from individual proteins within the same diffraction-limited volume, which greatly simplifies data acquisition and processing. We used DCC-SMLM to resolve the controversy surrounding the oligomeric state of two SLC26 multifunctional anion exchangers and to determine the oligomeric state of four members of the SLC17 family of organic anion transporters.
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Evaluation Summary:
The authors present a method for measuring the oligomerisation state of tagged membrane proteins by PALM co-localisation that is new, interesting and potentially very useful for identifying oligomerization states of unknown proteins in native cells. While the authors develop a basic theory and apply the method to a set of candidate proteins with solid results, their implementation could be refined and improved, which would help to better delineate the full scope and the limitations of their method. An open-source software tool would help other researchers to adopt this analysis. The work is relevant for cell biologists, especially those studying membrane proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
The authors present a method for measuring the oligomerisation state of tagged membrane proteins by PALM co-localisation that is new, interesting and potentially very useful for identifying oligomerization states of unknown proteins in native cells. While the authors develop a basic theory and apply the method to a set of candidate proteins with solid results, their implementation could be refined and improved, which would help to better delineate the full scope and the limitations of their method. An open-source software tool would help other researchers to adopt this analysis. The work is relevant for cell biologists, especially those studying membrane proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Tan et. al develop an SMLM method for determining the number of subunits in oligomeric proteins where each subunit is labeled with two fluorescent markers of separable colors in fixed cells. Rather than counting each subunit, their approach infers the number of subunits based on the statistical likelihood of observing the protein at all in one of the color channels, and uses the other color channel to estimate the fraction of labeled proteins that are not observed. Their approach requires first calibrating an experimental system using labeled proteins of known stoichiometry to determine the probability of observing each marker in that setup, which thereafter can be used to determine the number of subunits in similarly labeled proteins of unknown stoichiometry. They apply their approach to several anion …
Reviewer #1 (Public Review):
Tan et. al develop an SMLM method for determining the number of subunits in oligomeric proteins where each subunit is labeled with two fluorescent markers of separable colors in fixed cells. Rather than counting each subunit, their approach infers the number of subunits based on the statistical likelihood of observing the protein at all in one of the color channels, and uses the other color channel to estimate the fraction of labeled proteins that are not observed. Their approach requires first calibrating an experimental system using labeled proteins of known stoichiometry to determine the probability of observing each marker in that setup, which thereafter can be used to determine the number of subunits in similarly labeled proteins of unknown stoichiometry. They apply their approach to several anion exchangers/transporters of unknown stoichiometry and validate their result with native gels. The approach is limited to populations of fairly uniform stoichiometry, but has some marked advantages over other methods such as bleach step counting in that it is feasible to use fluorophores with low probabilities of detection and can readily be applied to proteins in cells at higher densities. These advantages will be very useful to experimentalists for understanding the oligomeric assembly of proteins in native cells. Some clarification of the methods and assumptions are needed for a more general readership.
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Reviewer #2 (Public Review):
Summary:
In contrast to many other SMLM methods which aim to count subunits in membrane protein complexes, the authors aim to deduce the average oligomerisation state from the probabilistic co-detection of at least 1 'reporter' fluorophore, which has relatively poor detection efficiency, with the detection of at least 1 fused 'marker' fluorophore. They derived a simple theoretical framework, calibrated the method using proteins of known oligomerisation state (validated against high-resolution clear native gel electrophoresis), and applied it to clarify the oligomerisation state of six SLC26 and SLC17 family member membrane proteins.
Major strengths and weaknesses:
The proposed concept is new and interesting. It has the potential to overcome some, though not all, limitations of existing techniques. For …
Reviewer #2 (Public Review):
Summary:
In contrast to many other SMLM methods which aim to count subunits in membrane protein complexes, the authors aim to deduce the average oligomerisation state from the probabilistic co-detection of at least 1 'reporter' fluorophore, which has relatively poor detection efficiency, with the detection of at least 1 fused 'marker' fluorophore. They derived a simple theoretical framework, calibrated the method using proteins of known oligomerisation state (validated against high-resolution clear native gel electrophoresis), and applied it to clarify the oligomerisation state of six SLC26 and SLC17 family member membrane proteins.
Major strengths and weaknesses:
The proposed concept is new and interesting. It has the potential to overcome some, though not all, limitations of existing techniques. For example, the concept is limited to measuring the average oligomerisation state and as such is not suitable to measure a distribution of (higher) oligomerisation states.
The implementation of the concept is suboptimal in both, theoretical and practical, aspects:
On the theory side, a rigorous probabilistic framework for the assignment of the most likely oligomerisation state is missing. This includes a sensitivity analysis of Equation 1 (or, better, of Equation 6) which highlights at which n or p this method is most sensitive. Also, no confidence intervals for fitted values of m and p were provided which could be used in such a sensitivity analysis.
On the practical side, the SMLM detection/processing details are not state-of-the-art (PSF fit with fixed SD 2D Gaussian; not using maximum likelihood estimation for fitting; DBSCAN algorithm to group raw (single-frame) data). In conjunction with setting a minimum value of 6 (PAmCherry) and 10 (mVenus) for the number of localisations per cluster, these together might contribute to the poor detection efficiency for PAmCherry of 0.12, which is in contrast to the reported maturation efficiency of the protein, and which the authors attribute to protein misfolding.Appraisal of whether the results support their conclusions:
The authors were successful in establishing their new concept. The conclusions about the oligomerisation state of SLC family membrane proteins are, despite the methodological shortcomings, convincingly supported by the data.
The authors' measured oligomerisation states of candidate membrane proteins which formed homo-dimers up to tetramers. They did not attempt to measure stoichiometries of different subunits. The title of the paper thus is misleading. Although the technique could be applied to measure the oligomerisation state of different subunits in independent samples using different expression constructs, and thus an average stoichiometry could be determined, it is not suitable to directly measure stoichiometries of different subunits in the same sample.
It is unlikely that the author's attribution of the very low detection efficiency to a 'misfolded' fraction of proteins is the only possible explanation.Discussion of the likely impact of the work
If this analysis was further improved (see limitations above) and offered as an open-source software tool, it could find wider-spread application and complement existing methods to measure the oligomerisation state of membrane proteins from monomers to tetramers using relatively standard PALM approaches.
A potential limitation is that labs which wish to adopt this method are required to perform a whole set of calibration measurements to experimentally determine the detection efficiency p (and the 'misfolding' parameter m), see Figure 4A, which is relatively laborious.
The presented results on the oligomerisation state of SLC family membrane proteins are of direct interest to researchers in this area of molecular cell biology. -
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