Characterisation of Amyloid Aggregation and Inhibition by Diffusion-Based Single-Molecule Fluorescence Techniques

Round 1

Reviewer 1 Report

Polanco and colleagues review the use of interesting single-molecule fluorescence methods to unravel the mechanism of amyloid fibrillization.

The review is mostly comprehensive, well organized, and excellently written.

My only comment is the oversight of some seminal works in the field for the introduction. For instance:

The use of FCS to study amyloid aggregation; and FCS coupled to lifetime filtering:

- Tjenberg et al. (1999), DOI: 10.1016/S1074-5521(99)80020-9.

- Sengupta et al. (2003), DOI: 10.1016/S0006-3495(03)75006-1

- Gerard et al. (2006), DOI: 10.1096/fj.05-5126fje

- Nath et al. (2010) DOI: 10.1016/j.bpj.2009.12.4290

- Paredes et al. (2012), DOI: 10.3390/ijms13089400

The use of pulsed interleaved excitation to study amyloid aggregation:

- Castello et al. (2015), DOI: 10.1021/acs.jpcb.5b01957

Author Response

We thank the reviewer for his/her nice comments on our work and for suggesting the introduction of relevant references in the introduction. All suggested references have been now included in the revised version of the manuscript.

Reviewer 2 Report

The review article “Characterisation of amyloid aggregation and inhibition by diffusion-based single-molecule fluorescence techniques” discusses the biophysical methods through which fibrillation can be observed. Thorough introductions of many methods, primarily conducted in the single-molecule concentration range, are followed by representative data that demonstrates the applicability of the techniques discussed. For example, using α-synuclein as a model, single-molecule FRET analysis is able to distinguish oligomeric populations by size. With these results, the reviewers were able to identify a small molecule that can potentially interfere with fibril formation and bind specifically to the toxic oligomeric species. The methods discussed in this review are also applicable to heterogeneous protein experiments, as interactions between tau and α-synuclein can also be detected and monitored.

Overall, the review is well written and provides an in-depth look into the methods that can be used to study aggregation and identify potential therapeutics. The following are a few minor adjustments that can be made to the manuscript:

1.       Aggregation, by definition, involves multiple particles. The authors should clarify to the readers how single-molecule techniques can be used to study a process that by definition cannot be a single particle. For example, the authors can emphasize that single-molecule refers to the label/dye being detected and not necessarily the whole system being detected.

2.       FCS and FCCS, strictly speaking, are typically not conducted at single-molecule concentrations (i.e., nM concentration range  vs. pM).

3.       TCCD at picomolar concentration requires strong affinity between the two interacting species.

4.       Clarification of which techniques can be conducted at non-single-molecule conditions (e.g., TCCD, FCS, FCCS) and which should be single-molecule (e.g., smFRET) will help the reader understand how and when these techniques can/should be used. In addition, to distinguish single-molecule from ensemble FRET, I recommend that the authors use the acronym smFRET, which is very common in the single-molecule field.

5.       In line 22 of the abstract, when stating “we review our recent contributions”, the authors should clarify that they are reviewing the field of single-molecule fluorescence techniques and including their contributions unless they really meant that what the authors are mainly presenting are just their work.

6.       Line 66 the term ‘microscopic’ is not ideal because it implies relation to microscopy. ‘Intermolecular’ may be a better substitute for ‘microscopic’

7.       The abbreviation αS is rarely used to describe α-synuclein. The abbreviation ‘α-syn’ is more  conventional.

Apart from these minor recommended changes, the review is insightful and provides a detailed look into the capabilities of single-molecule fluorescence techniques. Some of the sentences are dense and the readability of the review can still be improved.

Author Response

We thank the reviewer for his/her nice comments and feedback on our work. We have tried to address the points raised by the reviewer as explained below.

Point 1: Aggregation, by definition, involves multiple particles. The authors should clarify to the readers how single-molecule techniques can be used to study a process that by definition cannot be a single particle. For example, the authors can emphasize that single-molecule refers to the label/dye being detected and not necessarily the whole system being detected.

We have explained in the introduction that protein aggregation involves multiple types of protein species which can be better identified and characterised using single-molecule/particle approaches than with average-based in bulk techniques, and emphasize in multiple parts of the text that the molecules under study need to be fluorescently labelled in order to be identified in fluorescence single-molecule experiments. In order to make this latter point clearer, we have included a sentence at the beginning of section 2:

Line 154: “Fluorescence-based single-molecule methods typically require the attachment of particularly bright fluorophores to the macromolecule of interest ...”

In addition, we have clarified in section 3 and section 4 that in the experiments performed all the protein molecules were fluorescently-labelled and, therefore, all protein particles could be, in principle, detected in single-molecule experiments. In section 5, we already explained that only a subfraction of protein molecules had a fluorophore attached.

Line 369: “Equimolecular mixtures of aS labelled with either AlexaFluor488 (AF488) or AlexaFluor647 (AF647) (experiments performed with 100% of labelled protein molecules) were incubated…”

Line 489: “For this, 100% fluorescently labelled molecules were used. αS was labelled with AF488 at the C-terminal region of the protein; a region that remains highly flexible and disordered in all protein species, while the peptide was N-terminally labelled with Atto647N.”

Line 562: “We incubated the proteins at 25 μM with 1 μM of fluorescently-labelled variants (Atto647N-Tau and AF488-αS)…”

Point 2: FCS and FCCS, strictly speaking, are typically not conducted at single-molecule concentrations (i.e., nM concentration range  vs. pM)

The reviewer is right and FCS/FCCS are typically conducted at 1 nM concentrations, although they can be also performed at pM concentrations (as some of the examples shown in the manuscript). In any case, these techniques are referred to as single-molecule techniques by the scientific community, as they typically detect the diffusion of one molecule at a time that gets in and out of the confocal volume. We have clarified this point:

Line 273: “FCS, also referred to as fluorescence fluctuation spectroscopy, is a single-molecule technique (although typically conducted at nM concentrations) based on…”

Point 3: TCCD at picomolar concentration requires strong affinity between the two interacting species

The reviewer has highlighted an important limitation of the TCCD technique. We have tried to clarify this point in the revised version of the manuscript:

Line 208-209: “This technique is therefore highly sensitive for detecting small populations of interacting particles, although it typically requires high affinity complexes, …”

Point 4: Clarification of which techniques can be conducted at non-single-molecule conditions (e.g., TCCD, FCS, FCCS) and which should be single-molecule (e.g., smFRET) will help the reader understand how and when these techniques can/should be used. In addition, to distinguish single-molecule from ensemble FRET, I recommend that the authors use the acronym smFRET, which is very common in the single-molecule field.

TCCD, smFRET and FCS/FCCS are all considered single-molecule techniques. Indeed, the four techniques can be performed with the same protein sample at pM concentrations and examples can be found in section 4. We have followed the reviewer´s suggestion and use the acronym smFRET when referring to single-molecule FRET experiments.

Point 5: In line 22 of the abstract, when stating “we review our recent contributions”, the authors should clarify that they are reviewing the field of single-molecule fluorescence techniques and including their contributions unless they really meant that what the authors are mainly presenting are just their work.

We have now corrected the sentence.

Point 6: Line 66 the term ‘microscopic’ is not ideal because it implies relation to microscopy. ‘Intermolecular’ may be a better substitute for ‘microscopic

The term microscopic in this sentence has been now removed to avoid confusion.

Point 7: The abbreviation αS is rarely used to describe α-synuclein. The abbreviation ‘α-syn’ is more conventional.

Different abbreviations for alpha-synuclein can be found in the bibliography, and a significant number of them uses αS. We have adequately introduced the abbreviation at the beginning of the manuscript (line 80).

Reviewer 3 Report

Amyloid aggregation has long been a research focus since it is associated with numerous human disorders, including the most common neurodegenerative disorders Alzheimer’s and Parkinson’s disease. Due to the intrinsic heterogeneity and complexity of this process, conventional ensemble-based techniques could only provide limited information. Based on the extensive experience of application of diffusion-based single-molecule fluorescence techniques on amyloid aggregation investigation, Polanco and colleagues did an excellent review on how single-molecule fluorescence techniques could be employed to characterize the aggregation process in unprecedented details. This is a very informative and well-written review. I read it with lots of joys and strongly recommend for publication. I only have a few minor editorial points below.

Line 144: "detail" should be "detailed".

Line 182: Add "," before "making it possible to determine burst-integrated...".

Lines 190 and 191: To be consistent with the order of following subtitle, switch the position of "fluorescence correlation spectroscopy (FCS)" and "Forster resonance energy transfer (FRET)".

Line 344: Add "," before "using extrinsic dyes capable of binding...".

Line 400: Change "cross-beta" to "cross-B" for consistence.

Line 476: Delete "the" before "responsible for pathology...".

Line 655: "provide" should be "provided".

Lines 670-676: This senstence is too long and hard to understand. It needs to be divided into two or three sentences for a better readability.

Author Response

We thank the reviewer for his/her nice comments on our work and for spotting some editorial typos and proposing some formatting changes to improve the readibility of our manuscript. We have followed all the reviewer´s suggestions as outlined in his/her report and made the corresponding changes in the revised version of the manuscript.