PUFFFIN: A novel, ultra-bright, customisable, single-plasmid system for labelling cell neighbourhoods

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1. 

   Review Commons

   Mar 18, 2024

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   We thank all three reviewers for their positive comments on the value of our work and for and for their helpful suggestions.

   *Reviewer #1 (Evidence, reproducibility and clarity:

   This manuscript reports the development of a new bright fluorescent reporter that allows to label neighbouring cells. The system is based on upon secretion and uptake of s36GFP, a positively supercharged fluorescent protein. The authors also develop a Halo tag that will allow for straight forward colour exchange, as well as a customisable single plasmid construct with modular components.

   There are some minor suggestions that the authors may want to consider:

   1. The authors …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   We thank all three reviewers for their positive comments on the value of our work and for and for their helpful suggestions.

   *Reviewer #1 (Evidence, reproducibility and clarity:

   This manuscript reports the development of a new bright fluorescent reporter that allows to label neighbouring cells. The system is based on upon secretion and uptake of s36GFP, a positively supercharged fluorescent protein. The authors also develop a Halo tag that will allow for straight forward colour exchange, as well as a customisable single plasmid construct with modular components.

   There are some minor suggestions that the authors may want to consider:

   1. The authors conclude that "PUFFIN labelling is transferred rapidly between cells within minutes". They report in their time lapse experiments (Figure 2A,C) that sGFP can be detected within neighbours of secretors after 30 minutes when the cells are plated in a 50:1 non-labelled/secretor cell ratio, whereas it can be detected after 15 minutes when the cells are plated in a 1:9 ratio. Is there any synergistic effect on the signal when the proportion of secretors is increased or is this difference just because there is more signal, making it easier to visualise. *

   We have addressed this point with new experiments (new data shown in Figure 2E and Supp Figure S2A,B). This makes it clear that labelling can indeed be detected earlier when the proportion of secretors is higher. This is likely to be because higher secretor:acceptor ratios result in stronger labelling, which in turn makes it easier to detect labelled neighbours at very early time points - even within as early as 15 minutes. We also confirm that, even when secretors are very sparse (1:50 ratio), label becomes detectable in neighbours within 60 minutes.

   *Is there any reason why the main Figure legends lack a title, but the supplementary figures have one?

   1. In Figure 3, it may be helpful to label each option as A, B, C..
   2. In Figure 4E, the legends + JF646 and -JF646 may be switched around. Shouldn't the orange box should be (+) and the grey box should be (-)?

   We have modified / corrected the labelling as suggested and added titles to the main figure legends.

   *Reviewer #1 (Significance):

   This is a very valuable tool to address how cells change the behaviour of those in their environment. It will be very valuable for those interested in cell non-autonomous effects within a cell population or tissue. It will be especially valuable for live cell imaging; pulse chase experiments as well as omics approaches to understand cell behaviour in niches. *

   We thank this reviewer for their positive comments on the value of our work.

   *Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   The authors describe a new method, Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), to label with Fluorescent Proteins, neighboring cells. In brief, specific cells that express a nuclear mCherry are engineered to secrete a supercharged fluorescent protein (36GFP) fused to the ultra-bright green-fluorescent mNeonGreen (mNG) (s36GFP). Neighboring cells uptake s36GFP and can be easily visualized. The authors added the human serum albumin signal peptide which is efficiently cleaved to create s36GFP. The PUFFFIN system can also be customized for color-of-choice labelling using HaloTags. A shortcoming of the paper is that it is a method paper established in tissue culture cells with no biological applications. A test of the system in an in vivo model would improve the study. The authors should at least describe specific examples of how the method can be used to answer biological questions. *

   We agree that the paper would be improved by demonstrating a biological application of our system. We are currently working on experiments to address a biological question, and will be submitting a revised manuscript containing these data.

   *Reviewer #2 (Significance (Required)):

   This straightforward and elegant approach is an improvement of current methods that are based on synthetic receptor-ligand interactions as it does not require genetic modification of both 'sender' cells and 'responding' cells. The approach should prove to be an effective and flexible tool for illuminating cellular neighborhoods. An interesting potential application of the method is to effectively deliver proteins fused to s36GFP.

   A shortcoming of the paper is that it is a method paper established in tissue culture cells with no biological applications. A test of the system in an in vivo model would improve the study. The authors should at least describe specific examples of how the method can be used to answer biological questions.*

   We thank this reviewer for their positive comments on the value of our work. We agree that the paper would be improved by demonstrating a biological application of our system. We are currently working on experiments to address a biological question, and will be submitting a revised manuscript containing these data

   *Reviewer #3 (Evidence, reproducibility and clarity (Required)):

   In this manuscript, the authors introduced a novel cell-neighbor-labeling system named PUFFFIN. PUFFFIN, as well as 'PUFFHalo', offers an elegantly simple method for distinguishing between secretors and receivers, providing users with a versatile tool to label proximate neighbors through the uptake of s36GFP, subsequently permitting their isolation via FACS for subsequent analysis. In addition, this system could be very useful considering of its customizability by exchanging elements, such as tissue-specific promotors, color-of-choice (HaloTag), and genes of interest to cater to the diverse requirements of secretors. Overall, this system is well-designed and characterized, and the claims in this study are mostly supported by the data. However, this neighbor-labeling approach is not efficiently used to obtain biological insights. The following comments are intended to enhance the overall quality of the study:

   Major comments: *

   In Vidio1, it appears that certain nuclear mCherry+ cells did not secrete s36GFP-mNG during 19hrs recording window. However, in Figure1D and E, these GFP-mCherry+ cells were reported as having a 0% occurrence. This may be the result of either a delay in GFP secretion, or possible mCherry leakiness in unmodified cells. Please provide clarification. *

   There is indeed one mCherry+ cell in video 1 that fails to generate s36GFP-mNG signal. This cell, unlike most other cells in the movie, fails to divide or actively migrate during the 19h recording period, but instead is being passively “pushed around” by surrounding cells, and therefore looks to us very much like a dead or dying cell (levels of cell death to tend to be slightly higher than usual during live imaging). We have looked through our other videos and identified only one other example of an mCherry+ GFP-negative cell: this cell is clearly dying because the nucleus disintegrates over the course of the movie.

   We considered the possibility that some proportion of secretors may fail to generate signal even if they are healthy. We examined all our FACS analysis data. We detected at most 0.15% of such ‘failed secretors’, and most usually none. We conclude that any mCherry+ GFP- cells exist at extremely low frequencies and/or tend to be dying cells. Either way, they are very unlikely to interfere with interpretation of experimental data.

   *Additionally, including representative images of the co-culture experiment in Figure 1.E would enhance the presentation of the data. *

   These data have now been added to Supplemental Figure S1 C

   *Since the authors mention that s36GFP-mNG labeling was not detectable beyond four cell diameters, it would be helpful to include statistical data regarding the average distances or cell layers that GFP can travel, thus describing the permeation and labeling limit of s36GFP-mNG, adjacent to Figure2C. *

   We’ve now quantified the data and provide this information in a new panel (Figure 2D).

   *Please comment on the application prospect of this system utilizing in vivo. In addition, comment should be made on the difference of PUFFFIN system and recent reported CILP (PNAS 2023). *

   We have added discussion on prospects for using the system in vivo (new text lines 65-67). We have also described the CILP system in the revised introduction, explaining that it is an inducible version of the Cherry Niche system that we describe in our introduction (new text lines 291-294).

   *Minor comments:

   1. Please include the percentage of GFP+ and GFP- cells in Figure2.D, similar to what is provided in Figure S1.B. *

   This is a great suggestion so we have decided to add this information to all flow cytometry histograms within the paper, Figure 2D.

   *The '+' and '-' marks in Figure3.E appears to be mismatched with the results, please double-check and correct. *

   This has now been corrected.

   *I am curious about the interactions between secretors and 'receivers.' As the authors claim 'unbiased labeling' with this system, it's important to investigate whether the uptake abilities of receivers vary among different cell types. In other words, does the system exhibit cell-type preferences among receiver cells? This question could be optionally addressed through co-culture experiments involving secretors, receiver type A, and receiver type B. *

   We will perform additional experiments to address the reviewer’s question by directly comparing labelling efficiency across different receiver cell-types.

   Reviewer #3 (Significance (Required)):

   *This study reported a simple and sensitive system for labeling neighboring cells in vitro, which can be customized by replacing exchangeable components for customized need. With promising application in vitro, this system could be further developed and tested in vivo. Fluorescent protein labeling in neighboring cells has been a topic of study recently, and this manuscript introduced a new tool that is added to such resources, offering a user-friendly and customizable alternative. Overall, this system will be of interest to researchers working on neighbor-cell labeling and study of cell-cell communications. *

   We thank this reviewer for their positive comments on the value of our work.

   Read the original source
2. 

   Review Commons

   Mar 18, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   In this manuscript, the authors introduced a novel cell-neighbor-labeling system named PUFFFIN. PUFFFIN, as well as 'PUFFHalo', offers an elegantly simple method for distinguishing between secretors and receivers, providing users with a versatile tool to label proximate neighbors through the uptake of s36GFP, subsequently permitting their isolation via FACS for subsequent analysis. In addition, this system could be very useful considering of its customizability by exchanging elements, such as tissue-specific promotors, color-of-choice (HaloTag), and genes of interest to cater to the diverse requirements of secretors. Overall, this …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   In this manuscript, the authors introduced a novel cell-neighbor-labeling system named PUFFFIN. PUFFFIN, as well as 'PUFFHalo', offers an elegantly simple method for distinguishing between secretors and receivers, providing users with a versatile tool to label proximate neighbors through the uptake of s36GFP, subsequently permitting their isolation via FACS for subsequent analysis. In addition, this system could be very useful considering of its customizability by exchanging elements, such as tissue-specific promotors, color-of-choice (HaloTag), and genes of interest to cater to the diverse requirements of secretors. Overall, this system is well-designed and characterized, and the claims in this study are mostly supported by the data. However, this neighbor-labeling approach is not efficiently used to obtain biological insights. The following comments are intended to enhance the overall quality of the study:

   Major comments:

   1. In Vedio1, it appears that certain nuclear mCherry+ cells did not secrete s36GFP-mNG during 19hrs recording window. However, in Figure1D and E, these GFP-mCherry+ cells were reported as having a 0% occurrence. This may be the result of either a delay in GFP secretion, or possible mCherry leakiness in unmodified cells. Please provide clarification. Additionally, including representative images of the co-culture experiment in Figure 1.E would enhance the presentation of the data.
   2. Since the authors mention that s36GFP-mNG labeling was not detectable beyond four cell diameters, it would be helpful to include statistical data regarding the average distances or cell layers that GFP can travel, thus describing the permeation and labeling limit of s36GFP-mNG, adjacent to Figure2C.
   3. Please comment on the application prospect of this system utilizing in vivo. In addition, comment should be made on the difference of PUFFFIN system and recent reported CILP (PNAS 2023).

   Minor comments:

   1. Please include the percentage of GFP+ and GFP- cells in Figure2.D, similar to what is provided in Figure S1.B.
   2. The '+' and '-' marks in Figure3.E appears to be mismatched with the results, please double-check and correct.
   3. I am curious about the interactions between secretors and 'receivers.' As the authors claim 'unbiased labeling' with this system, it's important to investigate whether the uptake abilities of receivers vary among different cell types. In other words, does the system exhibit cell-type preferences among receiver cells? This question could be optionally addressed through co-culture experiments involving secretors, receiver type A, and receiver type B.

   Significance

   This study reported a simple and sensitive system for labeling neighboring cells in vitro, which can be customized by replacing exchangeable components for customized need. With promising application in vitro, this system could be further developed and tested in vivo. Fluorescent protein labeling in neighboring cells has been a topic of study recently, and this manuscript introduced a new tool that is added to such resources, offering a user-friendly and customizable alternative. Overall, this system will be of interest to researchers working on neighbor-cell labeling and study of cell-cell communications.

   Read the original source
3. 

   Review Commons

   Mar 18, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The authors describe a new method, Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), to label with Fluorescent Proteins, neighboring cells. In brief, specific cells that express a nuclear mCherry are engineered to secrete a supercharged fluorescent protein (36GFP) fused to the ultra-bright green-fluorescent mNeonGreen (mNG) (s36GFP). Neighboring cells uptake s36GFP and can be easily visualized. The authors added the human serum albumin signal peptide which is efficiently cleaved to create s36GFP. The PUFFFIN system can also be customized for color-of-choice labelling using HaloTags.

   A shortcoming of the …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The authors describe a new method, Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), to label with Fluorescent Proteins, neighboring cells. In brief, specific cells that express a nuclear mCherry are engineered to secrete a supercharged fluorescent protein (36GFP) fused to the ultra-bright green-fluorescent mNeonGreen (mNG) (s36GFP). Neighboring cells uptake s36GFP and can be easily visualized. The authors added the human serum albumin signal peptide which is efficiently cleaved to create s36GFP. The PUFFFIN system can also be customized for color-of-choice labelling using HaloTags.

   A shortcoming of the paper is that it is a method paper established in tissue culture cells with no biological applications. A test of the system in an in vivo model would improve the study. The authors should at least describe specific examples of how the method can be used to answer biological questions.

   Significance

   This straightforward and elegant approach is an improvement of current methods that are based on synthetic receptor-ligand interactions as it does not require genetic modification of both 'sender' cells and 'responding' cells. The approach should prove to be an effective and flexible tool for illuminating cellular neighborhoods. An interesting potential application of the method is to effectively deliver proteins fused to s36GFP.

   A shortcoming of the paper is that it is a method paper established in tissue culture cells with no biological applications. A test of the system in an in vivo model would improve the study. The authors should at least describe specific examples of how the method can be used to answer biological questions.

   Read the original source
4. 

   Review Commons

   Mar 18, 2024

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This manuscript reports the development of a new bright fluorescent reporter that allows to label neighbouring cells. The system is based on upon secretion and uptake of s36GFP, a positively supercharged fluorescent protein. The authors also develop a Halo tag that will allow for straight forward colour exchange, as well as a customisable single plasmid construct with modular components.

   There are some minor suggestions that the authors may want to consider:

   1. The authors conclude that "PUFFIN labelling is transferred rapidly between cells within minutes". They report in their time lapse experiments (Figure 2A,C) that sGFP can be …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This manuscript reports the development of a new bright fluorescent reporter that allows to label neighbouring cells. The system is based on upon secretion and uptake of s36GFP, a positively supercharged fluorescent protein. The authors also develop a Halo tag that will allow for straight forward colour exchange, as well as a customisable single plasmid construct with modular components.

   There are some minor suggestions that the authors may want to consider:

   1. The authors conclude that "PUFFIN labelling is transferred rapidly between cells within minutes". They report in their time lapse experiments (Figure 2A,C) that sGFP can be detected within neighbours of secretors after 30 minutes when the cells are plated in a 50:1 non-labelled/secretor cell ratio, whereas it can be detected after 15 minutes when the cells are plated in a 1:9 ratio. Is there any synergistic effect on the signal when the proportion of secretors is increased or is this difference just because there is more signal, making it easier to visualise.
   2. Is there any reason why the main Figure legends lack a title, but the supplementary figures have one?
   3. In Figure 3, it may be helpful to label each option as A, B, C..
   4. In Figure 4E, the legends + JF646 and -JF646 may be switched around. Shouldn't the orange box should be (+) and the grey box should be (-)?

   Significance

   This is a very valuable tool to address how cells change the behaviour of those in their environment. It will be very valuable for those interested in cell non-autonomous effects within a cell population or tissue. It will be especially valuable for live cell imaging; pulse chase experiments as well as omics approaches to understand cell behaviour in niches.

   Read the original source
5. 

   preLights

   Oct 9, 2023

   Excerpt

   PUFFFIN: Shining light on cell chat

   Read the original source
6. 

   Version published to 10.1101/2023.09.06.556381v2 on bioRxiv

   Sep 8, 2023
7. 

   Version published to 10.1101/2023.09.06.556381v1 on bioRxiv

   Sep 7, 2023