Super-resolution analysis of PACSIN2 and EHD2 at caveolae

Tamako Nishimura; Shiro Suetsugu

doi:10.1371/journal.pone.0271003

. 2022 Jul 14;17(7):e0271003. doi: 10.1371/journal.pone.0271003

Super-resolution analysis of PACSIN2 and EHD2 at caveolae

Tamako Nishimura ¹, Shiro Suetsugu ^1,^2,^3,^*

Editor: Christophe Lamaze⁴

PMCID: PMC9282494 PMID: 35834519

Abstract

Caveolae are plasma membrane invaginations that play important roles in both endocytosis and membrane tension buffering. Typical caveolae have invaginated structures with a high-density caveolin assembly. Membrane sculpting proteins, including PACSIN2 and EHD2, are involved in caveolar biogenesis. PACSIN2 is an F-BAR domain-containing protein with a membrane sculpting ability that is essential for caveolar shaping. EHD2 is also localized at caveolae and involved in their stability. However, the spatial relationship between PACSIN2, EHD2, and caveolin has not yet been investigated. We observed the single-molecule localizations of PACSIN2 and EHD2 relative to caveolin-1 in three-dimensional space. The single-molecule localizations were grouped by their proximity localizations into the geometric structures of blobs. In caveolin-1 blobs, PACSIN2, EHD2, and caveolin-1 had overlapped spatial localizations. Interestingly, the mean centroid of the PACSIN2 F-BAR domain at the caveolin-1 blobs was closer to the plasma membrane than those of EHD2 and caveolin-1, suggesting that PACSIN2 is involved in connecting caveolae to the plasma membrane. Most of the blobs with volumes typical of caveolae had PACSIN2 and EHD2, in contrast to those with smaller volumes. Therefore, PACSIN2 and EHD2 are apparently localized at typically sized caveolae.

Introduction

Caveolae are flask-shaped plasma membrane invaginations that are abundant in several cell types found in muscle, epithelial, and adipose tissues [1–3]. Caveolae play dual roles at the plasma membrane, as an endocytic apparatus and a membrane reservoir for buffering membrane tension. During endocytosis, the caveolar invagination is pinched off to form endocytic vesicles, while in tension buffering it is flattened to provide extra surface area to increase the membrane surface [1,4,5].

Caveolae are composed of a unique set of proteins and lipids. The caveolar membrane is rich in cholesterol, similar to the lipid rafts at the plasma membrane, where several receptors and signaling proteins are reportedly concentrated [6–9]. Caveolae are also a platform for signaling proteins that are regulated by the caveolar endocytic function. The structural caveolar proteins comprise caveolins and cavins [10,11]. Caveolin exists as three isoforms, and the caveolin-1 and caveolin-3 amino acid sequences are almost identical [12,13]. Caveolin-1 is ubiquitously expressed, while caveolin-3 is predominantly expressed in muscle. Mutations associated with diseases such as muscular dystrophy have been identified in caveolin-3 [14,15], consistent with the role of caveolae in the tension buffering of muscle cells [16]. There are four cavin isoforms, and they are essential for caveolae [11,17–20]. Cavins associate with caveolins and generate the characteristic striations on the caveolar surface, as observed by electron microscopy [21–24].

The endocytosis of caveolae is mediated by dynamin [25], as in clathrin-mediated endocytosis. The invaginated membrane of clathrin-coated pits is mainly produced by BAR domain proteins [26,27], which directly generate membrane curvatures and recruit structural proteins for membrane remodeling, including dynamin and Wiskott–Aldrich syndrome family proteins [28]. Dynamin mediates the pinching of invaginations to form vesicles, in cooperation with the actin cytoskeleton [29]. The BAR domains are divided into the BAR, N-BAR, and I-BAR domain subfamilies [30,31]. Among them, the F-BAR domain-containing protein PACSIN (Syndapin) is involved in caveolae [32–34]. Three isoforms of PACSIN have been described. PACSIN3 is a muscle-specific isoform, and its knockout results in caveolar biogenesis abnormalities [35]. PACSIN2 is a ubiquitous isoform involved in caveolae formation and endocytosis [34]. PACSIN1 is brain-specific, and its role in caveolae has not yet been clarified [34]. Importantly, PACSIN2 has membrane deforming ability, which is altered by the cholesterol content of the membrane, implying the important role of PACSIN2 in caveolar homeostasis [36]. Indeed, PACSIN2 is stably localized at caveolae, presumably at the neck of caveolar invaginations [33,34,37]. Furthermore, PACSINs have NPF sequences that bind to the EHD2 protein, which is localized at and stabilizes caveolae, presumably by mediating actin cytoskeleton anchoring [32,38,39]. Importantly, dynamin is recruited to caveolae only when PACSIN2 disappears, suggesting the regulation of dynamin binding to PACSIN2 during endocytosis [40].

Approximately 150 caveolin-1 molecules have been detected in mature caveolae [37,41]. Due to this abundance of caveolin-1, typical caveolae appear to have a certain caveolin-1 density, which can be measured by the nearest neighbor distance (NND) between caveolin-1s in single-molecule localization microscopy (SMLM), a method that can determine the coordinates of molecules at an accuracy equivalent to the protein size; that is, ~10 nm in the plane parallel to the focal plane [42]. Using SMLM data projected onto a two-dimensional plane, the membrane deformation can be monitored by the density estimations of caveolin-1 [42] or the distances between caveolin-1 and caveolae-localized molecules [43]. Furthermore, by introducing a cylindrical lens, the localization depth measurement; that is, the three-dimensional determination of the coordinates, could be achieved with an accuracy of ~50 nm in the depth direction [44,45]. The three-dimensional coordinates of caveolin-1 localization can be grouped into blob-shaped geometrical structures, which were previously classified into the typical caveolae or other caveolin-1 clusters [46,47]. In this study, we used three-dimensional SMLM to examine PACSIN2 and EHD2 localizations relative to caveolin-1. The PACSIN2 and EHD2 coordinates mostly overlapped with the caveolin-1 coordinates in the caveolin-1 blobs of caveolar volume, which are thought to correspond to typical caveolae. However, PACSIN2 was ~5 nm closer to the plasma membrane than EHD2 and caveolin-1, suggesting its role in connecting caveolae to the plasma membrane. Most of the blobs with volumes of typical caveolae had PACSIN2 and EHD2, whereas those with smaller volumes did not. Therefore, PACSIN2 and EHD2 are apparently localized at typically sized caveolae.

Results and discussion

First, we observed the single-molecule localizations in antibody-stained HeLa cells. SMLM uses total internal reflection to illuminate the fluorophore, and thus the observation is limited to the plasma membrane neighboring the glass surface on which the cells attach. HeLa cells were labeled with antibodies against caveolin-1 and PACSIN2 or EHD2. Two kinds of antibodies for each protein were used, Caveolin-1 (7C8) + Caveolin-1 (3238), EHD2 (G-3) + Caveolin-1 (3238), EHD2 (11440-1-AP) + Caveolin-1 (7C8), PACSIN2 (SAB-1402538) + Caveolin-1 (3238), and PACSIN2 (Senju) + Caveolin-1 (7C8). These antibodies were visualized with secondary antibodies that were doubly labeled with Alexa 647 combined with Cy3 or Alexa 405. The activation of Alexa 405 or Cy3 by the excitation light was converted to the activation of Alexa 647, enabling the observation of the two labels at the same wavelength for Alexa 647. Therefore, the wavelength aberration was negligible between the observations of the two labels. The Alexa 647 signals that were associated with the activation Alexa 405 or Cy3 signals were considered for the analysis, to avoid the non-specific observations. To enable three-dimensional observations, a cylindrical lens was utilized to examine the signal depths by observing the deformation of the SMLM spherical signal according to the distance to the focal plane. The typical images reconstructed from the coordinates showed the proximity localizations of clustered signals of PACSIN2 and caveolin-1, as well as EHD2 and caveolin-1 (Fig 1).

Fig 1 — Representative reconstituted image of caveolin-1 (green) with those of PACSIN2 or EHD2 (magenta) in HeLa cells. The merged image of the focal plane (XY) is shown on the left. The box indicates the region for the enlarged images on the right, and contains one blob composed of caveolin-1 presumably corresponding to a caveola, with the projection to show the signal distribution to the Z-direction for each caveolin-1, PACSIN2, or EHD2 image. The combinations of antibodies are: (A) Caveolin-1 (7C8) + Caveolin-1 (3238), (B) Caveolin-1 (3238) + EHD2 (G-3), (C) Caveolin-1 (7C8) + EHD2 (11440-1-AP), (D) Caveolin-1 (3238) + PACSIN2 (SAB-1402538), and (E) Caveolin-1 (7C8) + PACSIN2 (Senju).

The single-molecule localizations of caveolin-1 within an 80 nm distance from each other were grouped into four classes by the SuperResNet software, according to the number of signals, area, shape, and so on [46,47]. The clustering below 80 nm was considered to be reasonable because caveolae are typically 60–100 nm in diameter [10,48], and the number of observations of caveolin-1 was 4–15 within 100 nm of caveolae with this antibody labeling, according to our previous estimations for caveolar shapes [42]. The spatial distributions of these groups of signals were mostly spherical, and hence they are called blobs. The mapping of the blobs by t-distributed stochastic neighbor embedding (t-SNE) by Mahalanobis distance, cosine distance, and Euclidean distance resulted in the clear separation of the blobs, suggesting robust classification (Fig 2A). The number of blobs was consistent between observations (Fig 2B). The estimated size of caveolin-1 blobs in each class was also similar between observations, suggesting the accurate grouping of the signals (S1 Fig). As reported in the development of this method [47], the class 1 and class 2 blobs contained fewer caveolin-1 localizations and smaller volumes, while the class 3 blobs had volumes equivalent to those of typical caveolae (Fig 2C and 2D). The class 3 blobs typically contained abundant ~20 caveolin-1 signals. The volumes of the class 3 blobs were approximately ten times larger than those of the class 1 and 2 blobs, and were similar to the volumes of 60–100 nm spheres, strongly suggesting that class 3 corresponded to typical caveolae. The class 4 blobs contained superabundant localizations of ~100 signals, and thus might represent caveolae rosettes or clusters [49,50].

Fig 2 — (A) Typical clustering results of caveolin-1 signals of blobs from a cell into groups according to proximity by SuperResNet and their projections by t-SNE with Mahalanobis distance, cosine distance, and Euclidean distance. (B) The number of each class of caveolin-1 blobs per observation. (C) The average number of caveolin-1 signals from a blob of each class per observation. (D) The average volume of each class of caveolin-1 blobs per observation. In (B-D), the dot represents an average from an observation, which typically contained a cell. N = 6–10 observations for each combination of antibodies. (E) A caveolin-1 blob containing PACSIN2. Each signal of PACSIN2 and caveolin-1 is shown as a dot, and the signals within 80 nm of each other are connected by lines. The centroids of the caveolin-1 and PACSIN2 signals are illustrated by *, with spheres based on the radii of the standard deviations of caveolin-1 and PACSIN2. (F) The definition of colocalization by the overlap of the spheres of standard deviations. (G) Percentages of the colocalizations of blobs. The percentages of caveolin-1 blobs colocalized with PACSIN2 and EHD2 are shown for each antibody combination. The co-staining of two caveolin-1 antibodies indicated the blobs with two co-localized caveolin-1 antibodies.

To estimate the colocalization of the two antibody labels, the spatial distribution of signals was estimated by comparing the distance between the centroids of the two antibody blobs to the standard deviations of the signal coordinates of blobs (Fig 2E). The colocalization was determined by the overlap of the spatial distributions of the two kinds of signals; i.e., the centroid distance below the sum of each standard deviation (Fig 2F). Approximately half of the class 1 and class 2 blobs of caveolin-1 had signals from another antibody against caveolin-1, PACSIN2, and EHD2 (Fig 2G). Within each class, the blobs with colocalization had larger volumes and signals than those without colocalization (S2 Fig). Therefore, the number of molecules to be detected is related to the volume of the blobs and the number of observed signals, suggesting that the class 1 and 2 blobs might have an insufficient number of molecules for colocalization detection or might not have colocalization. Such difficulty in analyzing small structures would probably result from the antibody labeling, where only a small fraction of the proteins can be visualized, as has been shown that 1–8 signals per caveola by electron microscopic analysis by using various caveolin-1 antibodies [51]. Most of the class 3 and class 4 caveolin-1 blobs had the signals from another antibody to caveolin-1, PACSIN2, and EHD2, suggesting that caveolae with a typical size had PACSIN2 and EHD2.

We next examined the spatial distribution of the signals; i.e., the shape, of the class 3 blobs. The standard deviations in the XY plane, parallel to the glass surface or plasma membrane, and the Z direction in depth were similar for the caveolin-1 signals between various antibody combinations (Fig 3A and 3B). The standard deviations of the PACSIN2 and EHD2 signals did not appear to be largely different from those of caveolin-1. Therefore, PACSIN2, EHD2, and caveolin-1 had largely overlapped spatial localizations in caveolin-1 blobs.

Fig 3 — (A) Standard deviations of the blobs of each antibody staining in the XY plane, the plane parallel to the lens surface or coverslip of the cell attachment; i.e., the plasma membrane. (B) Standard deviations of the blobs of each antibody staining in the Z or depth direction. In (A, B), the pairs of antibodies used for co-staining are shown side-by-side. The dot represents an average from an observation, which typically contained a cell. N = 6–10 observations for each combination of antibodies. (C) The difference in the blob centroid depths of the indicated antibodies used for co-staining. A negative value indicates closer localization to the plasma membrane. The dot represents an average from an observation, which typically contained one cell. N = 6–10 observations for each combination of antibodies. The statistical difference in the depth of the PACSIN2 antibodies was evaluated by one-way ANOVA with post-hoc Holm-Bonferroni analysis and the p-values were indicated. (D) Domain diagrams of PACSIN2 and EHD2. The antigens for the antibodies, as well as the interaction sites between PACSIN2 and EHD2, are also illustrated. (E) A model of PACSIN2 F-BAR domain localization in caveolae. The PACSIN2 F-BAR domain, SH3 domain, EHD2, and caveolin-1 are illustrated in an approximate scale. The size of the caveola is obtained from ~2 times of the standard deviations. The PACSIN2 F-BAR domain is located close to the plasma membrane, but PACSIN2, EHD2, and caveolin-1 mostly overlap each other, resulting in the hypothetical unit of PACSIN2, EHD2, and presumably caveolin-1 in caveolae. The shaded part is hypothetical.

Next, we assessed the relative differences in the depth distributions by the distance of the centroid of caveolin-1 signals of the class 3 blobs to that of the associated PACSIN2 and EHD2 blobs. The negative distance in the z direction indicated a localization closer to the plasma membrane, i.e., the glass on which the cells were attached. The distances of the EHD2 centroids by the two kinds of antibodies to that of caveolin-1 were similar to each other and also to the centroid determined with another caveolin-1 antibody, indicating that the EHD2 localization was similar to that of caveolin-1 (Fig 3C). The monoclonal PACSIN2 antibody (SAB-1402538) recognizes the region before the SH3 domain of PACSIN2 that interacts with EHD2 (Fig 3D) [39]. The PACSIN2 centroid determined with the monoclonal antibody did not have a significant difference in depth from the EHD2 and caveolin-1 centroids (Fig 3C), which is consistent with the interaction of the region before the SH3 domain of PACSIN2 with EHD2. The polyclonal PACSIN2 antibody (Senju) recognizes the F-BAR domain, which binds to membrane [34]. Interestingly, the PACSIN2 centroid identified by this polyclonal antibody to the F-BAR domain was 5–10 nm closer to the plasma membrane than the EHD2 centroid (Fig 3C). The F-BAR domain of PACSIN2 is an arc-like rod of ~20 nm in length with a width of 5 nm [52]. The SH3 domains are globular domains with a diameter of ~5 nm [53], and EHD2 is also a globular protein of ~10 nm diameter [54]. Therefore, the PACSIN2 F-BAR domain was localized closer to the plasma membrane at a one protein distance, suggesting the role of PACSIN2 in connecting caveolae to the plasma membrane. Combined with the overall overlaps of PACSIN2, EHD2, and caveolin-1 localizations, these proteins would form a unit of PACSIN2, EHD2, and caveolin-1, in which the F-BAR domain of PACSIN2 faces the plasma membrane (Fig 3E).

This SMLM analysis suggested that PACSIN2 and EHD2 are localized in typical caveolae. We previously reported that PACSIN2 is localized to caveolae throughout the caveolar life cycle [32–34]. However, PACSIN2 appeared to tubulate membrane upon endocytosis and cholesterol depletion. The TIRF analysis indicated that PACSIN2 and caveolin-1 were co-localized, from the appearance of caveolin-1 at the plasma membrane, and the colocalization with dynamin occurred only at the last moment of endocytosis [40]. Upon cholesterol removal, PACSIN2 binding to the membrane was strengthened, supporting the production of membrane tubules, presumably for endocytosis [36]. Accordingly, caveolae with abundant cholesterol would exhibit a weaker affinity to PACSIN2, to prevent the elongation of caveolae for endocytosis. Therefore, the localizations of PACSIN2 and EHD2 at the entire caveola, with the exposure of PACSIN2 at the putative neck region, a boundary between the plasma membrane and the caveolar main body, would provide the potential for the extension of the caveola to such tubular structures upon endocytosis and cholesterol depletion.

Materials and methods

HeLa cell culture

HeLa cells were cultured as described previously [34] in Dulbecco’s modified Eagle’s medium (Nacalai), supplemented with 10% fetal calf serum, 63 μg/ml benzylpenicillin potassium, and 100 μg/ml streptomycin.

Antibodies

The anti-PACSIN2 rabbit polyclonal antibody (Senju) was affinity-purified from the serum of rabbits immunized with the F-BAR domain of PACSIN2 [34]. The anti-EHD2 rabbit polyclonal antibody (11440-1-AP, Proteintech), the mouse monoclonal anti-caveolin-1 (7C8, Santa Cruz Biotechnology, sc-53564, 1:100), the rabbit polyclonal anti-caveolin-1 (Cell Signaling, #3238, 1:200), the mouse monoclonal anti-PACSIN2 (Sigma, SAB-1402538, 1:100), and the mouse monoclonal anti-EHD2 (G-3, Santa Cruz, sc-515458, 1:100) antibodies were purchased.

STORM observation and analysis

The three-dimensional STORM setup with a cylindrical lens for depth measurement was purchased from Nikon and modified based on previous reports [42,44,45]. Dye preparation, secondary antibody labeling, and cell staining for STORM imaging (Nikon) were performed according to the manufacturer’s protocols, using combinations of Alexa Fluor 405 + Alexa Fluor 647 or Cy3 + Alexa Fluor 647 [44,45]. Alexa Fluor 405 and Cy3 are the activator dyes, and Alexa Fluor 647 is the reporter dye. The ratio of the activator dye: the reporter dye: antibody is 2–3: 0.6–1: 1. HeLa cells were cultured on Lab-Tek II chambered cover glasses (Nunc) that were pre-cleaned with 1M KOH for 1 hr. They were fixed in 3% paraformaldehyde (WAKO) + 0.1% glutaraldehyde (TAAB, electron microscopy grade) in HEPES-buffered saline (30 mM Hepes, pH 7.4, 100 mM NaCl, 2 mM CaCl₂) for 10 min at room temperature, reduced with 0.1% NaBH₄ in PBS for 7 min, and blocked in blocking buffer (3% BSA + 0.2% Triton X-100 in PBS) for 1 hr at room temperature. The cells were then stained with a 1:100 dilution of the primary antibodies in the blocking buffer for at least 1 hr at room temperature. After washing with wash buffer (0.05% Triton X-100, 0.2% BSA in PBS), the cells were incubated with secondary antibodies for 1 hr at room temperature and then washed. Finally, the cells were post-fixed with 3% paraformaldehyde + 1% glutaraldehyde in HEPES-buffered saline for 10 min at room temperature, and then stored in PBS at 4°C.

For image acquisition, the cells were soaked in 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, and 10% glucose supplemented with cystamine, glucose oxidase, and catalase, according to the manufacturer’s instructions. An N-STORM (Nikon) super-resolution microscope equipped with a 100×/1.49 objective lens (Apo TIRF 100× Oil DIC N2, Nikon) and an EMCCD camera (iXon Du-897, ANDOR) was used for imaging. One image for the activation laser (405 or 561 nm) and three sequential images for the reporter laser (647 nm) were obtained for 10,000 cycles (total 40,000 images) and analyzed with the NIS-Elements AR 4.60.00 software provided by Nikon.

The signals upon the reporter laser irradiation following the observation by the activation laser irradiation were considered to be the specific signals and analyzed further. The coordinates that were at almost identical positions (<20 nm) in the continuous observations were eliminated, because the Alexa dye emits signals multiple times [55–57]. The coordinates were converted into the VISP format by the ChriSTORM ImageJ plugin [58–60], as described in the Supplementary Data 1 and 2. In all figures, each dot represents one signal. The clustering of the caveolin-1 signals was performed by SuperResNet [46,47], where >4 caveolin-1 signals within 80 nm, a value determined as a 20% smaller size of caveolin-1, were connected to a cluster. The clusters were divided into 4 classes according to the SuperResNet analysis of caveolin-1 [46,47], by the numbers of signals, shape parameters, and so on. Typically, thousands of blobs were identified per cell. The clusters of caveolin-1 signals exhibited blob structures. The volume of the class 3 blob corresponded to the volume of a sphere with a 60–100 nm diameter, a typical size of a caveola, which ranges from 0.9–4 × 10⁶ nm³. The PACSIN2 and EHD2 signals were also grouped into clusters by SuperResNet. The overlaps of the signal distribution of the signals were considered for co-localization; i.e., the centroid distance below the sum of each standard deviation (Fig 2F). The center of mass of the clusters was considered for the depth difference. These calculations were performed with MATLAB. For each blob, the standard deviations in the XY and Z axes and the depth differences between the centroids of clusters of each antibody labeling were examined and then averaged per each observation, which typically contains a cell. The averages of the observations were then plotted in each Figure. In Fig 3C, the statistical evaluation was examined by One-way ANOVA with post-hoc Holm-Bonferroni analysis.

Supporting information

S1 Fig. The statistics of blobs.

The average number of caveolin-1 blobs per observation, the XY standard deviation, and the Z standard deviation for each class and each combination of antibodies are shown. The dot represents an average from an observation, which typically contained one cell. N = 6–10 observations for each combination of antibodies. The combinations of antibodies are as follows: a: Caveolin-1 (7C8) + Caveolin-1 (3238), b: Caveolin-1 (3238) + EHD2 (G-3), c: Caveolin-1 (7C8) + EHD2 (11440-1-AP), d: Caveolin-1 (3238) + PACSIN2 (SAB-1402538), and e: Caveolin-1 (7C8) + PACSIN2 (Senju).

(PDF)

Click here for additional data file.^{(490.7KB, pdf)}

S2 Fig. The statistics of blobs with or without colocalization.

The volume of a caveolin-1 blob and the average number of caveolin-1 signals per blob per observation, obtained for each class for each combination of antibodies, shown with or without the colocalization of the two stains. The dot represents an average from an observation, which typically contained one cell. N = 6–10 observations for each combination of antibodies described in S1 Fig. w: Caveolin-1 blobs with the colocalization; wo: Caveolin-1 blobs without the colocalization.

(PDF)

Click here for additional data file.^{(532.1KB, pdf)}

S1 Data. The compressed files of the original coordinates in the VISP files.

(ZIP)

Click here for additional data file.^{(90.9MB, zip)}

S2 Data. The compressed files of the original coordinates in the VISP files and the inventory of the files.

(ZIP)

Click here for additional data file.^{(87.3MB, zip)}

Acknowledgments

We thank the laboratory members for fruitful discussions.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by JSPS (https://www.jsps.go.jp/english/)(KAKENHI, JP 20H03252, JP20KK0341, JP21H05047) to SS and by JSPS (JP20K06625) to TN, and JST (https://www.jst.go.jp/EN/) CREST (JPMJCR1863) to SS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0271003.r001

Decision Letter 0

Christophe Lamaze

24 Jan 2022

PONE-D-21-39140Superresolution analysis of PACSIN2 and EHD2 at caveolaePLOS ONE

Dear Shiro,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

You will see from the verbatim comments of the reviewers below that while they found your work of potential interest they both raised significant criticims that need to be addressed before publication. The reviewers are particularly concerned by the quality of the manuscript and English literacy. Several experimental controls and explanations in Material and Methods are missing together with statistics validation, cryptic figure legends and so on.

I would therefore recommend to fully address the criticism of both reviewers who are each recognized experts in caveolae. I hope you will find these commenst useful and lokk forward to reading an improved manuscript in due time.

Best regards

Christophe Lamaze

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The manuscript by Shiro Suetsugu entitled "SUperresolution analysis of PACSIN2 and EHD2 aat caveolae" concerns the super-resolution analysis of PACSIN2 and EHD2 localization in Hela cells. The author describes using STORM for single molecule localization of these two proteins relative to caveolin-1 used here as a marker for caveolae. The author compares the three-dimensional distribution of PACSIN2 and EHD2 in regard to the localization of caveolin-1. He shows that when classified into mature or immature blobs, half of the immature blobs contained EHD2 and PACSIN2 while most of the mature blobs had EHD2 and PACSIN2 suggesting a progressive association of these two proteins with maturation of caveolae. The author also shows that PACSIN2 then EHD2 were closer to the membrane compared to caveolin1 suggesting that they are localized at the neck of caveole.

Overall the idea to analyze PACSIN2 and EHD2 by STORM is interesting and the work is of good quality. My enthusiasm is slightly dimished because there are hardly any pictures to validate the findings and the paper is very minimalist. In fig.1 the individual channels could at least be shown separately. What about using Cavin proteins or other markers as controls to validate the findings? or using expression of the same tagged proteins to validate the results with the antibodies?

The parameters used to separate the different objects in SuperResNet are not described in detail in the methods section and it is difficult to understand how the clustering was performed. The author separates the objects into different classes and make assumptions on the degree of maturation but this could not be the case. One could imagine easily that caveolae could be more heterogeneous and some more or less flattened caveolae could still be mature. The term "maturity" is not appropriate and it would better to use comparison in terms of curvature as it is directly connected to the height of the objects which is what is being best measured by STORM.

The author chose to group PACSIN2 and EHD2 into clusters that were +/- 80nm to be associated with the caveolin1 blob. How can the authors then claim that these objects could be localized at the neck if they are so far from the caveolin signal? DO any of the images suggest accumulation of EHD2 or PACSIN2 at the neck? maybe the author could show an example of STORM image to substantiate this finding.

The authors mention on p5 that caveolae typical diameter is 100 nm while in reality it is 60-70nm at the ultrastructural level

There are several typos throughout the MS (for example title on p5 "STROM observation", etc...) and the english is not always correct. Could be useful to have a native english person proof-read the manuscript.

Reviewer #2: In this short article, Dr Suetsugu sets out to establish the superresolution analysis of PACSIN2 and EHD2 at caveolae. EHD2 and PACSIN2 are in addition to the caveolins and cavins, the most recognised and studied caveolar proteins. The research topic is interesting and topical, but the manuscript appears at places hastily put together and lacks some necessary controls.

Major comments

1) Materials and methods section is not complete, and need to be substantially expanded in order to provide context to the study. As it is, the data presented are hard to interpret.

Some additional examples where the methods section need updating

“Under subtitle: “HeLa cell culture, transfection, and live imaging”

The section reads

141 HeLa cells were cultured as described previously [28] in Dulbecco’s modified Eagle’s 5 142 medium (DMEM), supplemented with 10% fetal calf serum (FCS).

There is no mention on transfection or live imaging. The manuscript does not use those techniques.

2) There are no controls for that the signal observed by the antibody labelling is specific. Super resolution is a very sensitive technique, and at such antibody validation under specific experimental conditions, including fixation etc is critical. Discussion, and analysis of the fixative used is necessary, as fixation is well known to change the morphology and preservation of caveolae. The fixative will likely also impact on epitope availability, which confounds the conclusions, including of the monoclonal CAV1 used. Is the CAV1 epitope available to the antibody throughout the caveolae bulb? A comparison with widely used and characterised poly clonal CAV1 might yield this necessary insight.

3) No details on the statistical tests carried out are available and the comparisons in the figure legends. Likewise N numbers should also be included, biological repeats etc.

4) The figure legends need more information, forinstance no information in Figure 1, on what the cell this is, it would also be helpful to have the images displayed in split and merged channels. In addition, to me the colour is magenta, and not red as stated above the figures.

5) Justification for the reason behind “where >4 caveolin-1 signals within 80 nm chosen”. And justification of why “clusters within 80 nm were considered to be the PACSIN2 or EHD2 clusters close to the Caveolin-1”. Results/conclusions might have been very? different if these distances had changed.

Minor comments

Figure 2, bulb, blob or blub are listed in the figure axis, are they meant to be the same?

Sub title, “STROM observation and analysis”, should be “STORM observation and analysis” In general there are rather widespread typo’s

Reference for caveosome is needed.

Reference for caveolae size is needed.

Line 163 The “with the provided NIKON Software” software needs to be named.

It would be helpful, if the IF images were shown in separate channels, as well as the merged. This is the commonly accepted way of representing dual colour images.

Line 66 correct “with the role of caveolae in caveolae formation [27, 28, 31].”

Therefore, PACSIN1 and EHD2 were suggested to be localized at the mature caveolae.

Is this Pacsin 2 instead of Pacsin1? There is as far I can see no data presented on PACSIN1

**********

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Reviewer #2: No

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PLoS One. 2022 Jul 14;17(7):e0271003. doi: 10.1371/journal.pone.0271003.r002

Author response to Decision Letter 0

2 May 2022

Q) –Reviewer #1: The manuscript by Shiro Suetsugu entitled "SUperresolution analysis of PACSIN2 and EHD2 at caveolae" concerns the superresolution analysis of PACSIN2 and EHD2 localization in Hela cells. The author describes using STORM for single-molecule localization of these two proteins relative to caveolin-1 used here as a marker for caveolae. The author compares the three-dimensional distribution of PACSIN2 and EHD2 in regard to the localization of caveolin-1. He shows that when classified into mature or immature blobs, half of the immature blobs contained EHD2 and PACSIN2 while most of the mature blobs had EHD2 and PACSIN2 suggesting a progressive association of these two proteins with maturation of caveolae. The author also shows that PACSIN2 then EHD2 were closer to the membrane compared to caveolin1 suggesting that they are localized at the neck of caveolae.

A) We appreciate your kind offer to revise our manuscript and extend the deadline. In this revised manuscript, we think we have experimentally addressed most of the concerns raised by the reviewers.

Q) Overall the idea to analyze PACSIN2 and EHD2 by STORM is interesting and the work is of good quality. My enthusiasm is slightly diminished because there are hardly any pictures to validate the findings and the paper is very minimalist. In fig.1 the individual channels could at least be shown separately. What about using Cavin proteins or other markers as controls to validate the findings? or using expression of the same tagged proteins to validate the results with the antibodies?

A) Thank you for your valuable comments. According to your advice, we have separated the merged picture into the individual channels, as shown in the new Fig. 1. Also, we replaced Figure 1 with those representing the analysis of the STORM signal coordinates of the class 3 blobs.

To validate the caveolin-1 localization, we tested the two anti-cavin-1 antibodies (CST #69036 and BD #611258). However, they did not work under the conditions that were used to stain the other proteins, and therefore, the cavin-1 antibody was not used to validate the caveolin-1 staining. However, we tested the co-immunostaining by using two caveolin-1 antibodies, the mouse monoclonal anti-caveolin-1 (7C8) antibody (used in the previous version of the manuscript) and the rabbit polyclonal anti-caveolin-1 (3238) antibody. These antibodies both detect the N-terminal cytoplasmic portion of caveolin-1. The co-localization criteria were set to the overlaps of the spatial distributions of the signals, as shown in the revised Figure 2F. The class 3 blobs, which had ~20 caveolin-1 signals and typical caveolar volume, exhibited good co-localization (Figure 2G). However, the overall co-localization of these caveolin-1 antibodies was only ~50% in the class 1 and class 2 blobs, which have ~8 caveolin-1 signals. When we compared the numbers of signals of caveolin-1 and the volumes of the blobs, the number of signals and the volumes of the co-localized blobs were larger than those without colocalization (Figure S2), suggesting that class 1 and 2 blobs were too small to have co-staining or were not co-localized. For the antibody-dependent STORM signal, we confirmed the previous loss of caveolin-1 signals by the caveolin-1 siRNA, using the same caveolin-1 (7C8) antibody (Tachikawa et al., 2017). Therefore, we think the STORM observation of caveolin-1 was reconfirmed by the co-staining with two caveolin-1 antibodies for the blobs of the size of typical caveolae.

We also tested the localizations of PACSIN2 and EHD2 by using mouse monoclonal antibodies in combination with polyclonal antibodies to caveolin-1, which is a different combination of antibodies from those in the previous manuscript; i.e., the epitopes are different. In addition, we observed PACSIN2 and EHD2 again with the antibodies used in the previous manuscript. On class 3 blobs, the polyclonal EHD2 antibody and monoclonal EHD2 antibody used in this study recognize overlapping regions of EHD2, and the localization of EHD2 relative to caveolin-1 was similar. In contrast, the monoclonal PACSIN2 antibody recognizes the linker region before the SH3 domain, which is close to the EHD2 binding site, whereas the polyclonal PACSIN2 antibody recognizes the F-BAR region. The position of the PACSIN2 antibody staining was ~5 nm different in depth relative to that of caveolin-1. Because two different antibodies to EHD2 gave similar localizations, the differential localizations of PACSIN2 antibodies were strongly suggested to result from the relative positions of PACSIN2.

Lastly, the antibody staining method in this paper is the combination of the activator and reporter dyes in the secondary antibody, for the detection of signals only when the activator signals were observed (Bates et al., 2007; Huang et al., 2008). The advantage of this method is the use of the same reporter dye to avoid chromatic aberration. The other methods using different wavelengths for the detection are not considered to be suitable for such analyses of co-localizations with nm accuracy. The use of fluorescent proteins requires two wavelengths for detection, and thus we did not use fluorescent proteins.

Q) The parameters used to separate the different objects in SuperResNet are not described in detail in the Methods section and it is difficult to understand how the clustering was performed. The author separates the objects into different classes and make assumptions on the degree of maturation but this could not be the case. One could imagine easily that caveolae could be more heterogeneous and some more or less flattened caveolae could still be mature. The term "maturity" is not appropriate and it would better to use comparison in terms of curvature as it is directly connected to the height of the objects which is what is being best measured by STORM.

A) In the revised manuscript, we described the details of the parameters. The signals were first filtered to cut the signals within 20 nm to avoid the possible blinking of the same antibody, which is also implemented in the NIKON software used for signal identification. The neighbor signals within 80 nm were labeled as clusters. The 80 nm value is the setting of the SuperResNet used in its publication (Khater et al., 2018), which also analyzed caveolae. We adapted these values because they are reasonable considering the sizes of caveolae, typically 60-70 nm in diameter. Furthermore, the number of observations of caveolin-1 was 4-15 with this antibody labeling according to our previous estimations (Tachikawa et al., 2017), and thus 80 nm for clustering appeared to be reasonable.

The four-class separation of clusters was also adopted from the paper (Khater et al., 2018) because it appears to extract the "mature" caveolae with ~20 caveolin-1 signals. However, according to the comments, we rephased the term maturation to the caveolin-1 signal abundance and the volumes of the blobs.

We also measured the spatial distribution of caveolin-1 signals in the XY plane and Z (depth) direction by calculating the standard deviation of the coordinates of each blob. The standard deviation is considered to correlated with the radius of the blob. The class 1 and class 2 caveolin-1 clusters had 30-40 nm standard deviations in both XY and Z, and the class 3 clusters had 60-70 nm standard deviations in both XY and Z. Therefore, it was difficult to discuss the curvature of the blobs for the flatness of caveolae. This might indicate that there are few "flat" caveolae under our culture conditions of DMEM supplemented with serum.

Q) The author chose to group PACSIN2 and EHD2 into clusters that were +/- 80nm to be associated with the caveolin1 blob. How can the authors then claim that these objects could be localized at the neck if they are so far from the caveolin signal? DO any of the images suggest accumulation of EHD2 or PACSIN2 at the neck? maybe the author could show an example of STORM image to substantiate this finding.

A) The 80 nm value was adopted because the clustering threshold is also 80 nm for caveolin-1. However, according to your comments, it would not be appropriate to use it to connect the two labelings. Therefore, we recalculated the co-localization by the overlap of the spatial distribution of the signals, as examined by the standard deviation (Figure 2F).

Our previous Figure 3 showed typical images of the blobs shown by the lines connecting each signal, and were not easy to discern. We removed the Figure and replaced it with a cartoon for the measurement of the centroid and the standard deviation for the co-localization in the revised Figures 2E and F.

The word "neck" might be too exaggerated because PACSIN2 is just localized at ~5 nm, which is the size of one protein. Therefore, we modified the description to state that caveolin-1, EHD2, and PACSIN2 co-localize at class 2 caveolin-1 blobs, and that PACSIN2, especially its F-BAR domain, is thought to exist closer to the plasma membrane.

Q) The authors mention on p5 that caveolae typical diameter is 100 nm while in reality it is 60-70nm at the ultrastructural level

A) Thanks for your indication. Various papers have described the diameter as 70-100 nm, 50-100 nm, etc. We rephrased the diameter to 60-100 nm.

Page 8： The volume of the class 3 blob corresponded to the volume of a sphere with a 60-100 nm diameter, a typical size of a caveola, which ranges from 0.9-4 × 106 nm3.

Q) There are several typos throughout the MS (for example title on p5 "STROM observation", etc...) and the english is not always correct. Could be useful to have a native english person proof-read the manuscript.

A) We have corrected the typo as suggested and had an English proof-reading.

References

Fujimoto, T., Kogo, H., Nomura, R., and Une, T. (2000). Isoforms of caveolin-1 and caveolar structure. Journal of cell science 113, 3509-3517.

Huang, B., Wang, W., Bates, M., and Zhuang, X. (2008). Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810-813. 10.1126/science.1153529.

Khater, I.M., Meng, F., Wong, T.H., Nabi, I.R., and Hamarneh, G. (2018). Super Resolution Network Analysis Defines the Molecular Architecture of Caveolae and Caveolin-1 Scaffolds. Sci Rep 8, 9009. 10.1038/s41598-018-27216-4.

Sinha, B., Köster, D., Ruez, R., Gonnord, P., Bastiani, M., Abankwa, D., Stan, R.V., Butler-Browne, G., Vedie, B., Johannes, L., et al. (2011). Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144, 402-413. 10.1016/j.cell.2010.12.031.

Tachikawa, M., Morone, N., Senju, Y., Sugiura, T., Hanawa-Suetsugu, K., Mochizuki, A., and Suetsugu, S. (2017). Measurement of caveolin-1 densities in the cell membrane for quantification of caveolar deformation after exposure to hypotonic membrane tension. Sci Rep 7, 7794. 10.1038/s41598-017-08259-5.

Q) Reviewer #2: In this short article, Dr Suetsugu sets out to establish the superresolution analysis of PACSIN2 and EHD2 at caveolae. EHD2 and PACSIN2 are in addition to the caveolins and cavins, the most recognised and studied caveolar proteins. The research topic is interesting and topical, but the manuscript appears at places hastily put together and lacks some necessary controls.

A) We appreciate your kind offer to revise our manuscript and extend the deadline. In this revised manuscript, we think we have experimentally addressed most of the concerns.

Major comments

Q) 1) Materials and methods section is not complete, and need to be substantially expanded in order to provide context to the study. As it is, the data presented are hard to interpret.

Some additional examples where the methods section need updating

"Under subtitle: "HeLa cell culture, transfection, and live imaging"

The section reads

141 HeLa cells were cultured as described previously [28] in Dulbecco's modified Eagle's 5 142 medium (DMEM), supplemented with 10% fetal calf serum (FCS).

There is no mention on transfection or live imaging. The manuscript does not use those techniques.

We appreciate your helpful comments. We deleted these sentences because we did not transfect cells or perform live imaging. We also added more details in the methods.

Q) 2) There are no controls for that the signal observed by the antibody labeling is specific. Super resolution is a very sensitive technique, and at such antibody validation under specific experimental conditions, including fixation etc is critical. Discussion, and analysis of the fixative used is necessary, as fixation is well known to change the morphology and preservation of caveolae. The fixative will likely also impact on epitope availability, which confounds the conclusions, including of the monoclonal CAV1 used. Is the CAV1 epitope available to the antibody throughout the caveolae bulb? A comparison with widely used and characterised poly clonal CAV1 might yield this necessary insight.

A) Thank you for your kind advice. We think our fixation conditions (3% paraformaldehyde + 0.1 % glutaraldehyde) are similar to or the same as those in publications on caveolae, including ours (Tachikawa et al., 2017). Therefore, we tested other antibodies to confirm our results. We tested the co-immunostaining by using two caveolin-1 antibodies, the mouse monoclonal anti-caveolin-1 (7C8)antibody used in the previous version of the manuscript and the rabbit polyclonal anti-caveolin-1 (3238) antibody, which both detect the N-terminal cytoplasmic portion of caveolin-1. These antibodies co-localized with the class 3 blobs, with ~20 caveolin-1 signals, where the co-localization criteria were set to be the overlap of the spatial distribution of the signals, as shown in Figure 2F. The overall co-localization of these caveolin-1 antibodies was only ~50% with the class 1 and class 2 blobs, which have ~8 caveolin-1 signals on average. When we compared the numbers of signals of caveolin-1 and the volumes of the blobs, these values were larger than those without colocalization (Figure S2). Therefore, the co-staining appears to reflect the number of caveolin-1 molecules and the volume of the structure. The class 3 blobs, which had ~20 caveolin-1 signals, exhibited good co-localization (Figure 2G). For the antibody-dependent STORM signal, we have reconfirmed the loss of caveolin-1 signals by the caveolin-1 siRNA by using the same caveolin-1(7C8) antibody (Tachikawa et al., 2017). Therefore, we think the STORM observation of caveolin-1 was again confirmed by the co-staining of two caveolin-1 antibodies.

We also tested the localizations of PACSIN2 and EHD2 by using mouse monoclonal antibodies in combination with the polyclonal antibodies to caveolin-1 on the class 3 blobs. This is a different combination of antibodies from those in the previous manuscript; i.e., the epitopes are different. We again observed PACSIN2 and EHD2, using the antibodies as in the previous manuscript. The polyclonal EHD2 antibody and monoclonal EHD2 antibody used in this study recognize the overlapping region of EHD2, and the relative localization of EHD2 to caveolin-1 was similar. In contrast, the monoclonal PACSIN2 antibody recognizes the linker region before the SH3 domain, which is close to the EHD2 binding site, whereas the polyclonal PACSIN2 antibody recognizes the F-BAR region. The relative position of PACSIN2 antibody staining was ~5 nm different in depth relative to caveolin-1. Because two different antibodies to EHD2 revealed similar localizations, the differential localization of PACSIN2 antibodies was strongly suggested to result from the relative positions of PACSIN2.

Q) 3) No details on the statistical tests carried out are available and the comparisons in the figure legends. Likewise N numbers should also be included, biological repeats etc.

A) We added more details in the Materials and Methods, as well as the Figure legends. For each blob, the standard deviations on the XY and Z axes and the depth difference between the centroid of the clusters of each labeled antibody labeling were examined and then averaged per observation, which typically contained one cell. The averages of the observations were plotted in each Figure, and were used for statistical evaluations by the Student's t-test.

Q) 4) The figure legends need more information, for instance no information in Figure 1, on what the cell this is, it would also be helpful to have the images displayed in split and merged channels. In addition, to me the colour is magenta, and not red as stated above the figures.

A) We improved the figure legends. The Hela cells are displayed with each channel image in the revised manuscript.

Q) 5) Justification for the reason behind "where >4 caveolin-1 signals within 80 nm chosen". And justification of why "clusters within 80 nm were considered to be the PACSIN2 or EHD2 clusters close to the Caveolin-1". Results/conclusions might have been very? different if these distances had changed.

A) The 80 nm value is the SuperResNet setting used in its publication (Khater et al., 2018), which also analyzed caveolae. We adapted these values because they are reasonable when considering the size of caveolae, typically 60-70 nm in diameter. Furthermore, the numbers of observations of caveolin-1 were 4-15 with this antibody labeling, according to our previous estimations (Tachikawa et al., 2017), and thus 80 nm for clustering appeared to be reasonable. This assumption is consistent with the labeling efficiency of the antibody, reported as 1-8 signals per caveola by electron microscopic analysis (Fujimoto et al., 2000).

The 80 nm value for the co-localizations was adopted because the clustering threshold is also 80 nm for caveolin-1. However, according to your comments, it would not be appropriate to use for connecting the two labels. Therefore, we recalculated the co-localization by using the criteria of the overlaps of the spatial distributions of the signals by standard deviations, as shown in the revised Figure 2F.

Minor comments

Q) Figure 2, bulb, blob or blub are listed in the figure axis, are they meant to be the same?

A) We have corrected these as "blob". We apologize for this mistake.

Q) Sub title, "STROM observation and analysis", should be "STORM observation and analysis" In general there are rather widespread typo's

A) We have corrected the word to "STORM".

Q) Reference for caveosome is needed.

A) We noticed that "caveolae rosette" is a more suitable word for describing the caveolae clusters on the plasma membrane, and adopted this phrase and added the references (Del Pozo et al., 2021; Sinha et al., 2011).

Q) Reference for caveolae size is needed.

A) We have added the references for the size, that are (Parton and Simons, 2007; Rothberg et al., 1992),

Q) Line 163 The "with the provided NIKON Software" software needs to be named.

A) We have added the name of the software.

Q) It would be helpful, if the IF images were shown in separate channels, as well as the merged. This is the commonly accepted way of representing dual colour images.

A) We have separated the merged picture into the individual channels, as shown in the new Fig. 1.

Q) Line 66 correct "with the role of caveolae in caveolae formation [27, 28, 31]."

A) We have replaced the first "caveolae" with "PACSIN2".

Q) Therefore, PACSIN1 and EHD2 were suggested to be localized at the mature caveolae.

Is this Pacsin 2 instead of Pacsin1? There is as far I can see no data presented on PACSIN1

A) As suggested, we have corrected the "PACSIN1" to "PACSIN2."

References

Bates, M., Huang, B., Dempsey, G.T., and Zhuang, X. (2007). Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749-1753. 10.1126/science.1146598.

Del Pozo, M.A., Lolo, F.N., and Echarri, A. (2021). Caveolae: Mechanosensing and mechanotransduction devices linking membrane trafficking to mechanoadaptation. Curr Opin Cell Biol 68, 113-123. 10.1016/j.ceb.2020.10.008.

Fujimoto, T., Kogo, H., Nomura, R., and Une, T. (2000). Isoforms of caveolin-1 and caveolar structure. Journal of cell science 113, 3509-3517.

Huang, B., Wang, W., Bates, M., and Zhuang, X. (2008). Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810-813. 10.1126/science.1153529.

Parton, R.G., and Simons, K. (2007). The multiple faces of caveolae. Nature reviews. Molecular cell biology 8, 185-194. 10.1038/nrm2122.

Rothberg, K.G., Heuser, J.E., Donzell, W.C., Ying, Y.-S., Glenney, J.R., and Anderson, R.G.W. (1992). Caveolin, a protein component of caveolae membrane coats. Cell 68, 673-682. 10.1016/0092-8674(92)90143-Z.

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PLoS One. doi: 10.1371/journal.pone.0271003.r003

Decision Letter 1

Christophe Lamaze

1 Jun 2022

PONE-D-21-39140R1Super-resolution analysis of PACSIN2 and EHD2 at caveolaePLOS ONE

Dear Shiro,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it should be published provided that you respond to the minor concerns raised by the two reviewers. Therefore, we invite you to submit a revised version of the manuscript that addresses the minor points raised during the review process.

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Christophe Lamaze

Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: The authors have now significantly improved their manuscript. They have improved both the figures and the text and proof-read their MS.

Minor points:

the authors must simplify the presentation of Figure 3D, this panel is too complicated for the reader and also modify Figure 3E schematic as caveolae are never forming tubule-like structures but they have an omega shape.

Reviewer #2: The manuscript has greatly improved.

However, there are some issues that need to be discussed and corrected before publication.

1) There are some incorrect uses of references in the introduction.

“there are four cavin isoforms, and they are essential for caveolae”. Ref 11, only covers CAVIN1, the three original “cavin family papers” should be referenced here to be precise doi: 10.1083/jcb.200903053,doi: 10.1038/ncb1887, doi: 10.1038/emboj.2009.46. In addition, Professor Pilch’s original papers on the discovery of CAVIN1 functional importance are not cited, the authors could also consider to include these for the statement on CAVIN1.

Ref 26 is the original of EHD2 localisation to caveolae, so the authors should also include this when being discussed.

2) Needs detailed discussion on the limitations of using primary/secondary Abs to study the fine scale localisation of components within structures as small as caveolae.

3) Are student T-tests (and what type was used?) appropriate for these type of analysis? The authors need to explain the justification for this. The use of correct statistical test is critical.

Minor comments

Line 42 “quite similar” is a subjective term, and the authors could consider changing this ot be more specific.

Line 81 Change “However, Pacsin2 was” to “However, Pacsin2 is”

Line 87, no mention of what type of cells, this is first introduced in line 89, introduction should be in line 87.

Line 115, ref style for Khater, 2018 is not correct.

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PLoS One. 2022 Jul 14;17(7):e0271003. doi: 10.1371/journal.pone.0271003.r004

Author response to Decision Letter 1

4 Jun 2022

Reviewer #1: The authors have now significantly improved their manuscript. They have improved both the figures and the text and proof-read their MS.

A) We would like to appreciate your careful comments on the improvement of our manuscript.

Minor points:

A) We improved the Figure 3D for simplicity and we modified Figure 3E into the omega shaped one.

Reviewer #2: The manuscript has greatly improved.

However, there are some issues that need to be discussed and corrected before publication.

A) We would like to appreciate your careful comments on the improvement of our manuscript. We modified it further according to your comments.

1) There are some incorrect uses of references in the introduction.

Ref 26 is the original of EHD2 localisation to caveolae, so the authors should also include this when being discussed.

A) We appreciate for pointing these important papers. We cited these papers (Bastiani et al., 2009; Hansen et al., 2009; Liu and Pilch, 2008; McMahon et al., 2009) for CAVINs. The ref26 (now ref 32) is also at line 63 for the discussion of EHD2 localization.

2) Needs detailed discussion on the limitations of using primary/secondary Abs to study the fine scale localisation of components within structures as small as caveolae.

A) According to this suggestion, we added several sentences.

Line 132-138: Therefore, the number of molecules to be detected is related to the volume of the blobs and the number of observed signals, suggesting that the class 1 and 2 blobs might have an insufficient number of molecules for colocalization detection or might not have colocalization. Such difficulty in analyzing small structures would probably result from the antibody labeling, where only a small fraction of the proteins can be visualized, as has been shown that 1-8 signals per caveola by electron microscopic analysis by using various caveolin-1 antibodies (Fujimoto et al., 2000).

3) Are student T-tests (and what type was used?) appropriate for these type of analysis? The authors need to explain the justification for this. The use of correct statistical test is critical.

A) The comparison can be justified only between the caveolin-1 (7C8) antibody-treated groups for the PACSIN2 and EHD2 localizations by the two different antibodies. Therefore, we believe a two-tailed t-test is suitable. However, we also performed one-way ANOVA with post-hoc Holm-Bonferroni analysis, and p values are replaced.

Minor comments

Line 42 “quite similar” is a subjective term, and the authors could consider changing this ot be more specific.

A) It was rephrased to “almost identical” with citation.

Line 42: Caveolin exists as three isoforms, and the caveolin-1 and caveolin-3 amino acid sequences are almost identical (Tang et al., 1996; Way and Parton, 1995).

Line 81 Change “However, Pacsin2 was” to “However, Pacsin2 is”

A) This part is the summary of the results of this paper, and we would like to keep “was” here.

Line 87, no mention of what type of cells, this is first introduced in line 89, introduction should be in line 87.

A) We corrected it accordingly.

Line 89: First, we observed the single-molecule localizations in antibody-stained HeLa cells.

Line 115, ref style for Khater, 2018 is not correct.

A) We formatted it.

Bastiani, M., Liu, L., Hill, M.M., Jedrychowski, M.P., Nixon, S.J., Lo, H.P., Abankwa, D., Luetterforst, R., Fernandez-Rojo, M., Breen, M.R., et al. (2009). MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes. J Cell Biol 185, 1259-1273. 10.1083/jcb.200903053.

Fujimoto, T., Kogo, H., Nomura, R., and Une, T. (2000). Isoforms of caveolin-1 and caveolar structure. Journal of cell science 113, 3509-3517.

Hansen, C.G., Bright, N.A., Howard, G., and Nichols, B.J. (2009). SDPR induces membrane curvature and functions in the formation of caveolae. Nat Cell Biol 11, 807-814. 10.1038/ncb1887.

Liu, L., and Pilch, P.F. (2008). A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization. J Biol Chem 283, 4314-4322. 10.1074/jbc.M707890200.

McMahon, K.A., Zajicek, H., Li, W.P., Peyton, M.J., Minna, J.D., Hernandez, V.J., Luby-Phelps, K., and Anderson, R.G. (2009). SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae function. Embo j 28, 1001-1015. 10.1038/emboj.2009.46.

Tang, Z., Scherer, P.E., Okamoto, T., Song, K., Chu, C., Kohtz, D.S., Nishimoto, I., Lodish, H.F., and Lisanti, M.P. (1996). Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J Biol Chem 271, 2255-2261. 10.1074/jbc.271.4.2255.

Way, M., and Parton, R.G. (1995). M-caveolin, a muscle-specific caveolin-related protein. FEBS Lett 376, 108-112. 10.1016/0014-5793(95)01256-7.

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PLoS One. doi: 10.1371/journal.pone.0271003.r005

Decision Letter 2

Christophe Lamaze

22 Jun 2022

Super-resolution analysis of PACSIN2 and EHD2 at caveolae

PONE-D-21-39140R2

Dear Shiro,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. I would like to personally congratulate you for this much improved revised version and for this great work.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: All good and ready to go! Congratulations to the authors on this much improved version describing super-resolution analysis of caveolae components.

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PLoS One. doi: 10.1371/journal.pone.0271003.r006

Acceptance letter

Christophe Lamaze

6 Jul 2022

PONE-D-21-39140R2

Super-resolution analysis of PACSIN2 and EHD2 at caveolae

Dear Dr. Suetsugu:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. The statistics of blobs.

(PDF)

Click here for additional data file.^{(490.7KB, pdf)}

S2 Fig. The statistics of blobs with or without colocalization.

(PDF)

Click here for additional data file.^{(532.1KB, pdf)}

S1 Data. The compressed files of the original coordinates in the VISP files.

(ZIP)

Click here for additional data file.^{(90.9MB, zip)}

S2 Data. The compressed files of the original coordinates in the VISP files and the inventory of the files.

(ZIP)

Click here for additional data file.^{(87.3MB, zip)}

Attachment

Submitted filename: RevisePlosOnev3 220418submit2.docx

Click here for additional data file.^{(34.3KB, docx)}

Attachment

Submitted filename: ReviwerComments220601v2.docx

Click here for additional data file.^{(32.6KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Super-resolution analysis of PACSIN2 and EHD2 at caveolae

Tamako Nishimura

Shiro Suetsugu

Roles

Abstract

Introduction

Results and discussion

Fig 1. SMLM analyses of PACSIN2, EHD2, and caveolin-1.

Fig 2. Clustering of caveolin-1 into blobs and co-localization with PACSIN2 or EHD2.

Fig 3. The geometry of colocalization in the blobs.

Materials and methods

HeLa cell culture

Antibodies

STORM observation and analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Christophe Lamaze

Roles

Author response to Decision Letter 0

Decision Letter 1

Christophe Lamaze

Roles

Author response to Decision Letter 1

Decision Letter 2

Christophe Lamaze

Roles

Acceptance letter

Christophe Lamaze

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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