Article activity feed

  1. Author Response:

    Reviewer #2 (Public Review):

    1. The novelty of the current observation of two types of links is overstated, for example, in the abstract: "Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer scale precise stacking of the ROS disks" (Line 25). In fact, both of these links have been shown before (Usukura and Yamada, 1981; Roof and Heuser, 1982; Corless and Schneider, 1987; Corless et al., 1987; Kajimura et al., 2000). These previous studies deserve to be recognized. Of special note is the paper by Usukura and Yamada whose images of the disc rim connectors are by no means less convincing than shown in the current manuscript. On the other hand, the novelty and impact of the data related to peripherin appears to be understated, particularly in the abstract.

    We changed the abstract line 27 to: “Our data confirm the existence of two previously observed molecular connectors …”, cite the recommended references in the introduction (lines 54-55), the results (lines 131-132), and the discussion (lines 282/285). To highlight the previous reports, we rephrased the sentence in lines 132-133, “In agreement with these previous findings, we observed structures that connect membranes of two adjacent disks …”; the discussion is rephrased in lines 280-281, “Similar connectors have been observed previously ...” and “… and their statistical analysis confirmed the existence of two distinct connector species.”, and in lines 291-292, “Based on previous studies combined with our quantitative analysis, we put forward a hypothesis for the molecular identity of the disk rim connector which agrees in part with recent models”.

    1. Notably, ROM-1 has not been found in peripherin oligomers larger than octamers (e.g. Loewen and Molday, 2000 and subsequent studies by Naash and colleagues). This should be discussed in the context of the current model.

    We agree that this is an important aspect. We pick subvolumes along all disk rims, and on average we obtain the ordered scaffold as shown in the manuscript. We expect heterogeneity in the data because of the different degrees of oligomerization and the exclusion of ROM1 from higher oligomers. Our analysis required substantial classification to achieve convergence to a stable average, indeed indicating heterogeneity in the rim structure. However, we could not resolve additional structures to sufficient quality. It might be that this heterogeneity is what ultimately limits our achievable resolution. We added these thoughts in the discussion starting in lines 377-378, “PRPH2-Rom1 oligomers isolated from native sources exhibit varying degrees of polymerization (Loewen and Molday, 2000), and ROM1 is excluded from larger oligomers (Milstein et al., 2020). We could not resolve this heterogeneity as additional structures to sufficient quality by subvolume averaging, but in combination with the inherent flexibility of the disk rim, this heterogeneity might be the reason for the restricted resolution of our averages.”

    1. The following statement should be reconsidered given the established role of cysteine-150 in peripherin oligomerization: "We hypothesize that the necessary cysteine residues are located in the head domain of the tetramers (Figure 5B), ..." It has been firmly established that only one cysteine (C150) located in the intradiscal loop is not engaged in intramolecular interactions and is essential for peripherin oligomerization.

    Thank you for this advice. We agree and rephrased our discussion in lines 368-371, “The intermolecular disulfide brides are exclusively formed by the PRPH2-C150 and ROM1-C153 cysteine residues, which are located in the luminal domain (Zulliger et al., 2018). We hypothesize that these disulfide bonds (Figure 5B), are responsible for the contacts across rows (Figure 3) ...”

    1. Line 340: "A model involving V-shaped tetramers for membrane curvature formation was proposed recently (Milstein et al., 2020), but it comprises two rows of tetramers which are linked in a head-tohead manner. Our analysis instead resolves three rows organized side-by side in situ (Figure 5A)." I am confused by this statement: doesn't your model also show long rows connected head-to-head? The real difference is that Milstein and colleagues proposed four tetramers per rim whereas the current data reveal three.

    Thank you for pointing out this imprecise description. The model proposed by Milstein and the model in the old version of our manuscript, both propose linkage between tetramers via their disk luminal domains. In our manuscript, we refer to the luminal domain as the head domain. However, to our understanding, the Milstein model suggests two rows of tetramers, where one tetramer in the first row is rotated 180° with respect to a tetramer in the second row (therefore head-to-head), while our data indicate that the V-shaped repeats which we originally hypothesized to be tetramers are only rotated ~63° with respect to one another and are therefore rather oriented side-by-side:

    Fig. 2: Comparison of models for the organization of the ROS disk rim as proposed in in Milstein et al., 2020 (top panel)

    and in our work (lower panel). We now rephrased lines 383-385, “Instead, our analysis in situ resolves three rows of repeats which are also linked by the luminal domain but are rather organized side-by-side (Figure 5A).”

    1. Line 347: "Our data indicate that the luminal domains of tetramers hold the disk rim scaffold together (Figure 3C), which is supported by the fact that most pathological mutations of PRPH2 affect its luminal domain (Boon et al., 2008; Goldberg et al., 2001). It is possible that these mutations impair the formation of tetramers, rows of tetramers, and their disulfide bond-stabilized oligomerization. These alterations could impede or completely prevent disk morphogenesis which, in turn, would disrupt the structural integrity of ROS, compromise the viability of the retina and ultimately lead to blindness." This is not an original idea, as many studies showed that disruptions in peripherin oligomerization lead to anatomical defects in disc formation and subsequent photoreceptor cell death.

    Thank you for pointing this out. Our data are indeed in good agreement with the results made by many groups and further expand on them. We rephrased the manuscript in several places to clarify this relationship: in the abstract lines 32-34, “Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.”; in the introduction lines 78-79, “… allowed us to obtain 3D molecular-resolution images of vitrified ROS in a close-to-native state providing further evidence for previously suggested mechanisms leading to ROS dysfunction”; and in the discussion lines 393-397, “In good agreement with previous work, it is possible that these mutations impair the formation of complexes, and their disulfide bond-stabilized oligomerization (Chang et al., 2002; Conley et al., 2019; Zulliger et al., 2018). Hence, these alterations could impede or completely prevent disk morphogenesis …”. Also, additional relevant publications are cited in line 395.

    1. In regards to the distance between disc rims and plasma membrane, the authors cite the data obtained with frogs (10 nm) but not a more relevant, previously reported measurement in mice (Gilliam et al, 2012). The value of 18 nm reported in that study is much closer to the currently reported value.

    We appreciate the reference to this excellent paper. We added it in lines 335-337, “This value was derived from amphibians (Roof and Heuser, 1982) and deviates considerably from recent results (18 nm, (Gilliam et al., 2012)) and from our current measurements in mice (~25 nm).” Our aim was to point out that a model for ROS organization that is often cited and is otherwise well-founded (BatraSafferling et al., 2006) makes a wrong assumption about distance in the context of the mammalian systems. 7. The authors are (correctly) being very careful in assigning the molecular identity of disc interior connectors to PDE6. However, they are more confident in assigning the disc rim connectors to GARP2, which is reflected in the labeling of these links in figure

    1. Their arguments are valid, but these links are not attached to peripherin (a protein considered to be the membrane binding partner for GARPs), which is not immediately consistent with this hypothesis. Perhaps it would be fair to re-label the corresponding links in figure 5 as "disc rim connectors".

    That is an excellent and fair suggestion. We changed Figure 5 accordingly.

    1. On a similar note, the disc rim connectors seem to be located where ABCA4 is presumed to be localized within the rim, which may not be just a coincidence. The authors already have tomograms obtained from ABCA4 knockout animals. Is it possible to analyze whether these links are preserved in these tomograms?

    We agree, this is an important question to address. Unfortunately, neither the biological preparation nor the tomograms of the ABCA4 knockout were as good in quality as for the WT. Still, we frequently see connectors at the disk rim, especially after denoising of the tomograms.

    Fig. 3: connectors at disk rims in WT (left) and ABCA4 knockout mice (right).

    Sometimes it appears the connectors between adjacent disks are linked via an intradisk densities, which was already observed in Corless et al., 1987. We thought that these densities could be ABCA4 and tried to find them with two approaches in our WT tomograms (data not shown). In the first approach using a segmentation similar to what we did for the connectors between disks, we found an order of magnitude fewer intradisk connectors than (inter)disk rim connectors. In the second approach, we used the positions of segmented (inter)disk rim connectors and classified rotational averages which focused on the disk luminal space next to the contact point of a connector with the disk membrane. Again, less than 10% of the disk rim connector subvolumes were assigned to classes with an additional luminal density. Both experiments indicate that disk rim connectors sometimes occur with an additional luminal density. In total, we found less than 100 of these intradisk densities, an observation which seems to be preserved in WT and ABCA4 KO. Based on this small number of positions/locations, however, we cannot draw any conclusion. Therefore, we did not add this point to the manuscript.

    Read the original source
    Was this evaluation helpful?
  2. Evaluation Summary:

    Pöge at al. present a study of the rod outer segment (ROS). These are specialised cilia of rod photoreceptor cells, essential for sensing light cues and initiating the vision process. The authors apply cryo-FIB milling to generate highly preserved rod samples and report high-quality cryo-tomographic data providing new insights into the ultrastructure of the ROS. The work reveals potential molecular scaffolds both in the lumen of the membrane stacks and on the surface of the stack providing the structural basis for ROS crucial ordered ultrastructure. The data presented here will be highly valuable for the field of phototransduction.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

    Read the original source
    Was this evaluation helpful?
  3. Reviewer #1 (Public Review):

    Pöge at al present a cryo-tomographic study of the rod outer segment (ROS). These are specialised cilia of rod photoreceptor cells, which are essential for sensing light cues and initiating the vision process. They are elongated structures filled with stacks of membranous discs which contain rhodopsin, the major light-sensing protein, and other proteins, some of which maintain the disc architecture.

    In this paper, they take advantage of the structural preservation of cryo-FIB milling, and of the unprecedented molecular details available in the resulting cryo-tomograms, to investigate the elements that maintain the elaborated disc architecture.

    The authors provide a general description of the rod ultrastructure, providing precise description of the membrane arrangement and geometry. They then focus their attention to two regions:

    1. Disc connections. Here the authors are able to identify connecting densities and to propose, based on their length and density distribution, that the connections at the disc rims and those on the 'flat' internal surface are of different protein composition. They discuss which protein complexes are likely to contribute to these two types of connectors, although the experimental evidence for this is weak and theirs remains a model to be tested.

    2. Proteins generating/maintaining curvatures at the disc rims. Here subtomogram averaging provides intermediate resolution structures that are entirely novel, and clearly show a regular array of proteins holding the high membrane curvature. As before, the authors tentatively propose the identity of the proteins forming these structures, and while their assignment seems entirely reasonable there is no direct experimental evidence to support it.

    Overall, the quality of the reconstructions presented allows the achievement of a level of insight into rod membrane architecture that will be of interest to scientists working in the fields of in situ structural biology, and of ROS and more generally cell ultrastructure.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    1. The novelty of the current observation of two types of links is overstated, for example, in the abstract: "Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer scale precise stacking of the ROS disks" (Line 25). In fact, both of these links have been shown before (Usukura and Yamada, 1981; Roof and Heuser, 1982; Corless and Schneider, 1987; Corless et al., 1987; Kajimura et al., 2000). These previous studies deserve to be recognized. Of special note is the paper by Usukura and Yamada whose images of the disc rim connectors are by no means less convincing than shown in the current manuscript. On the other hand, the novelty and impact of the data related to peripherin appears to be understated, particularly in the abstract.

    2. Notably, ROM-1 has not been found in peripherin oligomers larger than octamers (e.g. Loewen and Molday, 2000 and subsequent studies by Naash and colleagues). This should be discussed in the context of the current model.

    3. The following statement should be reconsidered given the established role of cysteine-150 in peripherin oligomerization: "We hypothesize that the necessary cysteine residues are located in the head domain of the tetramers (Figure 5B), ..." It has been firmly established that only one cysteine (C150) located in the intradiscal loop is not engaged in intramolecular interactions and is essential for peripherin oligomerization. \

    4. Line 340: "A model involving V-shaped tetramers for membrane curvature formation was proposed recently (Milstein et al., 2020), but it comprises two rows of tetramers which are linked in a head-to-head manner. Our analysis instead resolves three rows organized side-by side in situ (Figure 5A)." I am confused by this statement: doesn't your model also show long rows connected head-to-head? The real difference is that Milstein and colleagues proposed four tetramers per rim whereas the current data reveal three.

    5. Line 347: "Our data indicate that the luminal domains of tetramers hold the disk rim scaffold together (Figure 3C), which is supported by the fact that most pathological mutations of PRPH2 affect its luminal domain (Boon et al., 2008; Goldberg et al., 2001). It is possible that these mutations impair the formation of tetramers, rows of tetramers, and their disulfide bond-stabilized oligomerization. These alterations could impede or completely prevent disk morphogenesis which, in turn, would disrupt the structural integrity of ROS, compromise the viability of the retina and ultimately lead to blindness." This is not an original idea, as many studies showed that disruptions in peripherin oligomerization lead to anatomical defects in disc formation and subsequent photoreceptor cell death.

    6. In regards to the distance between disc rims and plasma membrane, the authors cite the data obtained with frogs (10 nm) but not a more relevant, previously reported measurement in mice (Gilliam et al, 2012). The value of 18 nm reported in that study is much closer to the currently reported value.

    7. The authors are (correctly) being very careful in assigning the molecular identity of disc interior connectors to PDE6. However, they are more confident in assigning the disc rim connectors to GARP2, which is reflected in the labeling of these links in figure 5. Their arguments are valid, but these links are not attached to peripherin (a protein considered to be the membrane binding partner for GARPs), which is not immediately consistent with this hypothesis. Perhaps it would be fair to re-label the corresponding links in figure 5 as "disc rim connectors".

    8. On a similar note, the disc rim connectors seem to be located where ABCA4 is presumed to be localized within the rim, which may not be just a coincidence. The authors already have tomograms obtained from ABCA4 knockout animals. Is it possible to analyze whether these links are preserved in these tomograms?

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    This manuscript provides an updated and more detailed view on mouse rod outer segment (ROS). Using cryoFIB milling and cryoET the authors imaged intact ROS. ROS comprises of highly ordered, densely packed membrane disks that anchor rhodopsin. ROS ordered ultrastructure is crucial for vision and the authors aimed in this study to determine the structural basis for achieving this order.

    The authors revealed potential molecular scaffolds both in the lumen of the membrane stacks and on the surface of the stack. On the outer surface, two distinct molecular connectors were observed, likely contributing to the precise stacking of the membrane disks. One class of connectors specific to the disk periphery/rim and one distributed on the rest of the membrane. Within the lumen and specific to the curved edge a continuous supermolecular assembly of three rows was observed. Based on previous literature, the authors speculate on the identity of the scaffold proteins they observed.

    The imaging methods and image processing applied in this study are state-of-the-art. The methods are described in detail so can be used as a resource for other groups applying similar techniques. The provided ROS ultrastructure with the averaging of the macromolecular assemblies of the disk rim are of the highest quality and will be highly valuable for the field of phototrunsduction.

    The weakness of this study is in the molecular identification of the components forming the macromolecular assemblies observed. The resolution obtained by subtomogram averaging doesn't allow molecular identification and there weren't any direct experiments attempting to identify the components. Thus the molecular basis in this study is highly speculative.

    Read the original source
    Was this evaluation helpful?