Reticular Adhesion Formation is Mediated by Flat Clathrin Lattices and Opposed by Fibrillar Adhesions

  1. Laura Hakanpää
  2. Amr Abouelezz
  3. An-Sofie Lenaerts
  4. Seyda Culfa
  5. Michael Algie
  6. Jenny Bärlund
  7. Pekka Katajisto
  8. Harvey T. McMahon
  9. Leonardo Almeida-Souza

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Abstract

Reticular adhesions (RAs) consist of integrin αvβ5 and harbor flat clathrin lattices (FCLs), long-lasting structures with similar molecular composition to clathrin mediated endocytosis (CME) carriers. Why FCLs and RAs colocalize is not known. Here, we show that FCLs assemble RAs in a process controlled by fibronectin (FN) and its receptor, integrin αvβ5. We observed that cells on FN-rich matrices displayed fewer FCLs and RAs. CME machinery inhibition abolished RAs and live-cell imaging showed that RA establishment requires FCL co-assembly. The inhibitory activity of FN was mediated by the activation of integrin α5β1 at Tensin1-positive fibrillar adhesions. Conventionally, endocytosis disassembles cellular adhesions by internalization of their components. Our results present a novel paradigm in the relationship between these two processes by showing that endocytic proteins can actively function in the assembly of cell adhesions. Furthermore, we show this novel adhesion assembly mechanism is coupled to cell migration via a unique crosstalk between cell matrix adhesions.

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    Referee #3

    Evidence, reproducibility and clarity

    Overview:

    This manuscript addresses the emerging nexus linking the machinery associated with clathrin endocytosis (clathrin-coated pits; CCPs), flat clathrin lattices (FCLs) and the recently discovered Reticular Adhesions (RAs). This is timely work, reflecting recent foci on the relationship between these structures and systems. Initially clearly identifying reductions in FCL and RA formation on fibronectin, the authors sought to clarify the mechanisms that suppress or prevent FCL / RA formation on this matrix. Knock-down of integrin avb5 (core RA component) suppressed both RA and FCL formation, suggesting a dependence of FCLs on this integrin. This was supported by acute avb5 inhibition via cilengitide (avb5 and avb3 inhibitor) which caused disassembly of existing RAs and FCLs.

    Notably, the inverse relationship also appears true, with suppression of core clathrin endocytic machinery (AP2 complex components) being sufficient to greatly deplete RA formation. Supporting this finding, overexpression of a dominant negative-acting protein fragment (AP180 c-terminal fragment) blocked both AP2 localisation to the plasma membrane and RA formation.

    To unmix this bi-directional dependence further, the authors used acute cilengitide treatment followed by washout and post-washout incubation to first deplete RAs (cillengitide) and then allow monitoring of en masse RA formation after cilengitide washout. This is an effective experiment, however, the analysis would benefit from greater depth, particularly relating to the order of events. Analysis of this aspect seems central to the thrust of the paper, and some statistical analysis of either static co-occurrence or dynamic ordering in large numbers of FCL / RA structures (i.e. hundreds) would be of value.

    The authors next focused on the observation that fibronectin suppressed both FCL and RA structures, by assessing the role of fibronectin-receptor integrin b1. Acute antibody mediated integrin b1 inhibition (mab13) and integrin b1 knock down both confirmed that in cells on fibronectin, suppression of integrin b1 is sufficient to permit massive upregulation of both FCL and RA formation. This is surprising and very interesting. It raises questions about the actual ECM requirements for avb5-mediated reticular adhesion formation. It would seem that fibronectin per se can support very efficient RA / FCL formation, but that normally concurrent integrin b1 activities would suppress this. Given this implication, it would be especially important to clarify the purity of FN ECM coating (as explored in questions 1-3 below) at the time of imaging. The discussion addresses a number of topics, and proposes a mechanistic model to explain the results presented. I don't find the mechanism very convincing, as the directionality of the dependence between FCLs and RAs is not clearly delineated by the experiments presented, in my opinion. That there is co-dependence is convincingly shown, but whether there is directionality, and what order of events underpins FCL then RA or RA then FCL formation, is unclear. Nonetheless, the evidence presented does generally support the idea of a shift in the way we consider the role of endocytic machinery in adhesion regulation, from a disassembly only related function to additional functions associated with adhesion formation and maintenance. Ideally, the mechanisms around this new assembly / maintenance function would be further delineated here, but regardless, this work does point in the direction of important new questions in this area. Further discussion about the potential role of this interdependent regulatory process in, for example, mitosis, seems unwarranted and should probably be removed.

    Questions:

    1. A technical question on the replating experiments onto specific matrix proteins; after coating surfaces with the purified ECM components or controls, what media were the cells replated in? Ideally this should be serum-free media, to ensure that the ECM components of FBS / FCS are not immediately added to the purified ECM components. This should be clarified in the methods.
    2. Related to above, I cannot see how long cells were plated onto the different ECM conditions. This would be relevant to know and should be clarified because cells will secrete ECM over time and thus the purity of the ECM components addressed is dependent on the length of time cells are incubated and imaged for after attachment.
    3. Similarly, it is noted that cells plated on 'glass' support RA formation. It should be clarified what ECM component is then actually responsible for cell adhesion and adhesion complex (RA, FA or other) formation
      • since this requires an ECM component of some type. Presumably, 'on glass' means whatever ECM is either derived from the media used during cell attachment / incubation (if that media contains serum, which is vitronectin rich), or whatever ECM is secreted by the cells themselves over the attachment / incubation period prior to imaging.
    4. In the cilengitide washout experiments, the evidence shown in figures seems to suggest that AP2-positive FCLs form in locations where avb5 (probably RAs) are already present, whereas avb5 positive structures do not form from AP2-only structures. Statistical analysis of this pattern (i.e. which protein is present first) would be valuable to address directionality. Notably, in Figure 3G, it appears that avb5 is present and increasing prior to the subsequent arrival of AP2.
      • a. I would suggest that the cilengitide experimental results(3E-G) be shown in a separate figure from the endocytosis inhibition results (3A-D).
    5. The integrin b1 inhibition and knock down results are clear and interesting. Clarifying the ECM components present during these experiments would be valuable to interpretation of the paper.

    Significance

    see above

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Hakanpää and colleagues report on the relationship between flat clathrin lattices (FCLs) and reticular adhesions, with FCLs being preposed to nucleate reticular adhesions. Overall the experimental work is high quality and the data are generally well presented.

    Major comments:

    The Introduction is very brief and doesn't cover the information required to understand the paper. There are three cellular structures to be understood: focal adhesions, reticular adhesions and FCLs. The intro jumps straight into the FCLs (the paper is written from a FCL point of view) but there is no information about the other two structures really particularly the differences between them. Furthermore, there is nothing in the intro about cellular adhesion or why this is even worth studying. The authors should fully revise this Intro, there is much room for improvement!

    Fig 4D I could not understand this plot and the legend did not describe it properly. What are the units of FCL frequency? I guess it is FCLs per some distance (10µm?), the images need a scale bar. OK, I read the description in the methods and now I see it is the proportion of total CCSs that are FCLs; so frequency is the wrong term. The legend says that there were 32 videos and there are 32 points on the plot but what we need to know is: where n = 1 cell, what did the FCL frequency do over time? A line is drawn on the graph, no info on what the line is and the fit is poor (R2 < 0.5). The authors should take their series of data points from individual cells and fit curves to each and describe the summary statistics of the parameters of these fits OR average the data and fit to that, using the 1 SD of the data for weighting the fit. Probably more data is required to do any meaningful fitting here. To my eye it looks like FCL frequency goes from 0.3 to a plateau of 0.5 at 10 min and that more timepoints between 0 and 10 min would have been useful.

    Fig 3F/G is a nice expt. It looks as though the ITGB5 signal is already creeping up when the AP2 arrives. I agree that they accelerate together, but the prior accumulation of integrin is at odds with the conclusion that AP2/clathrin nucleates the adhesion. This experiment is missing two controls: what is the behaviour of ITGB5 in AP2 negative regions? What happens to both signals in the continued presence of cilengitide? These controls are needed to conclude that AP2 is nucleating the adhesion.

    Minor comments:

    Fig 6 - several typos - "adaptor proteins" "engagement" "containing"

    Significance

    Previously, endocytosis (clathrin-mediated) was thought to decrease cellular adhesion by removal of integrins. This paper suggests that the same machinery can be used to build adhesions. This is a surprising conclusion that will be of interest to many cell biologists; the topic of clathrin and adhesion is being actively explored by several labs either from the adhesion or the endocytosis sides. I have been following this topic from a distance and don't know the details of all the published papers, but this paper does seem to add something new over the recent work from Taraska, Sonnenberg, Montagnac, Strömblad.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Hakanpää et al explored the connection between flat clathrin lattices and reticular adhesions and the regulatory mechanism underlying the formation of these two structures in the U2OS cells. The author provided evidence that the composition of the extracellular matrix plays critical roles in their formation and concluded that fibronectin and its receptor, β1 integrin, inhibit the assembly of FCLs and RAs. The author depleted several components of the clathrin mediated endocytosis machinery and could show that it blocked the formation of reticular adhesions.

    Major comments

    1. In Fig. 1, the author plated U2OS cells on surfaces coated with different ECM components and then measured the frequency of FCLs. How long were the cells allowed to attach to the surfaces before they were imaged? Were the cells serum-starved before seeding? Given the fact that cells attached well even on BSA-coated dishes, I guess the cells were allow to attach and spread for at least overnight. In this case, the ECM components (vitronectin is very abundant in the serum in a concentration of 200-400 ug/mL, fibronectin is another one) in the culture medium would coat the glass surface and this has profound effects on the adhesion status of the cells. Thus, more details of this experiment need to be included and more attention should be pay regarding the data interpretation. In Fig. 1C, it seems like the mere glass surface induced the most FCL formation. However, if the cells are grown on the glass overnight or for days, the major component of the surface would actually be vitronectin and fibronectin (maybe more) rather than glass, thus it is not accurate to say that 'VTN reduced FCL frequency to some extent compared to glass' (Line 67).
    2. Line 91-93. It is not accurate to claim that the reticular adhesions are the only type of cellular adhesion maintained during mitosis. In several studies, active integrin β1 are found along the retraction fibers (Dix et al, Dev Cell, 2018; Chen et al, NCB, 2022). The importance of αVβ5 integrin in the spatial memory during mitosis was only shown in the in vitro cell culture. In fact, mice lacking αVβ5 integrin or its ligand vitronectin are both viable and show no major defects during embryonic development (Zheng et al, PNAS, 1995; Huang et al, Mol Cell Biol, 2000), suggesting reticular adhesions are dispensable in cell division in vivo. I advise to change it into'RAs are composed of αVβ5 integrin and are maintained during mitosis in culture'.
    3. It has been shown that fibronectin and laminin coating inhibit formation of reticular adhesions (Lock et al, NCB, 2018, Fig. S7). This study should be cited in Fig. 1. I suggest to also include laminin in Fig. 1C to make the list of ECM components more complete.
    4. Fig. 5C, the fluorescent intensity of αVβ5 integrin is increased dramatically when integrin β1 was depleted compared to the control shRNA. Although the images were collected in the TIRF mode, it is important to measure αVβ5 and β1 integrin level by immunoblot to confirm the knockdown efficiency of β1 integrin and exclude the possibility that the increase of RA formation is not due to the compensation by upregulation of αVβ5.
    5. Antibody-blocking or depletion of β1 integrin both lead to accumulation of FCL and RA formation, indicating that the activation of β1 integrin is critical in the inhibition of FCL and RA. Activation of β1 integrin depends on talin and kindlin, which bind β5 integrin with a much lower affinity. Would depletion of talin or kindlin cause FCL and RA formation similar to inhibition of β1 integrin?

    Minor comments

    1. In most of the quantifications, only the number of the cells measured were mentioned in the legend. The number of replicate experiments is missing. It should be included in the legend as well.
    2. A recent study (https://doi.org/10.1242/jcs.259465) demonstrated the molecular mechanism underlying the localization of αVβ5 integrin in flat clathrin lattices. It should be mentioned in the introduction.

    Significance

    Although it is not novel that FCLs and RAs share same localization and might actually be the different parts of the same structure (Zuidema et al, JCS, 2022), the observation that inhibiting β1 integrin stimulates FCL and RA assembly is interesting as it indicates the counter-balance between the αVβ5 and α5β1 integrins. It is a pity that the author did not dig deeper into the mechanism underlying this interesting finding, which should greatly increase the impact of this study.

  5. Version published to 10.1101/2022.05.30.494022v2 on bioRxiv
  6. Version published to 10.1101/2022.05.30.494022v1 on bioRxiv