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Actin chromobody imaging reveals sub-organellar actin dynamics

Cara Schiavon, Tong Zhang, Leonardo Andrade, Melissa Wu, Tsung-Chang Sung, Yelena Dayn, Jasmine W. Feng, Omar A. Quintero, Robert Grosse, Uri Manor

Preprint posted on May 15, 2019 https://www.biorxiv.org/content/10.1101/639278v1

Nanobody technology allows live imaging of sub-organellar actin dynamics at previously unachieved temporal and spatial resolution, and exploring its relevance for organelle fission.

Selected by Mariana De Niz

Background

Close proximities between organelles have been described over a long time. However, for decades, most studies focused on the study of organelles as separate entities. Only in recent years has there been a revolution in the concept of inter-organelle interactions, and the functional relevance of such interactions for cellular homeostasis.

The study of membrane contact sites has emerged as a relatively novel field in cell biology aiming to understand the molecular mechanisms and physiological relevance of contacts between organelles. Contact sites, based on current definitions (discussed in (1)) include the existence of tethering forces arising from protein-protein or protein-lipid interactions; function, including transport of molecules; and a defined lipidome/proteome arising from the high concentration of specific lipids or proteins at the contact site of the membranes creating a “synaptic” arrangement. The first heterotypic contact sites described involved the endoplasmic reticulum (ER), with most other organelles (reviewed in (1) and (2)).

The research ‘path’ and motivation, leading to the work by co-1stauthors Schiavon/Zhang and colleagues (3), are thoroughly discussed by the senior author, Dr. Uri Manor, in an inspiring tweeter thread. In this thread, he refers to work preceding his, focusing on the study of ER-mitochondrial contact sites, and particularly, to the molecular basis suggesting that actin and myosin are important for the process of mitochondrial constriction and fission (4,5,6), (Figure 1, left). While various molecular players in this process have been identified, fluorescence imaging of actin dynamics directly at the mitochondria or ER, had not yet been achieved due to limitations in existing imaging methods.

In the pursuit of achieving a way to investigate sub-organellar actin dynamics, and further investigating working models on the role of actin at ER-mitochondria contact sites in driving mitochondrial fission, Schiavon/Zhang and colleagues generated fluorescent protein-tagged actin nanobodies (termed ‘actin chromobodies’) targeted to organelle membranes (2). This led to findings relevant to the cell biology field, discussed as key points below.

Figure 1. (Left panel) ER-associated actin accumulates at mitochondrial fission sites. Previously published model for how mitochondrial Spire1C and ER-anchored INF2 could mediate mitochondrial constriction via actin filament assembly. The actin filament elongation activity exerts pressure on the mitochondrial outer membrane. Tethering complexes play a role in maintaining association between ER and mitochondrial membranes (from ref. 6). (Right panel) ER-associated actin accumulates at most organelle fission sites, including the mitochondria, endosomes, peroxisomes, lysosomes and Golgi.

Key findings

Technical advance

  • The group generated actin chromobody (AC) probes, fused to organelle membrane targeting sequences to image sub-organellar actin dynamics exclusively within a 10nm distance from the target organelle membrane.

Key observations 

  • This work shows for the first time, mitochondria- and ER-associated actin filaments with unprecedented resolution under physiological conditions, validating previous models suggesting that actin accumulates at ER-mitochondria intersections (Figure 1).
  • AC-mito accumulations on mitochondria were dependent on F-actin and demonstrated the superior labelling capacity of actin nanobodies to highlight this enrichment.
  • Fixation caused dramatic loss of AC-mito enrichment, and a decrease in signal in AC-mito and AC-ER expressing cells. This is consistent with other recent findings (7) and highlights the value of live imaging, supporting the relevance of the work presented by the authors.

Functional relevance

  • Using their novel constructs, the authors tested the hypothesis that actin accumulates at all ER-organelle contact sites to drive their fission. Live imaging of cells co-expressing AC-ER and markers for mitochondria, endosomes, peroxisomes, lysosomes and the Golgi all revealed accumulation of ER-associated actin at fission sites for each of these organelles (Figure 1).

 

Open questions and what I like about this paper

I like this paper because of three key points:

  • Scientifically, I think it is extremely valuable for the cell biology field, as it provides a novel tool to investigate sub-organelle dynamics in a manner that was previously not possible. We can go back and re-address cell biology questions which were technically impossible to address before.
  • Experimentally, the work is very well designed, and the rationale for each step is extremely clearly explained.
  • In terms of the philosophy of open science, this is the best example I have seen, whereby the authors engaged in open discussion, and explained in detail each step of the scientific method leading to the published pre-print. I find this extraordinary.

Regarding questions to the authors on future directions (see author responses below).

  • What is the biological significance of the ER-associated actin bundles you found on the nucleus?
  • Your work and others’ has demonstrated that fixation can introduce important artefacts. What are cell biology processes described prior to this current knowledge, which you imagine might be worth re-evaluating using current tools for live microscopy?
  • You explore the actin nanobody technology greatly in the context of your paper, and address questions that had arisen years before, in your own work and others. There are other nanobodies, and this technology is gaining more visible implementation as a research tool for microscopy. Do you envisage using other nanobodies in parallel to actin, to further explore the biological significance of membrane-contact sites and organelle fission?
  • You highlight in your conclusion that the possibility to use membrane-anchored AC probes as ‘proximity sensors’ for sub-cellular actin dynamics provides a novel tool for studying the role of actin in a wide range of cell biological processes. What are the main processes you are interested in to follow up with your work?
  • Among the pathologies that involve changes in sub-organelle dynamics, is pathogen-mediated hijacking of host organelles and host cargo. This expands the idea of contact sites, to those occurring between membranes of the pathogen and the host organelles (including contact sites). This opens many exciting questions, including some relevant to host cell sub-organelle actin dynamics. What are the fastest transient actin dynamics you have recorded?

Acknowledgements

I am very grateful to Uri Manor, Cara Schiavon and Tong Zhang for their time and willingness to engage in this preLight highlight, and to Mate Palfy for his input and helpful comments.


References

  1. Scorrano L, De Matteis MA, Emr S, Giordano F, Hajnoczky G, Kornmann B, Lackner LL, Levine TP, Pellegrini L, Reinisch K, Rizzuto R, Simmen T, Stenmark H, Ungermann C, Schuldiner M, Coming together to define membrane contact sites, Nat Comm, 2019 10(1):1287, doi: 1038/s41467-019-09253-3
  2. Eisenberg-Bord M, Shai N, Schuldiner M, Bohnert M, A tether is a tether: tethering at membrane contact sites, Dev Cell, 2016, 39(4): 395-409. doi: 10.1016/j.devcel.2016.10.022
  3. Schiavon C, Zhang T, Zhao B, Andrade L, Wu M, Sung TC, Dayn Y, Feng JW, Quintero OA, Grosse R, Manor U, Actin chromobody imaging reveals sub-organellar actin dynamics, bioRxiv, 2019, org/10.1101/639278.
  4. Korobova F, Ramabhadran V, Higgs HN, An actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2, Science,339(6118): 464-467, doi: 10.1126/science.1228360.
  5. Korobova F, Gauvin TJ, Higgs HN, A role for myosin II in mammalian mitochondrial fission, Curr Biol, 2014, 24(4):409-414, doi: 10.1016/j.cub.2013.12.032.
  6. Manor U, Bartholomew S, Golani G, Christenson E, Kozlov M, Higgs H, Spudich J, Lippincott-Schwartz J, A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division, Elife, 2015, doi:10.7554/eLife.08828
  7. Pereira PM, Albrecht D, Culley S, Jacobs C, Marsh M, Mercer J, Henriques R, Fix your membrane receptor imaging: actin cytoskeleton and CD4 membrane organization disruption by chemical fixation, Front Immunol, 2019, 10:675 doi:10.3389/fimmu.2019.00675

Tags: cell biology, sub-organelle actin dynamics

Posted on: 24th May 2019

Read preprint (2 votes)




  • Author's response

    Uri Manor, Cara Schiavon, Tong Zhang shared

    1. What is the biological significance of the ER-associated actin bundles you found on the nucleus?

    UM: Others such as the Wirtz lab have shown a role for a perinuclear actin “cap” in health and disease. It is thought that this cap helps shape the nucleus, for example during cell migration wherein the cell must squeeze through small channels. This has obvious implications for both cancer and developmental biology research. It may also be relevant for progeria, cardiomyopathy, or muscular dystrophy – basically any/all diseases associated with altered nuclear morphology. To clarify: We believe the AC-ER probe is labelling this perinuclear cap with such clarity by virtue of the fact that the ER is contiguous with the nuclear membrane, allowing for sufficient proximity such that the perinuclear actin cap can be detected.

     

    2. Your work and others’ has demonstrated that fixation can introduce important artefacts. What are cell biology processes described prior to this current knowledge, which you imagine might be worth re-evaluating using current tools for live microscopy?

    UM: We believe that the ultrastructure of all cell biological processes are likely to be affected by chemical fixation to varying degrees. The easy answer is that we need to evaluate everything with either live cell imaging or with cryofixation (see for example Elizabeth Villa or Ben Engel’s work with cellular cryotomography for the insanely high potential of this approach, which we are also adopting in our lab). Obviously, that is not always possible.

    To try to be more specific, we think anything associated with smaller, dynamic membranes is likely to be affected by chemical fixation, even without detergents. The fact is that paraformaldehyde pokes holes in membranes, but we largely ignore this because we still need to use detergents to poke holes large enough for proteins to get in/out of the cell during immunofluorescence. Given the well-established role for actin in remodelling membranes, all of these actin-membrane associated processes are ripe for re-evaluation with advanced live imaging probes.

     

    3. You explore the actin nanobody technology greatly in the context of your paper, and address questions that had arisen in years prior, in your own work and others. There are other nanobodies, and more visible implementation as research tools for microscopy. Do you envisage using other nanobodies in parallel to actin, to further explore the biological significance of membrane-contact sites and organelle fission?

    UM: We are extremely excited about nanobody technology capabilities in this context as well as others – see for example the Lichtman lab’s recent Nature Methods paper showing that nanobodies can be used to label tissues without (detergent-mediated) permeabilization, facilitating correlative imaging approaches that would otherwise be impossible.

    We are developing similar nanobody probes for microtubules and intermediate filaments to study their potential role in organelle dynamics. It is worth pointing out that actin filaments are an easy, obvious target. But there are also many proteins that are what those in the field like to call “cytoplasmic”, as in: they are “everywhere”. The potential for using organelle-targeted nanobodies or any other biosensors to better understand the dynamics of specific proteins or signalling pathways on specific organelle surfaces is extremely exciting to us.

     

    4. You highlight in your conclusion that the possibility to use membrane-anchored AC probes as ‘proximity sensors’ for sub-cellular actin dynamics provides a novel tool for studying the role of actin in a wide range of cell biological processes. What are the main processes you are interested in to follow up your work?

    UM: We are exploring additional specific membrane targeting sequences to better tease apart how specific membrane domains are associated with key actin structures. One area we didn’t touch in our paper but we would love to explore is the potential role of ER-associated actin in endocytosis.

    Other processes that would be interesting to explore are: cilia, filopodia, and microvilli formation,  multi-nucleation, cell migration, cell division, neuronal cell processes such as synaptic plasticity, cell-cell junction formation, exocytosis, and peroxisome and autophagosome formation.

     

    5. Among the pathologies involving sub-organelle dynamic changes, is pathogen-mediated hijacking of host organelles and host cargo. This expands the idea of contact sites, to those occurring between membranes of the pathogen and the host organelles (including contact sites). This opens many exciting questions, including some relevant to host cell sub-organelle actin dynamics. What are the fastest transient actin dynamics you have recorded?

    UM: The idea of using these probes to study pathogen motility is extremely exciting to us – many pathogens target and alter mitochondrial dynamics, and it is almost certain that actin dynamics are involved in that. The fastest actin dynamics we detected so far were certainly with these probes at ER-organelle contacts (2ndplace would be lamellipodia ruffling). We have movies of peroxisomes moving around the cell (they’re really, really fast!), and the AC-ER probe shows actin associated with these peroxisomes nearly 100% of the time, but it is very dynamic. So that leads to another area of future study: Does actin polymerization play a direct role in organelle motility, and is there a specific role for the ER in organizing or directing that motility?

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