My favourite papers

A few months ago Richard Grant while still at Faculty of 1000 asked me if I would like to contribute to the F1000 Naturally Selected blog to write about my 10 favourite papers of all time. He also asked me to write something about the 3 favourite papers of my own. I am reposting it here for ease of access and to at least start with something I like!

This is what I wrote:

Apart from these ten, there are many others that I have read and thought “I wish I had done that” or “that’s an amazing piece of work”. There are also texts without which I cannot even imagine where I (or even more broadly we) would be today–from Hooke’s Micrographia to Alberts’ quite astonishing Molecular Biology of the Cell. However, these are the primary research papers that I believe shaped me scientifically. Compiling this list has reminded me of one vital point in doing science–it is the people, the scientists themselves that shape one’s career as much as the science itself. I urge others to consider not just searching PubMed or F1000 for topics, protein names, or disease states, but also for individual authors. To know a body of work from an outstanding scientist can have great value in itself; it can both inspire and be something to aspire to.

Identification of 23 complementation groups required for post-translational events in the yeast secretory pathwayNovick, P., Field, C. and Schekman, R.
Cell 21, 205-215, 1980
Order of events in the yeast secretory pathway.
Novick, P., Ferro, S. and Schekman, R.
Cell 25, 461-469, 1981
I have slightly cheated the system from the outset here but for me it is impossible to refer to one of these papers without the other. The 1980 paper provided the foundation for my interest in both membrane traffic but also more widely in molecular cell biology. It is one of the first studies that I encountered that enables us to begin to assign functions to individual components during complex cellular processes. The second (1981) paper has one of the best titles of any paper–following on from the pioneering (and Nobel prize-winning) work of Albert Claude, Christian de Duve, and George Palade, it does exactly what it says–defines the ordering of the secretory pathway using a series of temperature-sensitive yeast mutants. The amount of work that went in to being able to draw Figure 6 in the 1981 paper is astonishing. The simplicity of this figure reflects the importance of this work. It has taken many years of subsequent work to determine the molecular bases for the function of many of these mutants and yet the original sec mutant screen remains an astonishing accomplishment and provides the basis for much of my own lab’s research as well as a considerable amount of my teaching.

Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin.
Duden, R., Griffiths, G., Frank, R., Argos, P. and Kreis, T. E.
Cell 64, 649-665, 1991
This paper triggered an enormous amount of tutorial discussion when I was an undergraduate. I had the fortune of having an enthusiastic, entertaining, and well informed tutor–John Lagnado, now Honorary archivist at the Biochemical Society. I believe that John picked it out in order to start a discussion on the similarities and differences between distinct coat complexes that might operate during membrane trafficking. The fundamental questions in membrane trafficking that underpin my own work were sparked in no inconsiderable way by this paper–the concepts of selective recruitment of coats to different membranes, regulation of the stages of vesicle formation and cargo recognition have been a theme of my own work since. It also provided a great introduction to the work of Thomas Kreis.


Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER.
Lippincott-Schwartz, J., Yuan, L. C., Bonifacino, J. S. and Klausner, R. D.
Cell 56, 801-813, 1989
This is the paper that really reinforced the concept of dynamic cell biology to me. The ability to treat cells with pharmacological agents and then determine what had happened was something I really only knew in terms of basic biochemistry. I hadn’t given much thought to the morphology of, let alone the inter-relationships between, intracellular compartments. This paper spawned a whole series of related articles, one of which was to be a defining factor in me joining George Banting’s lab in January 2006 as a postdoc.


A mitotic form of the Golgi apparatus in HeLa cells.
Lucocq, J. M., Pryde, J. G., Berger, E. G. and Warren, G.
J. Cell Biol. 104, 865-874, 1987
The concept of changing organelle structure then sparked my interest in the fate of endomembranes during mitosis. This is where I came upon the beautifully detailed work of Graham Warren. This paper is one of the first I read in this area and forms a cornerstone of a series of papers that continues to inform my work to this day. This theme of mitotic inheritance of the Golgi of course became an intense debate in cell biology focused on a small number of labs, notably those of Warren and Lippincott-Schwartz. By the end of my PhD I was hooked on membrane traffic and so joined George Bantings’s lab in Bristol.

Tubulin dynamics in cultured mammalian cells.
Saxton, W. M., Stemple, D. L., Leslie, R. J., Salmon, E. D., Zavortink, M. and McIntosh, J. R.
J. Cell Biol. 99, 2175-2186, 1984
ER-to-Golgi transport vsualized in living cells.
Presley, J. F., Cole, N. B., Schroer, T. A., Hirschberg, K., Zaal, K. J. and Lippincott-Schwartz, J.
Nature 389, 81-85, 1997

I first became aware of the possibilities of live cell imaging from the pioneering work microinjecting fluorescently labelled proteins such as tubulin and actin. Papers such as Saxton et al., (1984) taught me a great lesson in reading not only the most recent but also the most relevant literature. These experiments provided keen insight into dynamic cell biological processes, but it was the ability to genetically encode a fluorescent marker that was a step change in my understanding of membrane traffic and organelle biology. The mid-1990s proved a transformative time for cell biology with the dawn of the GFP age. Like many, I was transfixed by the movies coming from this work. While the groundbreaking work of Shimomura, Chalfie, and Tsien led to the 2008 Nobel Prize for the identification and development of GFP as a probe, the work of et al., (1997) provides a fine example of the application of this technology that went on to direct my own work as a postdoc.


Visualization of ER-to-Golgi transport in living cells reveals a sequential mode of action for COPII and COPI.
Scales, S. J., Pepperkok, R. and Kreis, T. E.
Cell 90, 1137-1148, 1997

Choosing a postdoc lab is always hard but certain key papers provide a turning point. This one showcased to me the elegant and insightful experiments coming from the Kreis lab. The shockingly early death of Thomas Kreis in 1998 led to a change of plan, leading me instead to Rainer Pepperkok’s lab, newly established at the EMBL in Heidelberg. This paper, combining membrane dynamics with advanced light microscopy was a formative point in my time as a scientist and so has a very important place on a list such as this.


Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.
Klar, T. A., Jakobs, S., Dyba, M., Egner, A. and Hell, S. W.
Proc Natl Acad Sci U S A 97, 8206-8210, 2000

My exposure to the incredible environment of EMBL and the possibilities provided by fluorescence microscopy ensured that I was up-to-date with the latest developments in advanced cell imaging. This paper from Stefan Hell’s group in Gottingen was the first relating to super-resolution imaging that really showed me the possibilities of these long-explored approaches. This paper suggested to me the dawn of another age of work in my field–super-resolution imaging. This field has now exploded with commercial systems for quite astonishing techniques such as STED, PALM, STORM, and structured illumination providing great promise for the future of our work.


Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T.
Nature 411, 494-498, 2001

I cannot even begin to count the number of papers I have now read using RNA interference, and specifically small interfering RNAs. This paper coincided with me starting my own lab and very rapidly the possibilities for this approach in relation to my own work became abundantly clear. My knowledge of this technology came from this paper and the subsequent rapid translation of the possibility to actual knockdowns in my own lab (originally using in vitro synthesized RNAs thanks to an induction in the mechanics of this approach by Harry Mellor who was running the adjacent lab in Bristol). The potential of RNAi is of course astonishing but it is the power to learn about such things and apply them to ones’ own work in a short time that makes this one of the outstanding papers in my scientific lifetime.


Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility.
Vale, R. D., Reese, T. S. and Sheetz, M. P.
Cell 42, 39-50, 1985
Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein.Gill, S. R., Schroer, T. A., Szilak, I., Steuer, E. R., Sheetz, M. P. and Cleveland, D. W.
J. Cell Biol. 115, 1639-1650, 1991

The obvious aspect of dynamic cell biology is where the force comes from. How does one transduce the energy provided by ATP into force to move a vesicle, organelle or even a cell? The answer of course is (in the main) motor proteins. The discovery by the Sheetz lab of kinesin was transformative in this field. This was also the first of Ron Vale’s papers that I am aware of reading. I can’t think how many of his I have read since and I remain in awe of the astonishing output of his lab and, very much like Randy Schekman, the scientific family of former lab members that he has established around the world. The developments from this through the discovery of the many other members of the kinesin superfamily, the characterization of cytoplasmic dynein (to add to the wealth of literature on axonemal dynein) provided the basis for my own desire to understand more about how these motors could be coupled to membranes, and more specifically to the vesicle transport machinery.

The Gill paper was not the first to define an activator of dynein-based motility (it is in fact referred to as Activator I in Schroer, T. A., and Sheetz, M.P. (1991) J. Cell Biol. 115:1309-1318). However, it was the first to show the importance of this complex through identification of p150Glued as a major subunit and reinforces the importance of biochemistry to both discovery and understanding of function as well as the necessity to integrate multiple approaches (here biochemistry, molecular biology, and cell biology) to produce truly outstanding papers. The Glued mutant in flies had shown the importance of this subunit to multiple cellular processes. There is still significant debate as to the precise molecular role of dynactin but its importance in dynein-based motility is without question.


Kinesin and Dynein Move a Peroxisome in Vivo: A Tug-of-War or Coordinated Movement?
Kural, C., Kim, H., Syed, S., Goshima, G., Gelfand, V. I. and Selvin, P. R.
Science 308:1469-1472, 2005

I have chosen this paper as it is simply one of my favourites. It addresses, as many similar papers do, the fundamental question of bidirectional motility of cargo. Here, a relatively artificial system is used-–Drosophila cells, treated with cytochalasin D to enable simpler visualization of an organelle, the peroxisome. This combination allows incredibly accurate determination of the spatial position of the peroxisome (a reasonably round organelle of sub-resolution size) using 2D Gaussian fitting. This was the first paper that I read with nanometre tracking of organelles in live cells. This technique of FIONA–-Fluorescence Imaging with One Nanometer Accuracy–provided an outstanding example of the impact of physics on biology and of the genuine advantage that can be gained from such interdisciplinary working. While not the first to work in this way it is in my view one of the clearest papers of this type and, like many of those above, one that I refer to in my own lectures to our undergraduate students.


These are my own “personal best” papers:

This list includes the ones that gave me the greatest pleasure not only to publish but also to work on. There are several others which have a particular relevance for different reasons but these are the three that I have chosen as my “favourites”.

  1. Coupling of ER exit to microtubules through direct interaction of COPII with dynactin.
    Watson, P., Forster, R., Palmer, K.J., Pepperkok, R. and Stephens, D.J.
    Nature Cell Biology, 7 (1) 48-55, 2005This paper was essentially the culmination of my first three years of work as an independent investigator (as an MRC Career Development Fellow). In 2001, the core machinery of the COPII coat was well known and indeed had been reconstituted in vitro [1]. The aim of my lab was to identify any effectors or regulators of COPII function through direct interaction analyses. Many such components had already been described for the clathrin system. Our two hybrid based approach led to the usual multiple hits, the “winner” among them being the C-terminal domain of p150Glued. Our subsequent work overlapped well with an ongoing project in the Pepperkok lab where I had worked as a postdoc. Good relations and ongoing discussions led to us pooling our data to try and reach a higher level.A major feature of this work was to show that the inner layer of the COPII coat could act to recruit or retain a factor that clearly works downstream of the budding event. It also established a link between COPII vesicle budding and microtubules and hinted at the possibilities for functional coupling of the vesicle formation events to microtubule motors. This has led the lab to explore such events throughout the cell which has included some great collaboration with the Cullen lab on endosomal sorting [2, 3]. I am also fully aware how important this paper proved to be in my securing an MRC Senior Fellowship in the same year.


  2. Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development.
    Townley, A. K., Feng, Y., Schmidt, K., Carter, D. A., Porter, R., Verkade, P. and Stephens, D. J.
    J. Cell Sci. 121 3025-3034, 2008This is without question my favourite of the papers I have authored. This is in no small part because it includes such a diversity of techniques and really showcases what we can achieve with the Wolfson Bioimaging Facility in Bristol. Our experiments up to this point were largely using conventional molecular cell biology techniques to define the role of the outer layer of the COPII coat. Much to our surprise, secretion of small soluble/diffusible cargoes was unaffected. Instead, collagen secretion was blocked. We constructed a theory based around the need to physically encapsulate the large size of procollagen. This all took place around the same time that Boyadiev and colleagues defined the molecular defect in patients with cranio-facial-lenticulo dysplasia as a mutation in the inner layer COPII subunit, Sec23A [4]. This was recapitulated in a zebrafish mutant called crusher which resulted from a missense mutation in Sec23A [5]. All data indicated that these mutations led to a failure of Sec23 to correctly couple to Sec13-Sec31 (which was beautifully confirmed in two papers published in Developmental Cell [6, 7]). So, it seemed likely that suppression of Sec13 expression in zebrafish embryos should broadly recapitulate the craniofacial development defects of the crusher mutant.

    So, with no prior experience but outstanding help from Yi Feng in Paul Martin’s lab, we embarked on our first foray into zebrafish. It turned out that this was true and this added immensely to the understanding that we could derive from our studies. It is really the diversity of techniques that were new to my lab that marks this work out–transmission and scanning electron microscopy, zebrafish work, all combined with the more conventional cell imaging and biochemical approaches with which we were familiar. Pushing us all outside our comfort zone developed this work to a much higher level than we might otherwise have attained. A key lesson from this was always to develop new ways of working, in particular to ensure one applies the most appropriate approach to the problem at hand. It also underlines the great value in collaborating with friends and colleagues; this work would simply have not been possible without the help of the Verkade lab and Yi Feng from the Martin lab.

  3. Organization of human endoplasmic reticulum exit sites: requirements for the localization of Sec16 to transitional ER.
    Hughes, H., Budnik, A., Schmidt, K.J., Johnson, A., Noakes, C., Carter, D.A., Verkade, P., Watson, P., and Stephens, D.J.
    J. Cell Sci., 122:2924-2934, 2009This paper runs a close second in terms of favourites. It shows that Sec16 localizes adjacent to other COPII components, thereby suggesting its role as a scaffold for assembly of the COPII coat. It also serves as a reminder of the big contribution that lab members make whatever their career stage and the importance of teamwork to achieve at a higher level. This paper is slightly odd in that the first 3 authors contributed equally to this work–something that is very easy to justify when one considers the data. Noakes and Johnson were undergraduate students in the lab, while Pete Watson had recently left to establish his own lab in Cardiff.Another reason why this work stands out is that we were asked at the review stage to clarify membrane curvature–did Sec16 localize to concave or convex curved membranes? Previous work from the Balch lab in particular suggested concave sites would be more likely [8]. The reviewer suggested we might answer this either through super-resolution light microscopy or electron tomography. The latter was an in-house possibility and so we opted for that. An amazing effort by Katy Schmidt working with Judith Mantell in the EM Facility and Paul Verkade’s invaluable input produced a result that I did not think would be technically possible in the time–a 3D reconstruction of transitional ER that we could unequivocally identify because of immunogold labelling. Section thickness precluded penetration of the label and so the reconstructed membranes were traced from expose parts of the section accessible to the antibody label. While a serious challenge, we did at least address the reviewer’s point and the paper is undoubtedly better for it.

    This work, and the second paper in this list, really showcase our work at the highest level. I am immensely proud of both papers–not only for the impact of the findings but for the quality of the imaging. It gives current and future lab members a lot to live up to! Both also demonstrate vividly the importance of electron microscopy to our work–something that some colleagues will appreciate I had been trying to do for some time.

References

  1. Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S.Y., Hamamoto, S., Schekman, R., and Yeung, T. (1998). COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell 93, 263-275.
  2. Traer, C.J., Rutherford, A.C., Palmer, K.J., Wassmer, T., Oakley, J., Attar, N., Carlton, J.G., Kremerskothen, J., Stephens, D.J., and Cullen, P.J. (2007). SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment. Nat. Cell Biol. 9, 1370-1380.
  3. Wassmer, T., Attar, N., Harterink, M., van Weering, J.R., Traer, C.J., Oakley, J., Goud, B., Stephens, D.J., Verkade, P., Korswagen, H.C., et al. (2009). The retromer coat complex coordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Dev. Cell 17, 110-122.
  4. Boyadjiev, S.A., Fromme, J.C., Ben, J., Chong, S.S., Nauta, C., Hur, D.J., Zhang, G., Hamamoto, S., Schekman, R., Ravazzola, M., et al. (2006). Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking. Nat. Genet. 38, 1192-1197.
  5. Lang, M.R., Lapierre, L.A., Frotscher, M., Goldenring, J.R., and Knapik, E.W. (2006). Secretory COPII coat component Sec23a is essential for craniofacial chondrocyte maturation. Nat. Genet. 38, 1198-1203.
  6. Bi, X., Mancias, J.D., and Goldberg, J. (2007). Insights into COPII Coat Nucleation from the Structure of Sec23*Sar1 Complexed with the Active Fragment of Sec31. Dev. Cell 13, 635-645.
  7. Fromme, J.C., Ravazzola, M., Hamamoto, S., Al-Balwi, M., Eyaid, W., Boyadjiev, S.A., Cosson, P., Schekman, R., and Orci, L. (2007). The Genetic Basis of a Craniofacial Disease Provides Insight into COPII Coat Assembly. Dev. Cell 13, 623-634.
  8. Bannykh, S.I., Rowe, T., and Balch, W.E. (1996). The organization of endoplasmic reticulum export complexes. J. Cell Biol. 135, 19-35.
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