Biologists are passionate about papers. Here we ask an ASCB member to pick two journal articles that were important, either personally or scientifically, and to answer—briefly and informally—three questions: Why did you pick this paper? What's it about? What does it mean to you?
Elisa M. Konieczko is at Gannon University in Erie, PA, and a member of the ASCB Public Information Committee.
"A technique that was central to my thesis work"
Janet M. Larkin, Michael S. Brown, Joseph L. Goldstein, and Richard G.W. Anderson. 1983. Depletion of Intracellular Potassium Arrests Coated Pit Formation and Receptor-Mediated Endocytosis in Fibroblasts. Cell. 33:273-285
1) This paper described a technique that was central to my thesis work. It was easy to learn, simple, dramatic in its effect on cells and reversible. I used this technique to examine the entry of various molecules, including vesicular stomatitis virus, in two different cell lines, one of which was susceptible to the virus and one of which was not. The posters that I presented at ASCB meetings using this technique generated great interest and many questions from meeting attendees. These posters were among the most visited of my graduate career.
2) Larkin et. al. developed a technique to drastically reduce receptor-mediated endocytosis (RME) in mammalian cells by depleting cells of intracellular potassium (K+). The depletion could be achieved slowly or rapidly and was completely reversible. Depletion of intracellular K+ was achieved slowly by incubating cells for 3 hours in an isotonic K+ free buffer, or rapidly by incubating cells for 5 min with hypotonic medium, followed by a 30 min incubation in an isotonic K+ free buffer. Once K+ levels in cells had been depleted by ~60 %, RME of various ligands was reduced by 70 to 95 %. The technique did not affect the binding of the ligand to the receptor, or disrupt the structure of intracellular organelles. However, both the number of clathrin coated pits and the rate of RME were markedly decreased. Addition of KCl to the culture medium restored K+ levels and RME to normal.
3) Prior to this paper, the most common technique to inhibit receptor-mediated endocytosis was to reduce the temperature of cells in culture to 4 °C. It was also possible to disrupt RME by treating cells with chemicals, such as oubain. However, the use of chemicals often disrupted many cellular processes and/or organelles, and often was not reversible. Larkin and her colleagues created a simple, reversible, and highly effective technique that could be used to study any molecule that was internalized by coated pits and RME.
"I use this paper ... to demonstrate how a concept [students] take for granted is, in fact, something that was developed in their lifetime."
Thomas Söllner, Sidney W. Whiteheart, Michael Brunner, Hediye Erdjument-Bromage, Scott Germanos, Paul Tempst, and James E. Rothman. 1993. SNAP Receptors Implicated in Vesicle Targeting and Fusion. Nature. 362:318-324.
1) This paper was published during my first postdoctoral fellowship. It sparked many energetic discussions in the departmental labs where I worked. It was quickly added to graduate seminars and the departmental journal club. The experiments presented in the paper were elegant and clear. More importantly, the authors used this data to propose a model that explained vesicle to target specificity of intracellular membrane fusion. That model, which described v-SNARES and t-SNARES, is now widely accepted and a central dogma of intracellular membrane traffic taught in cell biology classes and described in cell biology textbooks. This paper was also cited by the Nobel Foundation in awarding Jim Rothman the 2013 Nobel Prize in Medicine or Physiology.
2) Söllner et. al. isolated membrane vesicles from bovine brains containing N-ethylmaleimide-sensitive fusion protein (NSF) and NSF attachment proteins (SNAPs), and used an affinity purification procedure based on the natural binding properties of these proteins to isolate the target proteins, SNAP receptors (SNARES). These SNARE proteins were sequenced and identified as syntaxin B, syntaxin A, SNAP-25, and VAMP/synaptobrevin-3. The authors incorporated SNAP-25, NSF, and other components into 20S fusion particles, demonstrating that they identified SNAREs on the basis of their function. The final figure of the paper presents a model demonstrating how transport vesicles containing transmembrane v-SNARE proteins can attach to the t-SNARES on the target membranes through a 20S particle.
3) The experimental data here and the model proposed by Söllner and colleagues provided an elegant, logical, and unified explanation for the mechanism by which different types of intracellular vesicles generated by the cell could bind specifically to their correct target membrane. The experimental procedures used in this paper have been used by numerous other investigators to characterize many other v- and t-SNARE proteins. Additionally, the model presented in this paper has been experimentally confirmed in many different cell types and systems. I teach Cell Biology to undergraduates and many of them come into the class thinking that the material presented in textbooks has been known for many decades or longer. I use this paper by Söllner et. al. to demonstrate how a concept they take for granted is, in fact, something that was developed in their lifetime.