The phagophore in four dimensions—a study in wood

ABSTRACT

One of the key features of macroautophagy/autophagy is the dynamic nature of the membrane rearrangements that take place during expansion of the phagophore, the sequestering compartment that matures into an autophagosome. There are various ways to depict this process, but in most cases the method ultimately relies on a two-dimensional medium. Most people working in the field of autophagy realize that the typical ‘C’-shaped drawing of a phagophore is meant to represent a cup- or bowl-like structure that exists in the cell in 3 dimensions. However, explaining this concept to a lay person often leads to confusion and misinterpretation. Accordingly, we decided to generate a four-dimensional version of the expanding phagophore as a wood sculpture, that depicts this transient compartment in 3 dimensions over time.

Abbreviations: ER: endoplasmic reticulum

Introduction

One image that is quite compelling, even though it is nearly 2 decades since its first appearance, is the video generated by Noboru Mizushima that monitors GFP-ATG5/APG5 by time lapse fluorescence microscopy [1]. One interpretation of the video is that the phagophore expands sequentially and then just prior to, or upon, completion GFP-ATG5 is released from the membrane; the latter fits with the observation that most ATG proteins do not remain associated with the completed autophagosome. The mechanism of autophagosome formation remains unclear, but one aspect that does not appear to be controversial is that the sequestering vesicle does not form in one step from a pre-existing single piece of membrane. Rather, the phagophore expands, and this process is one key element to autophagic function; the phagophore can sequester cargo of various sizes.

Although the origin of the membrane(s) that forms the phagophore is not established, several studies support the concept of sequential expansion. For example, three-dimensional electron tomography suggests that at least in some circumstances the phagophore is connected with, and expands from, the endoplasmic reticulum (ER) [2,3]. In this case, the model is not that a portion of the ER pinches off to generate an autophagosome, but rather that membrane is extruded from the ER, via an omegasome [4]. A role for the ER in supplying membrane to the autophagosome seems extremely likely, and this source has been proposed for several decades, starting with morphological studies in mammalian cells, and continuing with molecular genetic analyses in yeast [2,5–7]. Other reports, however, suggest that the ER is not the sole source of the autophagosome membrane. Interestingly, morphological analyses in mammalian cells suggest multiple donor sources [8], and more recent molecular studies now lend support to this idea. For example, the ER-Golgi intermediate compartment [9], the Golgi complex [10], trans-Golgi network [11,12], mitochondria [13], perivacuolar mitochondrial reservoirs [14], the plasma membrane [15], recycling endosomes [16,17], and various ER-organelle contact sites [18] may all contribute to phagophore expansion.

Regardless of the donor membrane and the exact mechanism of autophagosome formation, it is difficult to convey the nature of the expansion process with two-dimensional drawings. Accordingly, we attempted to create a sculpture in wood that illustrates this key aspect of macroautophpagy—the expanding phagophore. The sculpture depicts the phagophore in three dimensions, and also as it changes/expands over time, hence the four-dimensional aspect.

Results

Daniel J. Klionsky (DJK): I first observed your sculptures at the Ann Arbor Street Art Fair. Something about the wooden vessels (I hesitate to refer to them simply as ‘bowls’) you had on display made me think about the phagophore. I imagine you were surprised when I proposed ‘The Expanding Phagophore’ project and showed you the cover from the 4 November 2004 issue of Science that has a three-dimensional computer-generated image of macroautophagy (Figure 1). What thoughts did you have in contemplating how to design this piece?

Figure 1.

A detail from the cover of science, Vol. 306, No. 5698. Design by D.J. Klionsky and B.A. Rafferty, computer modeling and rendering by B.A. Rafferty.

John C. Sewell (JCS): I thought about the project for a while, and came up with a plan. As I reviewed the images in the material you gave me, I became more aware of the ‘grain pattern’ formed by the rows and layers of the little structures [the phospholipids] that make up the walls of the bodies we want to illustrate. Certain orientations of wood grain pattern would offer a very similar result in a carving of any of the three forms that you proposed. At times in the past I had used a different method of surface treatment from that for any of the pieces that you saw. Rather than lacquered surfaces like those on the pieces in my display booth, I accentuate the grain pattern more by burning and then sandblasting the surface, both inside and out. The grain pattern becomes more visible and more tactile, as the softer grain (every other row) is removed to a greater depth, leaving the harder rows raised to the touch. The insides of my vessels at the show were treated in this way, but were then darkened to black with the addition of coats of clear lacquer. I could also ‘pickle’ the surface, adding white, or some light color, to the soft grain and leaving the hard grain darker and more defined with the addition of clear lacquer. Whether left raw or pickled, treating the wood surface in this manner would help to emphasize the continuity of the rows of autophagosomal wall components over the rim of the vessel into the interior surface.

DJK: Can you describe in a little more detail how you went about making these pieces?

JCS: I usually carve such forms from a log with a maximum diameter only slightly larger than the diameter of the finished sculpture. In this case, however, I chose my largest logs of catalpa from which to produce these pieces. Each measured well over two feet in diameter. Doing this gave me grain lines that were less curved, thus more accurately portraying the rows of structures that make up the walls of the phagophore. It made for a more complex project, but I believe the results warrant the choice. So, instead of just rounding off the corners of a log to get a rounded shape, I excavated the relatively small bowl form from a large block of wood. I spent quite a bit of time studying and measuring the logs to decide where to locate the bowls before ever making a cut.

DJK: Going back to the methods for finishing the work, can you explain exactly what you mean by ‘burning’ the surface?

JCS: The burning of the surface of the sculptures is part of the process required to produce the surface I described. This generates dark lines that contrast with alternating lighter lines (Figure 2). The grain lines are definitely brought out by this process, not hidden by black. I then sand blast away the charcoal. This leaves a raised pattern of every other wood grain ring. These raised lines represented the tree growth for the summer of each year of the tree’s life, and are the densest material in the tree. The wood growth during the spring is more porous for carrying water, so it burns out more deeply, leaving the texture.

Figure 2.

The expanding phagophore. Design and construction by John C. Sewell. Photograph by Deborah Gill.

Apparently because of its being from the large trunk logs and having such comparatively wide rings of summer growth, the wood surface held onto more dark color than I expected; however, I think it is appropriate for the bodies that we’re portraying. I think the narrow light lines better define the division between the rows of bodies that make up the double walls of the phagophore.

DJK: Unlike standard bowls, the phagophore would need to be suspended. Can you describe your approach to placing the three intermediate structures on a stand?

JCS: I often use non-ferrous metals in combination with the wood in my sculptures. To suspend the forms, I decided to make a stand for each from brass rod. They can be polished to a bright gold color, but I decided to keep them with the dark natural patina that they already had; I was afraid that the bright color of the brass would be too distracting and would take away from the sense that the bodies are in suspension. Initially, I had planned to make a separate stand for each; however, when you mentioned a single stand for all three, that idea sounded better to me, so that’s what I did. It was more of a project, but the result is much better than with the former plan. It really looks like a sculpture presentation. It is also more functional. Each body can be easily rotated or removed completely for examination without disturbing the base, and then returned to its position on the base with reasonable ease. I used your suggestion, and created a curved one-piece wood base. Though it complements the three sculptures and does not compete, it does have a sculptural quality of its own. And, the curve does enhance a sense of movement in the phagophores and a flow from one growth stage to the next.

The bodies are mounted with the alignment through the center of each one. They each have a socket in the bottom to receive the end of the 3/4”-diameter mounting rod (pipe) that is set at 90 degrees to the base. When mounted, they stand ‘suspended’ well above the base. It was difficult to get a good photo to show the complete piece without something getting distorted due to the perspective. For example, the support rods look a little splayed, but they are actually all mounted at 90 degrees to the base. And the base looks smaller than it is from the overhead perspective. The base is 33 inches long, and the maximum height is 22 inches.

Discussion

DJK: Now that you have completed this project, would you ever take on something like this again?

JCS: Yes. It was an interesting, stimulating, and challenging project. These are all ingredients that I like to have included in my work. It was nice to assimilate a better understanding of this ‘smaller’ part of our world, in the process of my studying and developing my impression of these autophagic structures.

Funding Statement

This work was supported by the National Institute of General Medical Sciences [GM053396].

Disclosure statement

No potential conflict of interest was reported by the authors.