Simultaneous live-imaging of peroxisomes and the ER in plant cells suggests contiguity but no luminal continuity between the two organelles

Abstract

Transmission electron micrographs of peroxisomes in diverse organisms, including plants, suggest their close association and even luminal connectivity with the endoplasmic reticulum (ER). After several decades of debate de novo peroxisome biogenesis from the ER is strongly favored in yeasts and mammals. Unfortunately many of the proteins whose transit through the ER constitutes a major evidence for peroxisome biogenesis from the ER do not exhibit a similar localization in plants. Consequently, at best the ER acts as a membrane source for peroxisome in plants. However, in addition to their de novo biogenesis from the ER an increase in peroxisome numbers also occurs through fission of existing peroxisomes. In recent years live-imaging has been used to visualize peroxisomes and the ER but the precise spatio-temporal relationship between the two organelles has not been well-explored. Here we present our assessment of the peroxisome-ER relationship through imaging of living Arabidopsis thaliana plants simultaneously expressing different color combinations of fluorescent proteins targeted to both organelles. Our observations on double transgenic wild type and a drp3a/apm1 mutant Arabidopsis plants suggest strong correlations between the dynamic behavior of peroxisomes and the neighboring ER. Although peroxisomes and ER are closely aligned there appears to be no luminal continuity between the two. Similarly, differentially colored elongated peroxisomes of a drp3a mutant expressing a photoconvertible peroxisomal matrix protein are unable to fuse and share luminal protein despite considerable intermingling. Substantiation of our observations is suggested through 3D iso-surface rendering of image stacks, which shows closed ended peroxisomes enmeshed among ER tubules possibly through membrane contact sites (MCS). Our observations support the idea that increase in peroxisome numbers in a plant cell occurs mainly through the fission of existing peroxisomes in an ER aided manner.

Keywords: ER; EosFP; drp3a; live-imaging; membrane-contact-sites; peroxisome.

Figure 1

Representative images of cells from double transgenic Arabidopsis plants expressing peroxisome and ER-targeted probes that suggest correlative behavior of both organelles. (A) Twenty-two time-lapse images that follows a single YFP-PTS1 highlighted peroxisome (with red dot) against the backdrop of green fluorescent ER (Movie S1). (B) A merge of all the frames from “A” traces the path of the single peroxisome. Every frame capturing the peroxisome movement has an accompanying subtle change in the subtending ER. (C) The movement per frame in the time-lapse image sequence in “A” and the path seen in “B” displayed as distance moved in μm shows the erratic nature of peroxisome motility. (D) Movement of six different peroxisomes followed over 120 s shows the range of variation in their rate of movement and suggests that no two peroxisomes move at the same rate or for the same distance. (E) The movement of three peroxisomes (a, b, and c) tracked along with changes in the organization of neighboring ER tubules over 13 sequential images from Movie S2. Frames are separated by ca. 8 s. In contrast the peroxisomes “a” and “b” exhibit oscillations ranging between 2 and 8μm (frames 1–8) alongside short ER-tubules that extend and retract from an ER island. Arrows in frames 6–9 point to the movement of peroxisome “b” wherein the ER tubule harboring it fuses with another tubule to create the familiar ER polygon, whereupon the peroxisome moves again in the pattern defined by the newly organized polygon (frames 7–10). Finally the oscillating peroxisomes “a” and “b” are drawn into a cytoplasmic ER strand and move away rapidly from their previous locations (frames 9–13). Peroxisome “c” shows the least movement (white line across the 13 frames) and remains lodged on a broad patch of ER membrane. An ER body has been outlined in red (bottom half of frames) to provide a comparative estimation of ER reorganization in another area of the cell. Size bars = 2.5μm.

Figure 2

Concomitant changes take place in peroxisome and ER motility. (A) Graphical representation of changes in motility of 10 peroxisomes over 12 min as the ambient temperature around cells in 4°C treated seedlings rises to about 23°C. At 3 min following exposure to room temperature peroxisomes begin to exhibit short oscillations (double sided arrows) in synchrony with extension-retraction of ER tubules. The range of movement increases over time (6- and 9-min time points) until by 12 min both the ER and peroxisomes exhibit normal motility including long saltations. (B) Both peroxisomes and the ER in cells treated with 1μM latrunculin-B stop moving and large blobs of ER (^*) surrounded by peroxisomes start appearing. (C) Sequential images taken at intervals of after washing away latrunculin-B show the gradual recovery of the ER accompanied by the circumambulation of peroxisomes apparently embedded in the ER (arrowhead in panel 8; path shown by circular arrows in panel 10) during the first 3–4 min. (D) Treatment with lat-B for more than 10 min usually leads to the formation of large ER globules (^*) surrounded by static peroxisomes. Such disorganized ER does not reorganize easily into normal cytoplasmic streaming of organelles. Size bars = 5μm. ^*indicates an ER globule; arrowheads point to peroxisomes.

Figure 3

The dynamic behavior of tubular green fluorescent protein highlighted peroxisomes in the apm1-1 mutant and differential coloring of peroxisomes using a green to red photoconvertible mEosFP. (A) GFP-highlighted abnormally elongated peroxisomes in the apm1-1 mutant organize into random shapes including open and closed polygons (sequential frames 1–4) within min. (B) A single tubular peroxisomes in the apm1-1 morphs sequentially (1–4) into a closed polygon that is highly reminiscent of shapes presented by polygons making up the cortical ER mesh. (C) The color of peroxisomes can be changed rapidly from green to red by using the photoconvertible mEos fluorescent protein and irradiating the organelles with violet-blue light. (D) A time-lapse image sequence shows the intermingling and subsequent separation of green (non-photo-converted) and red (photo-converted) tubular peroxisomes in a drp3-3 mutant line. The bottom left (Panel 1) shows laterally aligned green and red tubules. In subsequent panels 2–7 the green tubules glides over the red one until the two separate (panel 7). Arrowheads in panels 2–6 shows areas of overlap suggesting close interaction between the tubules. Note that the possible interactions do not appear to result in any exchange of fluorescent proteins. Other smaller tubules morph continuously, seem to interact transiently (panel 3), before separating but do not exchange fluorescent protein either. Scale bar: (A,B,D) = 5μm; (C) = 2.5μm.

Figure 4

Confocal visualization of green fluorescent tubular peroxisomes and red fluorescent luminal ER in living hypocotyl cells of the apm1-1 mutant of Arabidopsis. (A) Representative frames from a time lapse image sequence (Movie S3) showing the correlated behavior of red fluorescent cortical ER tubules around a tubular (green) peroxisome. The tubular peroxisomes lie in an ER lined channel and tubule extension and retraction (frames 2–5), formation of incomplete as well as complete polygonal arrangements (frames 6–8), appear to be defined by the surrounding ER (for animation see Movie S3). (B) The behavior of three peroxisome clusters and contiguous ER shows how tubular peroxisomes such as “a” undergo considerable contortions (frames 2, 6–7) while remaining confined to a small region of the ER. During the same period another elongated peroxisome “b” moves forward, retracts and moves again (arrows in green panels) along an ER strand. Note changes in ER organization concomitant with changes in peroxisome behavior (Movie S4). (C) A time-lapse sequence showing changes in the morphology of a tubular peroxisome (arrowhead frame 1) due to wrapping (frames 1, 2) and unfolding (frames 3–5) around a spindle shaped ER body and other neighboring ER tubules (see Movie S5). (D) Representative sequential images from a time-lapse series showing a tubular peroxisome (frame 1) extending over several ER polygons breaking (frames 2–3) and being pulled apart (frames 4–7) through the reorganization of its neighboring ER. Scale bars = 10μm.

Figure 5

Iso-surface rendering of confocal image stacks depicting the relationship between tubular peroxisomes and the neighboring ER tubules in the apm1-1 mutant of Arabidopsis. (A) A region of the cell showing a tubular peroxisome (panels 1, 3) extended within an ER-lined channel (panel 2). Arrowheads point to potential membrane contact sites. Scale bar = 5μm. (B) A volume rendered stack of 12 images suggests that the tubular peroxisomes in the apm1-1 mutant are enmeshed and embedded in the ER. Changes in ER organization would be expected to create similar alterations in the morphology of the associated peroxisomal tubule. (C) Several tubules threaded between the tubules making u the cortical ER. Note that while some areas of the ER overlap regions of the tubule the tubular peroxisomes can also surround portions of the ER (e.g., arrowhead). (D). A tubular peroxisome interwoven in the cortical ER mesh. Note embedded chloroplasts (blue; outlined square) and the possibility of finding membrane contact sites (arrowheads).