The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes - PubMed (original) (raw)

The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes

Luca Jahreiss et al. Traffic. 2008 Apr.

Abstract

Macroautophagy, a constitutive process in higher eukaryotic cells, mediates degradation of many long-lived proteins and organelles. The actual events occurring during the process in the dynamic system of a living cell have never been thoroughly investigated. We aimed to develop a live-cell assay in which to follow the complete itinerary of an autophagosome. Our experiments show that autophagosomes are formed randomly in peripheral regions of the cell. They then move bidirectionally along microtubules, accumulating at the microtubule-organizing centre, in a similar way to lysosomes. Their centripetal movement is dependent on the motor protein dynein and is important for their fusion with lysosomes. Initially, autophagosomes dock on to lysosomes, independent of lysosomal acidification. Two kinds of fusion then occur: complete fusions, creating a hybrid organelle, or more often kiss-and-run fusions, i.e. transfer of some content while still maintaining two separate vesicles. Surprisingly, the autophagolysosomal compartment seems to be more long lived than expected. Our study documents many aspects of autophagosome behaviour, adding to our understanding of the mechanism and control of autophagy. Indeed, although the formation of autophagosomes is completely different from any other vesicular structures, their later itinerary appears to be very similar to those of other trafficking pathways.

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Figures

Figure 1. Autophagosomes are formed randomly throughout the cytosol upon serum starvation

A) Location of initial autophagosomes (GFP–LC3-positive vesicles) and of newly appearing autophagosomes (see arrowheads for location of their appearance) during a 5-min video. B) Distance of initial and newly appearing autophagosomes from the MTOC of three different cells after 30- to 120-min starvation in serum-free CO2-independent medium (black bar indicates mean distance of autophagosomes from the MTOC, and plus sign indicates mean distance from the MTOC to the cell periphery). AP, autophagosome. Scale bar represents 10 μm.

Figure 2. A high proportion of autophagosomes are actually autophagolysosomes

A) Cells transfected with mCherry–LC3 and lgp120–GFP show a high degree of colocalization. B) Colocalization of mCherry–LC3 with lgp120 is somewhat higher than colocalization of GFP–LC3 with lgp120. Scale bars represent 10 μm.

Figure 3. lgp120/LC3 colocalization correlates with level of autophagic activity

A) Dimethyl sulphoxide (DMSO)-treated control with some colocalization (see magnification and arrowheads). B) Twenty-four hours rapamycin (Rap) treated with high level of colocalization (see magnification and arrowheads). C) Twenty-four hours bafilomycin (Baf) treated with low colocalization (see magnification and arrowheads). D) Quantification of percentage of double-labelled vesicles (mean of three experiments of four to five cells each, mean standard deviations; _t_-test: p (DMSO, Rap) = 0.008, p (DMSO, Baf) = 0.001). E) Quantification of colocalization of cells transfected with mCherry–LC3 and lgp120–GFP and stained for CIMPR. Cells were treated with 400 n

bafilomycin for 24 h (20 cells each, bars represent means with standard deviations, p (LC3+/CIMPR+/lgp120+) = 6 × 10−12, p (LC3+/CIMPR−/lgp120+) = 1 × 10−7). Scale bars represent 10 μm.

Figure 4. Autophagosomes engage in different types of both homotypic and heterotypic fusions

A) Homotypic fusion between two GFP–LC3-positive autophagosomes (arrowhead). Scale bar represents 3 μm. B) Homotypic fusion through membrane protrusion (arrowhead). Scale bar represents 3 μm. C) Complete autophagosome–lysosome fusion resulting in colocalization of mCherry–LC3 and lgp120–GFP in the hybrid organelle (arrowheads). For better visualization of double labelling, images were split into two separate channels (red and green) while using false colour for a better measure of intensity (blue < green < yellow < red). When observing an autophagosome interacting with a lysosome, membrane content exchange would be visible as an increase in intensity in the red channel at the location of the lysosome and vice versa. Scale bar represents 10 μm. D) Kiss-and-run fusion event resulting in some mCherry–LC3 inside lgp120–GFP-positive vesicle (arrowheads). Scale bar represents 10 μm.

Figure 5. Docking and fusion are two independent steps in the maturation of the autophagosome

A) Low LC3/lgp120 colocalization in cell treated with bafilomycin for 24 h. B) Enlargement of area in (A); autophagosome docked onto lysosome (arrowheads) with common movement, but no fusion (see separate intensities and Figure 4). Scale bars represent 10 μm. These data are representative of five independent experiments.

Figure 6. Autophagosomes move bidirectionally towards and away from the nucleus dependent on microtubules

A) Autophagosomes are distributed with a bias towards the MTOC. Scale bar represents 10 μm. B) Fast autophagosome movement towards the nucleus (N) (arrowheads). Scale bar represents 5 μm. C) Fast autophagosome movement away from the nucleus (N) (arrowheads). Scale bar represents 5 μm. D) Fast movements with a velocity above 0.1 μm/second are abolished by microtubule depolymerization by nocodazole. E) Microtubule-dependent movements (i.e. velocity ≥ 0.1 μm/second) are biased towards the nucleus [p = 0.02 (chi-squared test), cumulative result from six cells, 10 vesicles each].

Figure 7. Dynein mediates the movement of autophagosomes and autophagolysosomes towards the nucleus

A) Reduction in fast movements upon dynein inhibition (n = 6; Mann–Whitney _U_-test: p (EHNA, PBS) = 0.02). Six cells were analyzed in detail. In each cell, we determined the distribution of single movements in between each frame (11.5 seconds per frame) for 10 autophagosomes (any LC3-positive vesicle) per cell. Autophagosomes were tracked for as long as they were visible, and all their movements were recorded. As mentioned in the text, the same autophagosome can show both fast movements and periods of immobility during the observation period. The averaged distributions of movements per frame of fast movements (as characterized in Figure 6) over the six cells are shown. B) Data from (A) were analyzed as follows. For each frame, we determined if the direction of the movement was towards or away from the nucleus. The numbers were recorded for each cell, and the means and standard deviations for the six cells analyzed are shown. Dynein inhibition by EHNA decreases movement towards nucleus more strongly [n = 6; _t_-test: p (towards) = 0.02, p (away) = 0.08)]. Towards the nucleus (Towards), away from the nucleus (Away). C and D) Dynein knockdown decreases paths of autophagosomes (AP, LC3+/lgp120−) (C) and autophagolysosomes (APLS, LC3+/lgp120+) (D) towards the nucleus. The path of an autophagosome was defined as the distance covered from the beginning of tracking till the end, with the associated direction. For standardization between different vesicles and cells, these values were then divided by the duration of tracking and the mean diameter of the cell (the mean diameter of an ellipse approximating the cell shape). E) Dynein knockdown significantly changes the bias in direction of paths [defined in (D) above] from towards the nucleus to away from the nucleus (pooled data from 45 vesicles [three experiments, three cells each, five vesicles each), p (AP) = 0.00005 (chi-squared test), p (APLS) = 0.003 (chi-squared test)]. F) Efficient knockdown of dynein heavy chain [because of the high molecular weight of DHC (532 kD), endogenous wild-type huntingtin (343 kD) was used as a loading control].

Figure 8. Dynein knockdown redistributes autophagosomes towards the periphery and decreases autophagosome–lysosome fusion

A) Mostly perinuclear localization of autophagosomes/autophagolysosomes in control cells. B) Redistribution of autophagosomes/autophagolysosomes towards the periphery with a concomitant reduction in autophagosome–lysosome fusion (see magnification). C) Quantification of reduction in colocalization of LC3 with lgp120 [three experiments with 20 cells each, p < 0.0001 (odds ratio)]. D) Quantification of colocalization of cells transfected with control (Ctrl) or DHC siRNA, mCherry–LC3 and lgp120–GFP and stained for CIMPR (20 cells each, bars represent means with standard deviations, p (LC3+/CIMPR+/lgp120+) = 4 × 10−6, p (LC3+/CIMPR−/lgp120+) = 3 × 10−5). Scale bars represent 10 μm.

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The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes - PubMed (original) (raw)