Isolation of magnetic early and late endocytic compartments
A recent report has shown that negatively charged iron oxide nanoparticles bind to the surface of HeLa cells at 4°C and are subsequently internalized into the cells (27). Depending upon the duration of internalization, after binding at 4°C, the magnetic nanoparticles appear in compartments, presenting specific proteins of early or late endosomes. After 30 min of internalization, the magnetic particles colocalized with early endosome antigen-1 (EEA1) an effector of Rab5 that is associated with early endosomes (Figure 1A,B) and after 90 min with lysosome-associated membrane protein-1 (Lamp-1) that is associated with late endosomes and lysosomes (Figure 1C,D). The magnetic nanoparticles do not interfere with the morphology of the endosomes (27). Wilhelm et al. observed them in clathrin-coated endosomes after 10 min of chase at 37°C and in multivesicular endosomes after 60 min of chase. They do not affect the transport of cargo: internalized BSA was observed in late endocytic and lysosomal compartments after 90 min whether in the presence or absence of magnetic nanoparticles (unpublished data). Furthermore, the organelles loaded with magnetic nanoparticles acquire magnetic properties. Endocytic compartments loaded with magnetic nanoparticles are oriented in the magnetic field when HeLa cells were maintained in a magnetic field during fixation (Figure 1, see arrows).
Figure 1. Depending upon the time of their internalization, magnetic nanoparticles colocalized with early or late endosomes. HeLa cells were allowed to bind magnetic nanoparticles 1 h at 4°C. After washing, magnetic nanoparticles have been internalized at 37°C for 30 or 90 min. The cells were then incubated in a magnetic field for 20 min prior to being fixed under the magnetic field. The cells that have internalized the magnetic nanoparticles during 30 min (A and B) or 90 min (C and D) were observed by phase-contrast microscopy (A and C) or processed for IF with an anti-EEA1 antibody (B) or with an anti-Lamp-1 antibody (D) and imaged by confocal microscopy. Average projected Z stacks are shown. The black arrows in (A) and (C) represent the orientation of the magnetic field. Note that black dots in (A) and (C) that correspond to compartments loaded with magnetic nanoparticles are aligned in the magnetic field two-by-two in (A) or more in (B) (see small white arrows). The so formed chains of magnetic compartments codistribute with EEA1 or Lamp-1-labeled compartments in (B) and (D), respectively. Bar represents 3 μm.
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We therefore attempted to separate endosomes loaded with magnetic nanoparticles for various times from the nonmagnetic organelles after fractionation of HeLa cells. The purity of the membrane fraction isolated on the magnet was assessed by biochemistry, immunofluorescence (IF) and electron microscopy (EM). Depending upon the duration of internalization, the membrane fractions were enriched either in two specific markers of early endosomes, Rab5 and EEA1, or in two specific markers of late endocytic and lysosomal compartments, Lamp-1 and Rab7 (Figure 2A,B). After internalization for 15–45 min, the magnetic membrane fractions were enriched in Rab5, maximum enrichment being detected after 30 min internalization (Figure 2A,B). Early endosome antigen-1 was also detected in these fractions, and maximum enrichment of this marker was seen after 30–45 min of internalization (Figure 2A,B). By contrast, Rab7 and Lamp-1 were detected at maximal levels in the membrane fractions isolated after 60–180 min internalization of the magnetic nanoparticles (Figure 2A,B).
Figure 2. Biochemical and structural characterization of magnetic endosomes. A) HeLa cells were allowed to bind magnetic nanoparticles for 1 h at 4°C and, after washing, to internalize them at 37°C for periods ranging from 15 to 180 min, as indicated. The cells were then washed and homogenized, and the MF was isolated as described in the Materials and Methods. Forty micrograms of proteins for each of PNS, NMF and MF was analyzed by immunoblotting with antibodies against early endosomal markers Rab5 and EEA1 and late endosome/lysosome markers Rab7 and Lamp-1. B) The amounts of Rab5, Rab7, EEA1 and Lamp-1 detected in 40 μg of proteins of the MF were quantified as described in Materials and Methods and normalized to the amount detected in 40 μg of proteins of PNS. The means of three independent experiments are shown, and error bars indicate the standard deviation. C) Electron micrographs of the MFs isolated after 30 and 90 min internalization of the magnetic nanoparticles. D) Forty micrograms of proteins from the PNS, NMF and MF isolated after 30 (30 min) or 90 min of internalization (90 min), as indicated, was analyzed by immunoblotting with mouse anti-EEA1, mouse anti-Lamp-1, rabbit anti-ER and rabbit anti-GMAP210 antibodies. The polyclonal rabbit anti-ER serum contains several immunoglobulin-G that recognizes several proteins from the ER (54). Only one of these proteins that has an apparent molecular weight of 66 kD is shown here. E–L) The MFs isolated after 30 and 90 min internalization of the magnetic nanoparticles have been observed by phase-contrast microscopy (E, I, G and K) or labeled with anti-EEA1 (F and J) or anti-Lamp-1 antibodies (H and L) after injection in a flow cell. Arrowheads mark isolated endosomes, and arrows mark aggregates. Bars represent 9.7 μm.
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We further compared the magnetic fraction isolated after 30 min internalization with that isolated after 90 min. The morphologies of the membrane vesicles contained in the fractions isolated after 30 and 90 min were characteristic of early and late endocytic compartments, respectively, when analyzed by EM (Figure 2C). The fraction isolated after 30 min presented small magnetic vesicles of 50–200 nm, whereas the fraction isolated after 90 min showed larger vesicles of 200–500 nm. These larger vesicles contained internal vesicles or multilamellar structures characteristic of late endosomes and lysosomes, respectively (Figure 2C).
After 30 and 90 min of internalization, the magnetic fractions were enriched for EEA1 or Lamp-1, respectively (Figure 2D). However, small amounts of proteins from the ER or the Golgi complex similar to that detected in the corresponding nonmagnetic fractions (NMFs) or the postnuclear fraction can be observed in these fractions (Figure 2D).
To assess the homogeneity of the magnetic fractions isolated after 30 and 90 min internalization for markers of early and late endocytic compartments, we immunolabeled them with anti-EEA1 and anti-Lamp-1 antibodies. After 30 min, 80% (n= 857) of the individual vesicles observed by phase-contrast microscopy were labeled with the anti-EEA1 antibody (Figure 2F arrowheads), while some aggregates were labeled with anti-Lamp-1 antibody (Figure 2H arrows). Whereas after 90 min, 86% (n= 562) of the individual vesicles were labeled with the anti-Lamp-1 antibody (Figure 2L arrowheads), while some aggregates were labeled with anti-EEA1 antibody (Figure 2J arrows). Few aggregates present in both fractions were also labeled with anti-ER and anti-Rab6 antibodies (that decorates the Golgi complex), but we never observed individual vesicles labeled by these two antibodies (Figure S1). It is very likely that these aggregates account for protein contaminants detected by Western blotting in the magnetic fractions.
Together, these experiments suggest that a large majority of individual magnetic vesicles isolated after 30 min of internalization corresponds to early endosomes, while the large majority of individual magnetic vesicles isolated after 90 min of internalization corresponds to late endosomes and lysosomes. We will refer to the membrane fraction enriched in early endosomes as ‘magnetic early endosomes’ and the membrane fraction enriched in late endosomes and lysosomes as ‘magnetic late endosomes’.
Magnetic early and late endosomes move with distinct properties along microtubules in vitro
To analyze the contributions of various microtubule-associated motors to the movements of magnetic early and late endosomes, we reconstituted in vitro the microtubule-based movements of these endocytic compartments. Rhodamine-labeled microtubules were allowed to adhere to the glass surface of a flow chamber coated with polylysine, and magnetic endosomes were then injected into the flow chamber. Both populations of magnetic endosomes bound to microtubules. Arrows in Figure 3A point to individual magnetic late endosomes bound to fluorescent microtubules. Upon injection of ATP into the chamber, the movements of the individual magnetic early and late endosomes were very often discontinuous; during pauses, they oscillated while still bound to the microtubules (see Movies S1–S3). In contrast, aggregates did not move in these experimental conditions. Both magnetic early and late endosomes moved bidirectionally along the microtubules, as previously observed for asialoorosomucoid-loaded endocytic vesicles isolated from rat liver (22,24). Figure 3B shows one magnetic late endosome changing direction (see also Movies S1, S2).
Figure 3. Magnetic early and late endosomes have different motile properties in vitro. A) Phase-contrast microscopy of magnetic late endosomes overlaid with the fluorescent microtubule image of a whole microscope field after injection of the magnetic late endosomes into the flow cell. Arrows point to endosomes bound to microtubules. B) A sequence of high-magnification phase-contrast images overlaid with the fluorescent microtubule image illustrating the movement of one late magnetic endosome. The images were taken from the area indicated by a white rectangle in (A). The asterisk on each frame marks the starting point of the endosome, and the arrows point to its successive positions. Note that this magnetic endosome moves back and forth. C) The time during which magnetic early and late endosomes displayed oscillatory movements was normalized (%) to the total duration of their movements observed during an observation period of 4 min for both populations of magnetic endosomes. Thirty-one and 32 moving compartments were observed in five and four independent experiments for early and late magnetic endosomes, respectively. Plots show the mean (%) of oscillatory endosomes ± standard deviation (SD). D) The numbers of magnetic early and late endosomes and kinesin-1-coated beads moving bidirectionally were normalized to the total number of magnetic endosomes or beads moving directionally and plotted as mean (%) ± SD. Four hundred and ninety-nine, 428 and 236 magnetic early and late endosomes and kinesin-coated beads were analyzed in 8, 12 and 2 independent experiments, respectively. *p < 0.001, **p = 0.002. E) Distribution of instantaneous velocities (IV) of magnetic early and late endosomes moving on microtubules in vitro. The total number of IVs computed was 834 and 866 for magnetic early and late endosomes, corresponding to 31 and 32 moving compartments observed in five and four independent experiments, respectively. The number of IVs with a given value was calculated as a percentage of the total number of IVs computed. F) Distribution of the duration of the movements performed by magnetic early and late endosomes, calculated as a percentage of the total movements analyzed (n= 143 and 156 for magnetic early and late endosomes, respectively). G) Distribution of the lengths covered by magnetic early and late endosomes as a percentage of the total movements analyzed (n= 143 and 156 for magnetic early and late endosomes, respectively).
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Endosomes might switch direction either by using alternately plus-end- and minus-end-directed motors to move along an individual microtubule or by using a single motor but switching from one microtubule to another microtubule of opposite polarity in a bipolar bundle. We observed that early and late magnetic endosomes moved bidirectionally on single-polarity-marked microtubules (unpublished data). However, the statistical analysis of these movements was precluded by the limited number of movements observed per field. To identify the polarity of these microtubules, we had to dilute them 10 times more than the rhodamine-labeled ones. Consequently, we observed only 0.5 movements per field with these fluorescent microtubules compared with 15–20 movements per field with the rhodamine-labeled ones. We thus evaluated the contribution of bipolar bundles to the observed bidirectional movements on rhodamine-labeled microtubules. We compared the movement of polystyrene beads coated with a single type of motor kinesin-1 that moves bidirectionally only on bipolar microtubule bundles (28) with those of the magnetic endosomes. Of the kinesin-1-coated beads, 14% exhibited bidirectional movements in our experiments compared with 56% of magnetic early endosomes and 37% of magnetic late endosomes (Figure 3D). Thus, the majority of bidirectional movements observed in our experiments were probably because of the magnetic endosomes being moved in opposite directions by two or more different motors rather than by endosomes switching between microtubules of opposite polarities.
The magnetic early endosomes were more likely to move bidirectionally (56%) than were the magnetic late endosomes (37%), while both populations spent a similar proportion of their time in oscillation (Figure 3C,D), and they present similar distributions for the length and the duration of their movements (Figure 3F,G). Magnetic early endosomes also moved more slowly than did the late endosomes. The velocity of moving magnetic endosomes is not constant. We have compared the distribution of the instantaneous velocities (velocity measured between frames) of early and late magnetic endosomes. Thirty-one percent of the instantaneous velocities computed for the magnetic early endosomes were below 0.2 μm/s, compared with 15% of those for late magnetic endosomes (Figure 3E). The mean velocity of magnetic early endosomes was 0.27 ± 0.07 μm/s, whereas the mean instantaneous velocity of the magnetic late endosomes was 0.49 ± 0.1 μm/s. The value for early magnetic endosomes is on the same range of movements of transferrin-loaded compartments (29). The mean velocity of the magnetic late endosomes was very similar to the velocity of lysosomes and CD63GFP-positive compartments measured in vivo (0.45 ± 0.01 μm/s and 0.5 μm/s, respectively) (6,30).
Nielsen et al. (25) have reported that addition of cytosol to an in vitro microtubule-based motility assay increased the number of movements per minute and per field compared with movement in the absence of cytosol. The addition of cytosol from HeLa cells (2 mg/mL) containing dynein and kinesin-1 that had the ability to move microtubules in a gliding assay, to our in vitro motility assay had no effect on the frequency, bidirectionality or velocity of movements of the magnetic early or late endosomes (unpublished data). This suggests that microtubule-associated motors did not dissociate from the endocytic compartments during their isolation by the magnetic nanoparticle method and that no additional proteins are required for their movement on microtubules in our in vitro assay.
Together, these observations indicate that magnetic early and late endosomes have distinct motile properties: magnetic early endosomes move more slowly and change direction more frequently than do magnetic late endosomes. These distinct properties may reflect differences in the motor proteins they hold.
Kinesin-1 and dynein move early but not late endosomes in vitro
Kinesin from the kinesin-1 family and dynein are generally thought to mediate bidirectional movements of the endocytic compartments. We therefore asked whether these two types of motors contribute to the movement of the magnetic early and late endosomes that we see in vitro. We first investigated whether these motor proteins were associated with magnetic endosomes by immunoblotting. Both the magnetic early and late endosomes contained kinesin-1 and cytoplasmic dynein [Figure 4A, lanes magnetic fraction (MF)]. None of these proteins was enriched in the magnetic fractions, however, when compared with PNS (PNS lanes) or NMF (NMF lanes), consistent with the fact that these motors are associated with various organelles. The proportion of kinesin-1, DYNC1H1 (dynein heavy chain) and DYNC1I1 (dynein intermediate chain) detected in the magnetic early endosomal fraction represented 4, 9 and 8% of the amount detected in the PNS, respectively. The proportion of the kinesin-1 heavy chain in the magnetic late endosomal fraction represented 3% of the amount detected in the PNS. The amount of proteins from the dynein complex was slightly lower in the magnetic late fraction than in the early fraction (Figure 4B). The proportion of DYNC1H1 and DYNC1I1 (detected with the two different antibodies, 74.1 and 70.1, respectively) in the magnetic late endosomes represented 34, 45 and 32%, respectively, of the amount detected in the magnetic early endosomes (Figure 4B) and 2% of the total amount detected in the PNS.
Figure 4. Detection of kinesin-1, kinesin-2 and proteins of the dynein complex in magnetic early and late endosomes. A) Forty micrograms of protein from PNS, NMF and MF enriched in magnetic early and late endosomes, as indicated, was analyzed by immunoblotting with rabbit anti-kinesin-1, mouse anti-kinesin-2 (K2.4), mouse anti-DYNC1H1 and mouse anti-DYNC1I1 (70.1 and 74.1) antibodies. B) The amounts of kinesin-1, kinesin-2, DYNC1H1 and DYNC1I1 (detected with either antibody 70.1 or antibody 74.1) in 40 μg of protein from the MFs were estimated by quantification of the immunoblots using the procedure described in the Materials and Methods. The values were normalized to the amounts detected in 40 μg of proteins of the PNS as determined by the same method. Results of three independent experiments are shown as mean ± standard deviation. The p values are shown.
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To determine the specific involvement of dynein and kinesin-1 in the motility of individual magnetic endosomes in the in vitro assay, we next monitored their movements in the presence of antibodies that specifically block the activity of kinesin-1 or DYNC1 (dynein complex). The monoclonal antibody SUK4 inhibits kinesin-1 activity (4,12,31); addition of SUK4 to the motility assay did not affect significantly the number of magnetic early and late endosomes displaying directed movements when compared with no treatment or with movement in the presence of antibodies against Lamp-1 or EEA1 (Figure 5A). By contrast, SUK4 induced a dose-dependent decrease (by up to 37%) in the proportion of magnetic early endosomes moving in a bidirectional fashion, whereas this proportion remained unchanged for late magnetic endosomes (Figure 5B).
Figure 5. Antibodies against kinesin-1 and DYNC1I1 impair the movement of magnetic early endosomes but not of magnetic late endosomes in vitro. A and B) Microtubule-based movements of magnetic early and late endosomes in vitro were examined in the absence of antibody (−AB) or after addition of 12.5 μg/mL control antibody (anti-Lamp-1 antibody for magnetic early endosomes and anti-EEA1 antibody for magnetic late endosomes, as indicated) and 6.25 μg/mL [SUK4 (a)] and 12.5 μg/mL [SUK4 (b)] antibody against kinesin-1. C and D) Microtubule-based movements of early and late magnetic endosomes were examined in vitro without antibody or after addition of 25 μg/mL control antibody [as in parts (A) and (B)] and 12.5 μg/mL [74.1 (a)] and 25 μg/mL [74.1 (b)] antibody 74.1 against DYNC1I1. The number of magnetic early and late endosomes moving directionally in the presence of antibodies per field observed during 4 min is expressed as a percentage of the number of untreated magnetic endosomes moving under the same experimental conditions (n= 495 and n= 195 for magnetic early and late endosomes, respectively, analyzed with SUK4 antibody; n= 292 and n= 242 for magnetic early and late endosomes, respectively, analyzed with 74.1 antibody) (A and C). The proportion of magnetic endosomes moving bidirectionally relative to the number of those moving directionally was plotted for magnetic early and late endosomes treated with antibodies as mean (%) ± standard deviation (SD) (B and D). Errors bars indicate the SD of the mean for number of movements observed per fields. *p < 0.04, **p < 0.004.
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Monoclonal antibody 74.1 against DYNC1I1 has been reported to block cytoplasmic dynein activity by dissociating the dynein–dynactin complex (32). Addition of antibody 74.1 to the motility assay did not significantly affect the movement of magnetic late endosomes, but it decreased slightly the number of magnetic early endosomes displaying directed movements compared with the untreated magnetic endosomes (−AB) or with endosomes treated with the antibody against Lamp-1 (Figure 5C). By contrast, antibody 74.1 induced a marked dose-dependent decrease in the proportion of magnetic early endosomes moving in a bidirectional fashion, whereas this proportion remained unchanged for late magnetic endosomes (Figure 5D).
Consistent with previous observations, these data suggest that kinesin-1 and dynein are involved in moving early endosomes and contribute to their bidirectional movements (3,5), but, unexpectedly, these motors appear not to be the major motors that move late endosomes. Both antibody treatments inhibiting dynein and kinesin-1 function decreased the proportion of bidirectional movements of early magnetic endosomes, but they decreased slightly the total number of movements. This suggests that dynein and kinesin-1 are mostly associated with early magnetic endosomes that display also other motors that contribute to their movements.
Disruption of dynein function may affect the cellular distribution of late endocytic compartments indirectly
Our observations that dynein appears not to move late endosomes contradict the interpretations of previous studies showing that knock down of DYNC1H1 expression or dissociation of the dynein–dynactin complex delocalized the late endocytic compartments to the cell periphery, suggesting, therefore, that dynein is involved in moving late endosomes toward the perinuclear minus end of microtubules (2,3,9). To reconcile our study with the interpretation of the previous experiments, we investigated the impact of silencing DYNC1H1 expression upon the distribution of the Golgi complex and the lysosomes as well as upon the movements of the lysosomes. We used a mixture of four different small interfering RNA (siRNA) duplexes that target different regions of the messenger RNA encoding human DYNC1H1. Immunoblot analyses showed 85% depletion of DYNC1H1 4 days after siRNA transfection, whereas no depletion of DYNC1H1 was detected after transfection with control siRNA (Figure 6A). The level of DYNC1I1 was also significantly reduced while the level of the light chains LC81 and Tctex1 remained similar to that of untreated cells or of cells transfected with control siRNA (Figure 6A). The level of LC82 was also not affected (unpublished data). The observed reduction of DYNC1H1 and DYNC1I1 suggests that dynein complex is disrupted in these conditions, releasing the light chains in the cytosol.
Figure 6. DYNC1H1 siRNA impairs the distribution of the Golgi complex and Lamp-1 but has a minor effect on centrifugal and centripetal movements of lysosomes. A) Immunoblots of DYNC1H1, DYNC1I1, LC81 and Tctex1 in HeLa cells in absence of treatment (‘non treated’) or 4 days after transfection with control siRNA (‘cont. siRNA’) and DYNC1H1 siRNA (‘DYNC1H1 siRNA’). Immunoblotting of EEA1 serves as a control for loading of DYNC1H1 and tubulin as a control for loading of intermediate and light chains. B) The amount of DYNC1H1 detected in the cells transfected with control siRNA or DYNC1H1 siRNA was quantified as described in the Materials and Methods and normalized to the amount detected in cells transfected with control siRNA. The mean of three independent experiments is shown, error bars indicate standard deviation and the p value is shown on the graph. C–F) Cells 4 days after transfection with control siRNA (C and E) or with DYNC1H1 siRNA (D and F) were labeled for IF microscopy with an anti-Golgi antibody (C and D) or with an anti-Lamp-1 antibody (E and F). The outlines of the cells in the corresponding phase-contrast images are indicated. Bars represent 5 μm. G) Comparison of the number of cells with a Golgi complex in the perinuclear region, as shown in (C), with the number of cells with a dispersed Golgi complex, as shown in (D), after transfection with the control siRNA (n= 526) or with the DYNC1H1 siRNA (n= 635). The p value is shown on the graph. H and I) BODIPY-TR pepstatin A was internalized for 90 min in HeLa cells previously transfected with control siRNA or DYNC1H1 siRNA. The centripetal and centrifugal movements of the pepstatin-A-labeled compartments were then analyzed by time-lapse microscopy as described in the Materials and Methods. The average distances covered by pepstatin-A-labeled compartments to the center of the cell (centripetal) or to the cell periphery (centrifugal) during 4 min and the average of their instantaneous velocities in both directions are represented in (H) and (I), respectively. The p values are shown in the figure (H).
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Consistent with a previous report, in 79% of the cells transfected with DYNC1H1 siRNA, the Golgi complex was dispersed throughout the cytoplasm (compare Figure 6C and D and see Figure 6G) (33). Also, 81% of the transfected cells had a large proportion of Lamp-1 staining (indicating late endosomes and lysosomes) concentrated at the cell periphery, whereas in 96% of the cells transfected with the control siRNA, the majority of Lamp-1 staining was concentrated in the perinuclear region with the reminder scattered throughout the cell (compare Figure 6E and F and see Figure 7H).
Figure 7. Latrunculin A treatment reversed the delocalization of Lamp-1-labeled compartments caused by DYNC1H1 siRNA transfection. A–D) Cells 4 days after transfection with control siRNA (A and C) or with DYNC1H1 siRNA (B and D) were labeled for IF microscopy with an anti-tubulin antibody (A and B) or with a fluorescent phalloidin (C and D). Bars represent 5 μm in (A) and (B) and 10 μm in (C) and (D). E) Comparison of the number of cells with a cortical actin cytoskeleton, as shown in (C), with the number of cells showing an increase in the number of actin cables, as shown in (D), after transfection with the control siRNA (n= 250) or with the DYNC1H1 siRNA (n= 259). The p values are shown. F–G) Cells transfected with DYNC1H1 siRNA were treated for 30 min with 0.5-μm latrunculin A prior to processing and then analyzed for the distribution of actin filaments (F) or Lamp-1 (G). Bars represent 10 μm, and arrows point to the perinuclear accumulation of Lamp-1. H) Comparison of the number of cells with a perinuclear pool of Lamp-1-labeled compartments, as shown in Figure 5E, with the number of cells with a high concentration of Lamp-1-labeled compartments at the cell periphery, as shown in Figure 5F after transfection with the control siRNA (n= 337) or with the DYNC1H1 siRNA (n= 411), or transfection with the DYNC1H1 siRNA followed by treatment with 0.5 μm latrunculin A for 30 min (n= 220). *p < 0.001.
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We next analyzed the movement of the late endosomes/lysosomes upon depletion of the dynein heavy chain. We have previously shown that internalized pepstatin A conjugated covalently to BODIPY-TR cadaverine hydrochloride (BODIPY-TR pepstatin A) fills compartments that codistribute with Lamp-1 (30). Internalized BODIPY-TR pepstatin A codistributed also with Lamp-1-labeled compartments in cells transfected either with control siRNA or with DYNC1H1 siRNA and largely accumulated at the periphery of the cells transfected with DYNC1H1 siRNA (unpublished data). We compared the movements of these compartments after transfection of DYNC1H1 siRNA with those observed after transfection of control siRNA. Although the average velocities and the average distances covered by BODIPY-TR pepstatin-A-marked compartments of both centripetal and centrifugal movements were slightly reduced in cells transfected with DYNC1H1 siRNA, they were still undergoing directional movements in both centrifugal and centripetal directions between the cell center and the periphery (Figure 6H,I and Movie S4). These movements were totally inhibited by nocodazole treatment, supporting the fact that they were microtubule dependent (Movie S6).
We then reasoned that the delocalization of late endosomes/lysosomes and the slight decrease of the velocity and distance covered by these compartments in both directions after depletion of DYNC1H1 may be because of an indirect effect. For example, the release of the light chains in the cytosol under DYNC1H1 depletion may contribute to delocalizing late endosomes and lysosomes by a mechanism independent of the function of the dynein complex. Recent experimental evidence suggests a link between the actin dynamics and the dynein light chains TcTex1 and LC8 independent of their role in the dynein complex (17,18). In addition, actin filaments are known to co-operate with microtubules in the movement of late endosomes and lysosomes and to contribute to their normal subcellular distribution (30,34). We postulated that the redistribution of late endocytic compartments upon the depletion of DYNC1H1 might, therefore, be because of reorganization of the actin network.
To test this hypothesis, we investigated the impact of DYNC1H1 depletion on the actin and microtubule networks. The distribution of the microtubules appeared similar in cells transfected with DYNC1H1 siRNA and in those transfected with the control siRNA (Figure 7A,B). Cells transfected with the control siRNA displayed an important actin cortical network while actin cables were barely visible when we focused at the base of the nucleus (Figure 7C). In contrast, 84% of the cells transfected with DYNC1H1 siRNA displayed an increased number of actin cables compared with control cells when we imaged both cell populations at the same focal plane while the actin cortical network was barely visible (compare Figure 7C with D and see also Figure 7E).
We used latrunculin A, a drug that binds actin monomers and as a consequence moves the filamentous actin/globular actin (G-actin) equilibrium in cells toward G-actin to determine whether the actin network contributes to the redistribution of Lamp-1-marked compartments under DYNC1H1 depletion (35). The actin cables in DYNC1H1-depleted cells were depolymerized to a large extent by treatment with latrunculin A (Figure 7F). The treatment of DYNC1H1-depleted cells with latrunculin A did not inhibit movement of pepstatin-A-loaded endosomes, and it led to the redistribution of Lamp-1-labeled compartments from the periphery to the perinuclear region similar to that observed in control siRNA-treated cells (compare Figure 7G with Figure 6E and Movie S5 with Movie S4). Of the cells transfected with DYNC1H1 siRNA and treated with latrunculin A, 83% showed a similar distribution of Lamp-1-positive endosomes to that found in control siRNA-treated cells (Figure 7H). Although to a less extent (40% of the microinjected cells with the antibody 74.1 and treated with latrunculin), the delocalization of the Lamp-1-labeled compartments induced by the microinjection of the 74.1 antibody was also reversed by actin depolymerization (unpublished data). Thus, depolymerization of actin filaments by latrunculin A treatment reversed the effect of dynein heavy chain depletion or dissociation of the dynein dynactin complexes.
The return of Lamp-1-positive compartments to the perinuclear region following latrunculin A treatment of DYNC1H1-depleted cells suggests that disruption of the dynein complex interferes with the perinuclear distribution of late endosomes and lysosomes not directly by preventing their transport along microtubules but indirectly by reorganizing the actin network. The slight decrease of the average velocity and the average distance covered by the lysosomes under depletion of DYNC1H1 both in centrifugal and in centripetal movements may reflect the increase of the cytoplasmic viscosity because of the increase of actin filaments.
Kinesin-2 moves early and late endosomes
Previous reports have shown that kinesin-2 is required for the normal cellular distribution of late endosomes and that it moves asialoorosomucoid-loaded late endosomes in an in vitro assay (10,22). We therefore investigated the contribution of kinesin-2 to the movement of HeLa cell endocytic compartments in vivo and in vitro.
We first addressed whether one of the kinesin-2 heavy chains, KIF3A, that is associated with endosomes (10) contributes to the movement of late endosomes/lysosomes in vivo. We studied the movements of pepstatin-A-filled compartments in cells expressing the motor-less green fluorescent protein (GFP) fusion protein, GFP–kif3A-C2, that is dominant negative for the function of kinesin-2 (36). We compared the movement of endosomes filled with BODIPY-TR pepstatin A in HeLa cells expressing GFP–kif3A-C2 with those occurring in cells expressing GFP and in untransfected cells. The tracks of the BODIPY-TR pepstatin-A-labeled endosomes recorded in GFP–kif3A-C2-expressing cells tended to be shorter than those in GFP-expressing cells (compare Figure 8A,B). The mean square displacements (MSDs) of the labeled endosomes were computed from their tracks. MSD fits with a power law <r2(t)> =k.Δtα in which the exponent α reflects the type of movement observed: directional, Brownian or confined (37–40). According to the value of the exponent, we determined the proportion of late endocytic compartments undergoing Brownian motion (exponent = 1), directional movement (exponent > 1) or confined movement (exponent < 1) in the different experimental conditions (40). Expression of GFP–kif3A-C2 reduced the proportion of labeled compartments undergoing directional movement (34% compared with 46% in GFP-expressing cells and 54% in untransfected cells) and increased the proportion of compartments undergoing confined movement (56% compared with 41% in GFP-expressing cells and 34% in untransfected cells) (Figure 8C). Thus, as suggested by the experimental evidence reported by Brown et al. (10), kinesin-2 is involved in the in vivo movement of the late endosomes/lysosomes.
Figure 8. Expression of the motor-less kinesin-2 protein impairs in vivo movement of late endosomes and lysosomes. A and B) BODIPY-TR pepstatin A was internalized for 1 h 30 min by GFP–kif3-C2-transfected HeLa cells or GFP-transfected cells or untransfected cells and the movements of the pepstatin-A-labeled compartments were subsequently analyzed by time-lapse microscopy. Eighty representative lysosomes tracks are shown for GFP-expressing cells (A) or GFP–kif3-C2-expressing cells (B). C) MSD analysis of tracked organelles allowed us to classify the movement as ‘confined’ (exponent < 0.95), ‘Brownian’ (0.95 < exponent < 1.05) or ‘directional’ (exponent > 1.05) (40). The percentage of organelles in each class in cells expressing GFP–kif3-C2 or GFP, as well as in untransfected cells, is represented as the mean ± standard deviation from 24 cells filmed in four independent experiments. The p values are shown on the graph.
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To determine whether kinesin-2 contributes directly or indirectly to the movements of late endosomes/lysosomes, we analyzed the role of kinesin-2 in the movement of magnetic endosomes in vitro. We verified the presence of this motor in the early and late magnetic endosomes by immunoblotting. Kinesin-2 was more abundant than kinesin-1 in both early and late magnetic endosomal fractions. The total amount of kinesin-2 estimated in the magnetic early and late endosomes represented 14% of that detected in the PNS (Figure 4A,B).
We next monitored the movements of magnetic early and late endosomes in the presence of an anti-kinesin-2 antibody that was previously shown to inhibit specifically the activity of kinesin-2 (22). In agreement with our analyses in vivo, we observed that addition of anti-kinesin-2 antibody to the in vitro motility assay decreased the directional movement of magnetic late endosomes by up to 40% (Figure 9A). Interestingly, the proportion of bidirectional movements displayed by the remaining moving endosomes increased by 50% (Figure 9B). Furthermore, this antibody inhibited the directed movement of early magnetic endosomes by 70% (Figure 9A). The proportion of bidirectional movements performed by the remaining moving early endosomes in these experimental conditions was similar to that in untreated endosomes or in endosomes treated with antibodies against Lamp-1 (Figure 9B).
Figure 9. An antibody against kinesin-2 inhibits the movement of magnetic early and late endosomes in vitro.The microtubule-based movements of magnetic early and late endosomes were examined in vitro without antibody (−AB) and addition of 25 μg/mL control antibody (anti-Lamp-1 antibody for early magnetic endosomes and anti-EEA1 antibody for late magnetic endosomes) or with 12.5 μg/mL [Kif3 (a)] or 25 μg/mL [Kif3 (b)] anti-kinesin-2 antibody (from BD Transduction Laboratories). The number of magnetic early and late endosomes moving directionally in the presence or absence of antibodies per field observed during 4 min is expressed as a percentage of the number of untreated magnetic endosomes moving under the same experimental conditions (n= 212 and n= 242 for magnetic early and late endosomes, respectively, from two independent experiments) (A). The proportion of magnetic endosomes moving bidirectionally relative to the number of those moving directionally was plotted for magnetic early and late endosomes treated or not treated with antibodies, as indicated (B). The p values are shown on the graphs.
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The absence of decrease of the proportion of bidirectional movements in presence of anti-kinesin-2 antibody may suggest that a substantial proportion of magnetic early and late endosomes bearing kinesin-2 does not have any other active microtubule-based motor. Alternatively, kinesin-2-bearing early magnetic endosomes may also present the dynein–dynactin complex. According to Deacon et al. (41), the function of these two motors may be coordinated by the p150-glued protein of the dynactin complex, and therefore, inhibition of the function of kinesin-2 may impair the function of the dynein . These results together indicate that kinesin-2 is involved directly not only in the movements of late endosomes but also in the movement of early endosomes.