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. 2007 Jun 1;178(11):7199-210.
doi: 10.4049/jimmunol.178.11.7199.

Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells

Jatin M Vyas  1 , You-Me Kim, Katerina Artavanis-Tsakonas, J Christopher Love, Annemarthe G Van der Veen, Hidde L Ploegh
Affiliations

Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells

Jatin M Vyas et al. J Immunol. .

Abstract

Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
LPS induces endolysosomal tubules in primary BMDCs in a time- and dose-dependent manner. A, Using a fixed concentration of LPS (100 ng/ml), BMDCs from mice expressing class II MHC-eGFP were exposed for varying times and cells were scored for the presence or absence of tubular compartments. B, BMDCs from class II MHC-eGFP knock-in mice were exposed to graded amounts of LPS for 2 h. Five hundred cells were scored for the presence or absence of endolysosomal tubules at each indicated dose of LPS. C, BMDCs expressing endolysosomal proteins of class II MHC-eGFP, CD63-mRFP1, CD82-mRFP1, and LAMP1-eGFP demonstrated tubule formation. D, Magnification of the tubular compartments in each delineated area is shown. Scale bars represent 5 µm.
FIGURE 2
FIGURE 2
CD63, CD82, class II MHC, and LAMP1 colocalize in endolysosomal tubules of LPS-activated BMDCs. BMDCs from class II MHC-eGFP were transduced with lentivirus encoding either CD63-mRFP1 (column A) or CD82-mRFP1 (column B) and stimulated with 100 ng/ml LPS to induce the formation of endolysosomal tubular compartments. Individual images show endolysosomal tubules composed of CD63 or CD82 (both red) and the class II compartment (green). Merged images are shown in the third row from the top. Magnification of the region of the cell demonstrating tubular compartments is shown in the bottom panel of each column. B6 BMDCs were transduced with lentiviruses expressing either CD63-mRFP1 or LAMP1-eGFP and exposed to 100 ng/ml LPS for 2 h before imaging (column C). Cells expressing both CD63 (red) and LAMP1 (green) were imaged. The merged image is shown in the third panel from the top and magnification of the tubules is seen in the bottom panel. Scale bars represent 5 µm.
FIGURE 3
FIGURE 3
Endolysosomal tubules colocalize with LysoTracker and soluble Ag. A, BMDCs expressing CD63-mRFP1 were loaded with 50 nM LysoTracker Green DND-26 (green) for 30 min and then washed twice in PBS. The cells were stimulated with 100 ng/ml LPS, and cells with endolysosomal tubules were imaged. Tubular compartments can be visualized using both CD63-mRFP1 and LysoTracker as evidenced by the merged image and the magnification of these structures. B, The endolysosomal tubules showed dynamic movement with heterogeneous staining of both signals over time. Images were obtained every two seconds. The arrows indicate the dynamic movement and heterogeneity of the endolysosomal tubules. C, Using LysoTracker Red DND-99 (red) and BMDCs from class II MHC-eGFP DCs, the colocalization of class II MHC and LysoTracker in tubular endosomes is seen. BMDCs expressing CD63-mRFP1 were incubated with 50 nM of OVA-Alexa Fluor 647 for 30 min before the addition of 100 ng/ml LPS and imaged 2 h later. D, Cells with endolysosomal tubules showed colocalization of CD63 and OVA (merged images and magnified image). E, Serial images were obtained every 2 s to demonstrate the dynamic behavior of the endolysosomal tubules containing CD63 and OVA-Alexa 647. Arrows denote one representative endolysosomal tubule.
FIGURE 4
FIGURE 4
Endolysosomal tubules do not require the proper trafficking of class II MHC molecules. BMDCs derived from mice expressing class II MHC-eGFP but lacking an invariant chain (Ii−/−) were transduced to express CD63-mRFP1. A, In the absence of LPS, CD63 was found in its normal vesicular compartment whereas class II MHC was found in a reticular pattern suggestive of ER retention. B, After LPS stimulation, the CD63 compartment formed endolysosomal tubules, but no class II MHC tubules were seen in the endolysosomal tubules. Scale bars represent 5 µm.
FIGURE 5
FIGURE 5
Endolysosomal tubule formation occurs in BMDCs lacking MyD88. BMDCs derived from mice expressing class II MHC-eGFP but lacking MyD88 were transduced to express CD63-mRFP1. After stimulation with the pathogenic yeast CN, endolysosomal tubular compartments that contain both class II MHC and CD63 are seen. Scale bars represent 5 µm.
FIGURE 6
FIGURE 6
Endolysosomal tubular compartments demonstrate saltatory and bidirectional movement in primary BMDCs. A, BMDCs expressing CD63- mRFP1 were exposed to 100 ng/ml LPS for 2 h and imaged using confocal microscopy. Two endolysosomal tubules (arrows) exhibit dynamic behavior with saltatory movements (time listed in seconds after the first picture (far left) shown). B, The velocity of the tips of endolysosomal tubules was determined by examining sequential frames captured at 1-s intervals in a time-lapse movie using Metamorph software. C, Some tubular compartments show both centripetal and centrifugal motion. LPS-stimulated CD63-mRFP1 expressing BMDCs with prominent endolysosomal tubules were visualized every 1.5 s. Two tubules (identified with two different arrows) show these types of movements. The tubule identified with the filled arrowhead moved away from the centroid of the cell while the tubule identified with the double bar arrowhead showed movement toward the centroid of the cell. LPS-activated BMDCs expressing CD63-mRFP1 were imaged using 0.2-µm z steps. D, The resulting set of images was deconvoluted using the iterative restoration algorithm in Volocity and images were integrated to develop a three-dimensional model (left). Cross-sectional image in yz demonstrating the location of endolysosomal tubular compartments is seen at the top of the three-dimensional model and xz is seen to the left of the model. Higher magnification of this image false colored is shown on the right. Scale bars represent 5 µm.
FIGURE 7
FIGURE 7
Endolysosomal tubules colocalize with polymerizing microtubules, and nocodazole disrupts tubules in class II MHC-eGFP-expressing DCs. A, BMDCs were transduced to express YFP-tubulin and CD63-mRFP1. After LPS stimulation, cells that formed endolysosomal tubules were imaged as shown. B, Administration of 10 µM nocodazole to class II MHC-eGFP tubulating cells; serial images at the times indicated are shown. Nocodazole administration was arbitrarily assigned the value of 0 s (t = 0′00″). C, LPS-stimulated BMDCs expressing CD63-mRFP1 and EB1-eGFP were imaged. CD63+ tubules are in close apposition to EB1 “comets.” D, Over time, the CD63 tubules (long arrow) followed the EB1 path (short arrows).
FIGURE 8
FIGURE 8
Model of endolysosomal tubules and interdependence with microtubules. Endolysosomal tubules express CD63, class II MHC, CD82, and LAMP1 membrane proteins. Endolysosomal tubules use motor proteins associated with microtubules for bidirectional movement and follow the polymerizing microtubules as determined by the tracking of EB1-GFP. Although kinesin and dynein may serve as the motor proteins, no direct interaction was shown in this study. Endolysosomal tubules do not appear to be associated closely with actin. Class II MHC molecules are delivered to the cell surface by this mechanism (9). Whether CD63, CD82, and/or LAMP1 is delivered to the cell surface using endolysosomal tubules remains to be determined.
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