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First published online 29 July 2008
doi: 10.1242/jcs.023721

Journal of Cell Science 121, 2652-2661 (2008)
Published by The Company of Biologists 2008
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The role of cathepsin X in the migration and invasiveness of T lymphocytes

Zala Jevnikar1, Natasa Obermajer1, Matthew Bogyo2 and Janko Kos1,3,*

1 Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia
2 Department of Pathology, Stanford University, Stanford, CA 94305-5324, USA
3 Department of Biotechnology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia


Figure 1
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  		Fig. 1. Protein, activity and mRNA levels of cathepsin X in stably transfected Jurkat T lymphocytes (A) Protein levels of cathepsin X in the cytosol of resistant clones as determined by ELISA. The highest cathepsin X productivity was observed in clone 17, which was selected for further studies. (B) The molar level of cathepsin X in stably transfected Jurkat T lymphocytes with the highest productivity was 24-fold higher compared with wild-type cells. The mean levels obtained for wild-type Jurkat T lymphocytes (1.5 ng of cathepsin X per mg of total cell proteins) have been normalised to a relative concentration of 1.0. (C) Cathepsin X activity was determined by DCG-04 active-site labelling and the identity of cathepsin X in the cytosol was confirmed by immunoblotting. Activity of cathepsin X was increased in cathepsin-X-overexpressing T lymphocytes compared with wild-type cells. (D) Cathepsin X expression on the cell surface was unchanged after cathepsin X overexpression, as measured by flow cytometry. The geometric mean was 4.24 for wild-type and 4.23 for cathepsin-X-overexpressing T lymphocytes.

 

Figure 2
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  		Fig. 2. Homotypic cell aggregation of cathepsin-X-overexpressing T lymphocytes. Jurkat T lymphocytes were incubated for 24 hours in complete RPMI. (A) Wild-type Jurkat T lymphocytes show weak aggregation (B) Upregulation of cathepsin X in Jurkat T lymphocytes resulted in formation of large cell aggregates. Addition of EDTA, 2F12 cathepsin-X-neutralising mAb or cathepsin-X-specific inhibitor AMS36 reduced homotypic aggregation of cathepsin-X-overexpressing Jurkat T lymphocytes. Scale bars, 100 µm.

 

Figure 3
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  		Fig. 3. Morphological changes in T lymphocytes migrating on ICAM1 (upper panel) and Matrigel (lower panel). (A) Cells were activated with PMA and incubated for 24 hours on immobilised ICAM1. Wild-type Jurkat T lymphocytes show no evident phenotypic changes whereas overexpression of cathepsin X in Jurkat T lymphocytes resulted in the development of extended uropods. White arrows indicate the uropods attached to ICAM1-coated surface and black arrows the uropods attached to other cells. (B) Wild-type Jurkat T lymphocytes remained in a spherical non-migratory state after 48 hours of incubation on Matrigel. Activation with PMA resulted in the development of cytoskeletal rearrangements in a small proportion of the wild-type cells. By contrast, in transfected cells cathepsin X induced the formation of a polarised phenotype with long uropods, independently of the activation with PMA. (C) In cathepsin-X-overexpressing cells the accumulation of GFP–{alpha}-actinin-1 at the lamellipod (white arrows) is more evident than in wild-type cells. GFP–{alpha}-actinin-1 is concentrated also at the adhesive tip of the uropod (black arrow) formed only in cathepsin-X-overexpressing cells. Scale bars, 100 µm (A,B) and 10 µm (C).

 

Figure 4
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  		Fig. 4. Effect of cathepsin X on T lymphocyte migration. Data are expressed as the mean ± s.d. of triplicate wells. The percentage of migration obtained for wild-type cells was normalised to 1. (A) Cathepsin-X-overexpressing Jurkat T lymphocytes migration on uncoated polycarbonate membranes. (B) Cathepsin-X-overexpressing Jurkat T lymphocyte migration on ICAM1 coated membranes. 2F12, cathepsin-X-neutralising mAb (1 µM); AMS36, epoxysuccinyl-based specific inhibitor of cathepsin X (2 µM).

 

Figure 5
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  		Fig. 5. Localisation of active LFA-1 and cathepsin X in the migrating T lymphocytes. Jurkat T lymphocytes were seeded on ICAM1 pre-coated slides and allowed to migrate for 15 minutes before labelling. Active LFA-1 was detected with Alexa_fluor-488-conjugated specific antibody mAb 24. (A) Wild-type Jurkat T lymphocytes. Active LFA-1 is localised manly in the perimembranous region of the spherical non-migratory state cells. Cathepsin-X-overexpressing Jurkat T lymphocytes showing polarised phenotype. Active LFA-1 is localised predominantly at the uropod region (white arrows). (B) Colocalisation of active LFA-1 and cathepsin X in upregulated migratory cells along the z axis. Active LFA-1 is localised at the mid-cell region, which is in contact with ICAM1-coated surface (0.0 µm) and at the uropod projecting above the surface (2.7 µm). (C) Colocalisation of active cathepsin X (green; Alexa-Fluor-488) and the lysosomal marker protein LAMP2 (red; Alexa-Fluor-546) in cathepsin-X-overexpressing T lymphocytes. Cathepsin X is colocalised in the lysosomes (yellow), it is also present in perimembrane region and uropod (green). Scale bars, 50 µm (A), 5 µm (B), 10 µm (C).

 

Figure 6
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  		Fig. 6. (A-D) Colocalisation of cathepsin X (green; Alexa-Fluor-488) and LFA-1 (red; Alexa-Fluor-633) in Jurkat T lymphocytes migrating on ICAM1 (A,B) and Matrigel (C,D). Cells were seeded on pre-coated slides and allowed to migrate for 15 minutes (ICAM1) or 48 hours (Matrigel) before labelling. (A) In wild-type Jurkat T lymphocytes no morphological changes were observed, cathepsin X and LFA-1 are colocalised mainly in the perimembranous region. (B) In cathepsin-X-overexpressing Jurkat T lymphocytes with a polarised migratory phenotype cathepsin X and LFA-1 are colocalised predominantly at the uropod. (C,D) A similar colocalisation profile was obtained in 3D Matrigel. In cathepsin-X-overexpressing cells (D) with a polarised migratory phenotype cathepsin X and LFA-1 are colocalised at the perimembrane region and at the uropod. Fluorescent dyes were imaged sequentially in all colocalisation experiments in a frame-interlace mode to eliminate cross talk between the channels. The threshold level for this display was set to 90, which corresponds to two-thirds of the maximal brightness level. Pixels above the threshold in both channels (blue to white colour) and the contour plot are shown for images that demonstrate colocalisation. Scale bars, 20 µm.

 

Figure 7
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  		Fig. 7. Effect of cathepsin X on T lymphocyte Matrigel invasion. The percentage of invasion obtained for wild-type cells was normalised to 1. Cathepsin X transfection increased Jurkat T lymphocyte invasion through Matrigel 8.3-fold compared with wild-type cells. The invasion of cathepsin-X-overexpressing Jurkat T lymphocytes was inhibited by 29% by 2F12 anti-cathepsin-X-neutralising mAb and by 33% by cathepsin-X-specific inhibitor AMS36 (Fig. 7). Data are expressed the mean ± s.d. of triplicate wells.

 

Figure 8
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  		Fig. 8. Degradation of DQ-collagen-IV by living cells that migrate through Matrigel. Cells were incubated for 48 hours on Matrigel mixed with DQ-collagen-IV. (A) Degradation of DQ-collagen-IV by living MCF-10A neoT cells; green fluorescent degradation products of DQ-collagen-IV are present extracellularly and intracellularly. (B) Wild-type Jurkat T lymphocytes show no morphology changes and no fluorescent degradation products. (C) Cathepsin-X-overexpressing Jurkat T lymphocytes show a characteristic migratory phenotype and, similar to wild-type Jurkat T lymphocytes, no collagen degradation is observed. Scale bars, 20 µm (A) and 10 µm (B,C).

 

Figure 9
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  		Fig. 9. Potential role of cathepsin X in the aggregation, migration and invasiveness of T lymphocytes. The potential physiological role of cathepsin X was investigated by its overexpression in T lymphocytes. Overexpressed cathepsin X induces morphological changes and promotes migration, invasiveness and homotypic aggregation by modulating the activity of the β2 integrin LFA-1 that is abundantly expressed in T lymphocytes.

 

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© The Company of Biologists Ltd 2008