|Proteolytic enzymes, and in particular matrix metalloproteinases (MMPs), have emerged as major regulators of proteolytic cell invasion in various cell types. In particular, the membrane-bound isoform MT1-MMP is a “master switch” proteinase, which cleaves multiple extracellular matrix (ECM) components, and acts as a central regulator of proteolytic cell invasion in a variety of settings, including monocyte diapedesis, T-cell homing, and cancer cell metastasis
To fine-tune MT1-MMP spatiotemporal activity, a complex regulatory network has evolved around this protease. Besides the activation of pro-MT1-MMP by convertases and the inhibition of MT1-MMP by endogenous elements (e.g. TIMPs), the regulation also depends on multiple intracellular trafficking events. For example, MT1-MMP has to be delivered to the cell surface, and the GTPase Rab8 has been implicated in this process. Surface-exposed MT1-MMP, in turn, can be internalized and recycled, a process that is regulated by both clathrin- or caveolae-dependent pathways.
In this project, we investigate the role of kinesins and RabGTPases as motor proteins and regulators, respectively, for the intracellular transport of MT1-MMP in primary human macrophages. We could show that MT1-MMP positive vesicles travel bidirectionally along microtubules. This process is driven by kinesin-1 and -2 motors, as well as by cytoplasmic dynein, and regulates cell surface exposure of MT1-MMP (Wiesner et al., 2010).
Furthermore, the impact of several specific RabGTPases on MT1-MMP trafficking and function in primary human macrophages could be demonstrated. The GTPases Rab5a, Rab8a and Rab14 critically regulate cell surface exposure of MT1-MMP, contact of MT1-MMP-positive vesicles with podosomes, as well as podosome-localised ECM degradation in 2D and 3D, and also 3D proteolytic invasion (Wiesner et al, 2013).
Currently, we are working on a further detailed model of the intracellular transport of MT1-MMP and its regulators in primary human macrophages.
Invasion of primary human macrophages into collagen I.
Time lapse video of primary human macrophages embedded in dense collagen I (2.5 mg/ml; dark area on the left), invading into less dense collagen I (2 mg/ml; lighter area on the right), which contains a chemoattractant. White bar scales 41 µm; video starts 9 h after cell seeding. Note mesenchymal morphology of invading cells characterised by numerous elongated protrusions. To see movie, click on the image. (1 image/15 min; frame rate: 10 f/s; sequence: 32 h 15 min; 1.2 MB)
Colocalisation of MT1-MMP-mCherry and GFP-Rab22a in living cells.
Confocal time-lapse video of a primary human macrophage expressing MT1-MMP-mCherry (red) and GFP-Rab22a (green). White bar scales 12 µm; white boxes show simultaneously detailed time lapse, marked within the cell. Note colocalisation of MT1-MMP-mCherry with GFP-Rab22a positive (small or giant) vesicles. To see movie, click on the image. (1 image/2 sec; frame rate: 10 f/s; sequence: 92 s; 397 KB)
Model of MT1-MMP trafficking in macrophages.
Surface-associated MT1-MMP is taken up by endocytosis, which is controlled by Rab5a. Parts of this pool are recycled back to the cell surface, either by fast recycling controlled by Rab14 or by slow recycling through recycling endosomes controlled by Rab22a. Trafficking of newly synthesized MT1-MMP to the cell surface is controlled by Rab8a and may occur by exocytotic vesicles or recycling endosomes. RabGTPase isoforms colocalising with MT1-MMP in macrophages are indicated at their respective compartments. RabGTPases that are major regulators of MT1-MMP-dependent cell invasion in 3D are highlighted in deep red (Rab21, colocalising with MT1-MMP vesicles, but without major impact on MT1-MMP trafficking, in light red; other major RabGTPases, including tested isoforms without impact on recycling or biosynthetic trafficking of MT1-MMP in grey). EE, early endosomes; LE, late endosomes; PM, plasma membrane; RE, recycling; TGN, trans-Golgi network. Click image to enlarge.
C. Wiesner et al. Blood., 2010
C. Wiesner et al. J. Cell Sci., 2013