A specific subset of RabGTPases controls cell surface exposure of MT1-MMP, extracellular matrix degradation and 3D invasion of macrophages

Abstract

SummaryThe matrix metalloproteinase MT1-MMP has a major impact on invasive cell migration in both physiological and pathological settings such as immune cell extravasation or metastasis of cancer cells. Surface-associated MT1-MMP is able to cleave components of the extracellular matrix, which is a prerequisite for proteolytic invasive migration. However, current knowledge on the molecular mechanisms that regulate MT1-MMP trafficking to and from the cell surface is limited. We have identified three members of the RabGTPase family, Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in primary human macrophages. Both overexpressed and endogenous forms show prominent colocalisation with MT1-MMP-positive vesicles, whereas expression of mutant constructs, as well as siRNA-induced knockdown, reveal that these RabGTPases are crucial in the regulation of MT1-MMP surface exposure, contact of MT1-MMP-positive vesicles with podosomes, extracellular matrix degradation in two and three dimensions, as well as three-dimensional proteolytic invasion of macrophages. Collectively, our results identify Rab5a, Rab8a and Rab14 as major regulators of MT1-MMP trafficking and invasive migration of primary human macrophages, which could be promising potential targets for manipulation of immune cell invasion.

A novel interaction between Rab7b and actomyosin reveals a dual role in intracellular transport and cell migration

“…However, their effects on cell migration have so far been investigated only in connection to the well-established role of Rabs in intracellular trafficking (Kawauchi et al, 2010;Mai et al, 2011;Linford et al, 2012;Allaire et al, 2013;Wiesner et al, 2013).…”

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

“…The sequence-verified targeting vector was linearized with ClaI digestion and electroporated into R1 embryonic stem cells. Positively targeted clones were screened by Southern blot analyses using 32 P-labeled 5Ј and 3Ј external probes that distinguished wild-type and targeted alleles upon EcoRI and SacI digestion. Blastocyst injections resulted in eight chimeric mice, all of which transmitted the targeted allele through the germ line.…”

“…The effect of Rabs on MMP intracellular trafficking and secretion has only been explored recently. In cultured human macrophages, Rab5a, Rab8a, and Rab14 modulate MT1-MMP trafficking for cell migration and invasion (32). In RAW 264.7 cells, Rab3D is required for MMP9 vesicles to associate with Kinesin 5B and to be transported along microtubules (27).…”

Spire-1 a novel contributor of invadosome and associated invasive properties

“…5E): Myc-Spire-1 indeed bound to GST-Rab3A as did the Cter domain, in contrast to the N-KIND domain, whereas none of the recombinant proteins or polypeptides co-eluted with GST alone (data not shown). Since p150-Spire-1 was reported to colocalize with Rab11 and to function along the exocytic pathway (Kerkhoff et al, 2001;Schuh, 2011) and since a subset of GTPases including Rab8A were recently implicated in the release of MTP1-MMP at the plasma membrane in primary macrophages (Wiesner et al, 2013), we checked whether any of these could be recruited at invadosomes. By analysing the distribution of GFP-Rab7A, GFPRab8A or GFP-Rab11A expressed in 3T3-Src + cells, we often observed GFP-Rab7A and GFP-Rab11A in the vicinity of invadosomes as vesicles, but only GFP-Rab8A was detected partially overlapping with F-actin-marked invadosomes (supplementary material Fig.…”