Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.

A bivalent, bispecific Dab-Fc antibody molecule for dual targeting of HER2 and HER3.

Dual targeting of surface receptors with bispecific antibodies is attracting increasing interest in cancer therapy. Here, we present a novel bivalent and bispecific antagonistic molecule (Dab-Fc) targeting human epidermal growth factors 2 and 3 (HER2 and HER3) derived from the Db-Ig platform, which was developed for the generation of multivalent and multispecific antibody molecules. Dab-Fc comprises the variable domains of the anti-HER2 antibody trastuzumab and the anti-HER3 antibody 3-43 assembled into a diabody-like structure stabilized by CH1 and CL domains and further fused to a human γ1 Fc region. The resulting Dab-Fc 2 × 3 molecule retained unhindered binding to both antigens and was able to bind both antigens sequentially. In cellular experiments, the Dab-Fc 2 × 3 molecule strongly bound to different tumor cell lines expressing HER2 and HER3 and was efficiently internalized. This was associated with potent inhibition of the proliferation and migration of these tumor cell lines. Furthermore, IgG-like pharmacokinetics and anti-tumoral activity were demonstrated in a xenograft tumor model of the gastric cancer cell-line NCI-N87. These results illustrate the suitability of our versatile Db-Ig platform technology for the generation of bivalent bispecific molecules, which has been successfully used here for the dual targeting of HER2 and HER3.

Spatiotemporal Control of Intracellular Membrane Trafficking by Rho GTPases.

As membrane-associated master regulators of cytoskeletal remodeling, Rho GTPases coordinate a wide range of biological processes such as cell adhesion, motility, and polarity. In the last years, Rho GTPases have also been recognized to control intracellular membrane sorting and trafficking steps directly; however, how Rho GTPase signaling is regulated at endomembranes is still poorly understood. In this review, we will specifically address the local Rho GTPase pools coordinating intracellular membrane trafficking with a focus on the endo- and exocytic pathways. We will further highlight the spatiotemporal molecular regulation of Rho signaling at endomembrane sites through Rho regulatory proteins, the GEFs and GAPs. Finally, we will discuss the contribution of dysregulated Rho signaling emanating from endomembranes to the development and progression of cancer.

DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-ß-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-ß-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.