The large GTPase Mx1 binds Kif5B for cargo transport along microtubules.

A highly specific transport and sorting machinery directing secretory cargo to the apical or basolateral plasma membrane maintains the characteristic polarized architecture of epithelial cells. This machinery comprises a defined set of transport carriers, which are crucial for cargo delivery to the correct membrane domain. Each carrier is composed of a distinct set of proteins to verify precise routing and cargo selection. Among these components, the dynamin-related GTPase Mx1 was identified on post-Golgi vesicles destined for the apical membrane of MDCK cells. In addition to the presence on late secretory compartments, Mx1 was also detected on compartments of the early secretory pathway. Vesicular structures positive for this GTPase are highly dynamic, and we have studied the influence of the microtubule cytoskeleton on this motility. Live-cell microscopy indicated that microtubule disruption using nocodazole inhibits long-range trafficking of these structures. Mx1 directly or indirectly interacts with α-tubulin and the kinesin motor Kif5B as assessed by coimmunoprecipitation. In agreement with these observations knock out of Mx1 or a mutation in the unstructured L4 loop of Mx1 decreases the efficiency of apical cargo delivery. Interestingly, the L4 loop mutant still interacts with Kif5B; however, it causes vesicle elongation. This suggests that Mx1 aids in vesicle fission and stabilizes the interaction between Kif5B, microtubules and apical transport carriers.

The large GTPase Mx1 is involved in apical transport in MDCK cells.

In epithelial cells apical proteins are transported by specific transport carriers to the correct membrane domain. The composition of these carriers is heterogeneous and comprises components such as motor proteins, annexins, lectins, Rab GTPases and cargo molecules. Here, we provide biochemical and fluorescence microscopic data to show that the dynamin-related large GTPase Mx1 is a component of post-Golgi vesicles carrying the neurotrophin receptor p75(NTR) . Moreover, siRNA-mediated depletion of Mx1 significantly decreased the transport efficiency of apical proteins in MDCK cells. In conclusion, Mx1 plays a crucial role in the delivery of cargo molecules to the apical membrane of epithelial cells.

Sec16 alternative splicing dynamically controls COPII transport efficiency.

The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments.

A Natural Combination Extract of Viscum album L. Containing Both Triterpene Acids and Lectins Is Highly Effective against AML In Vivo.

Aqueous Viscum album L. extracts are widely used in complementary cancer medicine. Hydrophobic triterpene acids also possess anti-cancer properties, but due to their low solubility they do not occur in significant amounts in aqueous extracts. Using cyclodextrins we solubilised mistletoe triterpenes (mainly oleanolic acid) and investigated the effect of a mistletoe whole plant extract on human acute myeloid leukaemia cells in vitro, ex vivo and in vivo. Single Viscum album L. extracts containing only solubilised triterpene acids (TT) or lectins (viscum) inhibited cell proliferation and induced apoptosis in a dose-dependent manner in vitro and ex vivo. The combination of viscum and TT extracts (viscumTT) enhanced the induction of apoptosis synergistically. The experiments demonstrated that all three extracts are able to induce apoptosis via caspase-8 and -9 dependent pathways with down-regulation of members of the inhibitor of apoptosis and Bcl-2 families of proteins. Finally, the acute myeloid leukaemia mouse model experiment confirmed the therapeutic effectiveness of viscumTT-treatment resulting in significant tumour weight reduction, comparable to the effect in cytarabine-treated mice. These results suggest that the combination viscumTT may have a potential therapeutic value for the treatment AML.