Plasma membrane Ca2+ pump isoform 4 function in cell migration and cancer metastasis.

The Ca2+ ion is a universal second messenger involved in many vital physiological functions including cell migration and development. To fulfil these tasks the cytosolic Ca2+ concentration is tightly controlled, and this involves an intricate functional balance between a variety of channels and pumps of the Ca2+ signalling machinery. Among these proteins, plasma membrane Ca2+ ATPases (PMCAs) represent the major high-affinity Ca2+ extrusion systems in the cell membrane that are effective in maintaining free Ca2+ concentration at exceedingly low cytosolic levels, which is essential for normal cell function. An imbalance in Ca2+ signalling can have pathogenic consequences including cancer and metastasis. Recent studies have highlighted the role of PMCAs in cancer progression and have shown that a particular variant, PMCA4b, is downregulated in certain cancer types, causing delayed attenuation of the Ca2+ signal. It has also been shown that loss of PMCA4b leads to increased migration and metastasis of melanoma and gastric cancer cells. In contrast, an increased PMCA4 expression has been reported in pancreatic ductal adenocarcinoma that coincided with increased cell migration and shorter patient survival, suggesting distinct roles of PMCA4b in various tumour types and/or different stages of tumour development. The recently discovered interaction of PMCAs with basigin, an extracellular matrix metalloproteinase inducer, may provide further insights into our understanding of the specific roles of PMCA4b in tumour progression and cancer metastasis.

The Plasma Membrane Ca2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton.

We demonstrated that the plasma membrane Ca2+ ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell-cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca2+ concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca2+ overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.

The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I.

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.

P38 MAPK Promotes Migration and Metastatic Activity of BRAF Mutant Melanoma Cells by Inducing Degradation of PMCA4b.

Metastatic melanoma is the most aggressive type of skin cancer. Previously, we identified the plasma membrane Ca2+ pump isoform 4b (PMCA4b or ATP2B4) as a putative metastasis suppressor in BRAF mutant melanoma cells. Metastasis suppressors are often downregulated in cancer, therefore, it is important to identify the pathways involved in their degradation. Here, we studied the role of p38 MAPK in PMCA4b degradation and its effect on melanoma metastasis. We found that activation of p38 MAPK induces internalization and subsequent degradation of PMCA4b through the endo/lysosomal system that contributes to the low PMCA4b steady-state protein level of BRAF mutant melanoma cells. Moreover, BRAF wild type cell models including a doxycycline-inducible HEK cell system revealed that p38 MAPK is a universal modulator of PMCA4b endocytosis. Inhibition of the p38 MAPK pathway markedly reduced migration, colony formation and metastatic activity of BRAF mutant cells in vitro partially through an increase in PMCA4b and a decrease in ß4 integrin abundance. In conclusion, our data suggest that the p38 MAPK pathway plays a key role in PMCA4b degradation and inhibition of this pathway-by increasing the stability of PMCA4b-may provide a potential therapeutic target for inhibition of melanoma progression and metastasis.

Vps8 overexpression inhibits HOPS-dependent trafficking routes by outcompeting Vps41/Lt.

Two related multisubunit tethering complexes promote endolysosomal trafficking in all eukaryotes: Rab5-binding CORVET that was suggested to transform into Rab7-binding HOPS. We have previously identified miniCORVET, containing Drosophila Vps8 and three shared core proteins, which are required for endosome maturation upstream of HOPS in highly endocytic cells (Lorincz et al., 2016a). Here, we show that Vps8 overexpression inhibits HOPS-dependent trafficking routes including late endosome maturation, autophagosome-lysosome fusion, crinophagy and lysosome-related organelle formation. Mechanistically, Vps8 overexpression abolishes the late endosomal localization of HOPS-specific Vps41/Lt and prevents HOPS assembly. Proper ratio of Vps8 to Vps41 is thus critical because Vps8 negatively regulates HOPS by outcompeting Vps41. Endosomal recruitment of miniCORVET- or HOPS-specific subunits requires proper complex assembly, and Vps8/miniCORVET is dispensable for autophagy, crinophagy and lysosomal biogenesis. These data together indicate the recruitment of these complexes to target membranes independent of each other in Drosophila, rather than their transformation during vesicle maturation.

Investigating Non-selective Autophagy in Drosophila.

Drosophila melanogaster is a popular model organism in molecular genetics and cell biology. Various Drosophila tissues have been successfully used for studying autophagy, and our knowledge about the genetic regulation of this process is constantly growing. It is important to use assays that distinguish between non-selective autophagy and the selective forms. Here we introduce a selection of proven methods, which, taking into account their limitations, are suitable to measure non-selective autophagy in Drosophila fat and other tissues.

Drosophila Arl8 is a general positive regulator of lysosomal fusion events.

The small GTPase Arl8 is known to be involved in the periphery-directed motility of lysosomes. However, the overall importance of moving these vesicles is still poorly understood. Here we show that Drosophila Arl8 is required not only for the proper distribution of lysosomes, but also for autophagosome-lysosome fusion in starved fat cells, endosome-lysosome fusion in garland nephrocytes, and developmentally programmed secretory granule degradation (crinophagy) in salivary gland cells. Moreover, proper Arl8 localization to lysosomes depends on the shared subunits of the BLOC-1 and BORC complexes, which also promote autophagy and crinophagy. In conclusion, we demonstrate that Arl8 is responsible not only for positioning lysosomes but also acts as a general lysosomal fusion factor.

Rab2 promotes autophagic and endocytic lysosomal degradation.

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.