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Invadosomes degrade extracellular matrix and facilitate cell invasion but their molecular composition is not fully understood. Here, the authors combine laser capture and mass spectrometry to map the proteome of invadosomes, showing that they rely on internal translational activity to maintain their structure.

Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to the cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localized Golgi in a retromer-dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by the toxin, revealing a new pathway mediating BoNT/A cellular toxicity.

Breast tumours are embedded in a collagen I-rich extracellular matrix (ECM) network, where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required for cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required macropinocytosis-dependent ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the up-regulation of the intracellular levels of several amino acids, most notably tyrosine and phenylalanine. This resulted in elevated tyrosine catabolism on ECM under starvation, leading to increased fumarate levels, potentially feeding into the tricarboxylic acid (TCA) cycle. Interestingly, this pathway was required for ECM-dependent cell growth and invasive cell migration under amino acid starvation, as the knockdown of p-hydroxyphenylpyruvate hydroxylase-like protein (HPDL), the third enzyme of the pathway, opposed cell growth and motility on ECM in both 2D and 3D systems, without affecting cell proliferation on plastic. Finally, high HPDL expression correlated with poor prognosis in breast cancer patients. Collectively, our results highlight that the ECM in the tumour microenvironment (TME) represents an alternative source of nutrients to support cancer cell growth by regulating phenylalanine and tyrosine metabolism.

Abstract Background Phosphatase and tensin homolog (PTEN) is one of the most frequently inactivated tumor suppressors in breast cancer. While PTEN itself is not considered a druggable target, PTEN synthetic-sick or synthetic-lethal (PTEN-SSL) genes are potential drug targets in PTEN-deficient breast cancers. Therefore, with the aim of identifying potential targets for precision breast cancer therapy, we sought to discover PTEN-SSL genes present in a broad spectrum of breast cancers. Methods To discover broad-spectrum PTEN-SSL genes in breast cancer, we used a multi-step approach that started with (1) a genome-wide short interfering RNA (siRNA) screen of ~ 21,000 genes in a pair of isogenic human mammary epithelial cell lines, followed by (2) a short hairpin RNA (shRNA) screen of ~ 1200 genes focused on hits from the first screen in a panel of 11 breast cancer cell lines; we then determined reproducibility of hits by (3) identification of overlaps between our results and reanalyzed data from 3 independent gene-essentiality screens, and finally, for selected candidate PTEN-SSL genes we (4) confirmed PTEN-SSL activity using either drug sensitivity experiments in a panel of 19 cell lines or mutual exclusivity analysis of publicly available pan-cancer somatic mutation data. Results The screens (steps 1 and 2) and the reproducibility analysis (step 3) identified six candidate broad-spectrum PTEN-SSL genes (PIK3CB, ADAMTS20, AP1M2, HMMR, STK11, and NUAK1). PIK3CB was previously identified as PTEN-SSL, while the other five genes represent novel PTEN-SSL candidates. Confirmation studies (step 4) provided additional evidence that NUAK1 and STK11 have PTEN-SSL patterns of activity. Consistent with PTEN-SSL status, inhibition of the NUAK1 protein kinase by the small molecule drug HTH-01-015 selectively impaired viability in multiple PTEN-deficient breast cancer cell lines, while mutations affecting STK11 and PTEN were largely mutually exclusive across large pan-cancer data sets. Conclusions Six genes showed PTEN-SSL patterns of activity in a large proportion of PTEN-deficient breast cancer cell lines and are potential specific vulnerabilities in PTEN-deficient breast cancer. Furthermore, the NUAK1 PTEN-SSL vulnerability identified by RNA interference techniques can be recapitulated and exploited using the small molecule kinase inhibitor HTH-01-015. Thus, NUAK1 inhibition may be an effective strategy for precision treatment of PTEN-deficient breast tumors.

The enzymes GALNTs add GalNAc sugar to Ser and Thr residues, forming the Tn glycan. GALNTs are activated by trafficking from Golgi to ER, a process driven by the Src kinase and negatively regulated by ERK8. This GALNTs activation (aka GALA) pathway induces high Tn levels and is a key driver of liver tumor growth. Recently, Tabak and colleagues have contested our previous data that EGF stimulation can induce GALNTs relocation. Here, we show that relocation induced by EGF is actually detectable in the very images acquired by Tabak et al. Furthermore, we show that over-expression of EGFR strongly enhances EGF-induced relocation and that EGFR appears required to drive relocation induced by ERK8 depletion. Direct co-localisation of GALNT with the ER marker Calnexin is observed after EGF stimulation. We furthermore propose that quantification of O-glycosylation of the ER resident protein PDIA4 provides a mean to quantify GALA independently of imaging. In sum, we demonstrate that the claimed non-reproducibility was due to experimental imaging conditions, that EGFR is indeed a driver of GALA and propose additional markers to facilitate the study of this pathway.

Enterovirus 71 (EV71) infection causes a spectrum of symptoms including neurological disease. To improve our understanding of EV71-host interactions, Wu et al. here perform a genome-wide RNAi screen, which implicates cell cycle regulation and ER-associated degradation as important factors in EV71 replication.

The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed us to identify at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/29 and Syb; STX1, SNAP24/29 and YKT6; STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes.

Abstract The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome‐ and phosphatome‐wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto‐myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

Endoplasmic reticulum (ER)-mitochondrial contact sites play a pivotal role in exchange of lipids and ions between the two organelles. How size and function of these contact sites are regulated remains elusive. Here we report a previously unanticipated, but conserved role of the small GTPase Sar1 in the regulation of ER-mitochondrial contact site size. Activated Sar1 introduces membrane curvature through its N-terminal amphiphatic helix at the ER-mitochondria interphase and thereby reducing contact size. Conversely, the S. cerevisiae N3-Sar1 mutant, in which curvature induction is decreased, caused an increase in ER-mitochondrial contacts. As a consequence, ER tubules are no longer able to mark the prospective scission site on mitochondria, thereby impairing mitochondrial dynamics. Consistently, blocking mitochondrial fusion partially rescued, whereas deletion of the dynamin-like protein enhanced the phenotype in the sar1D32G mutant. We conclude that Sar1 regulates the size of ER-mitochondria contact sites through its effects on membrane curvature.

The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC's regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC's action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.