ATR mediates a checkpoint at the nuclear envelope in response to mechanical stress.

ATR controls chromosome integrity and chromatin dynamics. We have previously shown that yeast Mec1/ATR promotes chromatin detachment from the nuclear envelope to counteract aberrant topological transitions during DNA replication. Here, we provide evidence that ATR activity at the nuclear envelope responds to mechanical stress. Human ATR associates with the nuclear envelope during S phase and prophase, and both osmotic stress and mechanical stretching relocalize ATR to nuclear membranes throughout the cell cycle. The ATR-mediated mechanical response occurs within the range of physiological forces, is reversible, and is independent of DNA damage signaling. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown. We propose that mechanical forces derived from chromosome dynamics and torsional stress on nuclear membranes activate ATR to modulate nuclear envelope plasticity and chromatin association to the nuclear envelope, thus enabling cells to cope with the mechanical strain imposed by these molecular processes.

An essential step of kinetochore formation controlled by the SNARE protein Snap29.

The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. Here, we identify a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis-segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. We propose that part of the outer kinetochore is functionally similar to membrane fusion interfaces.

Segregation of the Qb-SNAREs GS27 and GS28 into Golgi vesicles regulates intra-Golgi transport.

The Golgi apparatus is the main glycosylation and sorting station along the secretory pathway. Its structure includes the Golgi vesicles, which are depleted of anterograde cargo, and also of at least some Golgi-resident proteins. The role of Golgi vesicles remains unclear. Here, we show that Golgi vesicles are enriched in the Qb-SNAREs GS27 (membrin) and GS28 (GOS-28), and depleted of nucleotide sugar transporters. A block of intra-Golgi transport leads to accumulation of Golgi vesicles and partitioning of GS27 and GS28 into these vesicles. Conversely, active intra-Golgi transport induces fusion of these vesicles with the Golgi cisternae, delivering GS27 and GS28 to these cisternae. In an in vitro assay based on a donor compartment that lacks UDP-galactose translocase (a sugar transporter), the segregation of Golgi vesicles from isolated Golgi membranes inhibits intra-Golgi transport; re-addition of isolated Golgi vesicles devoid of UDP-galactose translocase obtained from normal cells restores intra-Golgi transport. We conclude that this activity is due to the presence of GS27 and GS28 in the Golgi vesicles, rather than the sugar transporter. Furthermore, there is an inverse correlation between the number of Golgi vesicles and the number of inter-cisternal connections under different experimental conditions. Finally, a rapid block of the formation of vesicles via COPI through degradation of ϵCOP accelerates the cis-to-trans delivery of VSVG. These data suggest that Golgi vesicles, presumably with COPI, serve to inhibit intra-Golgi transport by the extraction of GS27 and GS28 from the Golgi cisternae, which blocks the formation of inter-cisternal connections.

The SPCA1 Ca2+ pump and intracellular membrane trafficking.

The Golgi apparatus (GA) is a dynamic store of Ca(2+) that can be released into the cell cytosol. It can thus participate in the regulation of the Ca(2+) concentration in the cytosol ([Ca(2+) ](cyt) ), which might be critical for intra-Golgi transport. Secretory pathway Ca(2+) -ATPase pump type 1 (SPCA1) is important in Golgi homeostasis of Ca(2+) . The subcellular localization of SPCA1 appears to be GA specific, although its precise location within the GA is not known. Here, we show that SPCA1 is mostly excluded from the cores of the Golgi cisternae and is instead located mainly on the lateral rims of Golgi stacks, in tubular noncompact zones that interconnect different Golgi stacks, and within tubular parts of the trans Golgi network, suggesting a role in regulation of the local [Ca(2+) ](cyt) that is crucial for membrane fusion. SPCA1 knockdown by RNA interference induces GA fragmentation. These Golgi fragments lack the cis-most and trans-most cisternae and remain within the perinuclear region. This SPCA1 knockdown inhibits exit of vesicular stomatitis virus G-protein from the GA and delays retrograde redistribution of the GA glycosylation enzymes into the endoplasmic reticulum caused by brefeldin A; however, exit of these enzymes from the endoplasmic reticulum is not affected. Thus, correct SPCA1 functioning is crucial to intra-Golgi transport and maintenance of the Golgi ribbon.

MIF/CD74 axis is a target for novel therapies in colon carcinomatosis.

BACKGROUND: Strategies aimed at obtaining a complete cytoreduction are needed to improve long-term survival for patients with colorectal cancer peritoneal carcinomatosis (CRC-pc). METHODS: We established organoid models from peritoneal metastases of two naïve CRC patients. A standard paraffin inclusion was conducted to compare their 3D structure and immunohistochemical profile with that of the corresponding surgical samples. RNA expression levels of the CRC stem cell marker LGR5 was measured by in situ hybridization. The secretome of organoids was profiled by mass spectrometry. Energy homeostasis of organoids was interfered with 4-IPP and metformin. Biochemical and metabolic changes after drug treatments were investigated by western blot and mass spectrometry. Mitochondria impairment was evaluated by electron microscopy and mitotraker staining. RESULTS: The two organoids recapitulated their corresponding clinical samples in terms of 3D structure and immmunoistochemical profile and were positive for the cancer stem cells marker LGR5. Proteomic analyses of organoids highlighted their strong dependence on energy producing pathways, which suggest that their targeting could be an effective therapeutic approach. To test this hypothesis, we treated organoids with two drugs that target metabolism acting on AMP-activated protein kinase (AMPK), the main regulator of cellular energy homeostasis, which may act as metabolic tumour suppressor in CRC. Organoids were treated with 4-IPP, an inhibitor of MIF/CD74 signalling axis which activates AMPK function, or metformin that inhibits mitochondrial respiratory chain complex I. As a new finding we observed that treatment with 4-IPP downregulated AMPK signalling activity, reduced AKT phosphorylation and activated a JNK-mediated stress-signalling response, thus generating mitochondrial impairment and cell death. Metformin treatment enhanced AMPK activation, decreasing the activity of the anabolic factors ribosomal protein S6 and p4EBP-1 and inducing mitochondrial depolarization. CONCLUSION: We provide evidence that the modulation of AMPK activity may be a strategy for targeting metabolism of CRC-pc organoids.

Multiple functions of the SNARE protein Snap29 in autophagy, endocytic, and exocytic trafficking during epithelial formation in Drosophila.

How autophagic degradation is linked to endosomal trafficking routes is little known. Here we screened a collection of uncharacterized Drosophila mutants affecting membrane transport to identify new genes that also have a role in autophagy. We isolated a loss of function mutant in Snap29 (Synaptosomal-associated protein 29 kDa), the gene encoding the Drosophila homolog of the human protein SNAP29 and have characterized its function in vivo. Snap29 contains 2 soluble NSF attachment protein receptor (SNARE) domains and a asparagine-proline-phenylalanine (NPF motif) at its N terminus and rescue experiments indicate that both SNARE domains are required for function, whereas the NPF motif is in part dispensable. We find that Snap29 interacts with SNARE proteins, localizes to multiple trafficking organelles, and is required for protein trafficking and for proper Golgi apparatus morphology. Developing tissue lacking Snap29 displays distinctive epithelial architecture defects and accumulates large amounts of autophagosomes, highlighting a major role of Snap29 in autophagy and secretion. Mutants for autophagy genes do not display epithelial architecture or secretion defects, suggesting that the these alterations of the Snap29 mutant are unlikely to be caused by the impairment of autophagy. In contrast, we find evidence of elevated levels of hop-Stat92E (hopscotch-signal transducer and activator of transcription protein at 92E) ligand, receptor, and associated signaling, which might underlie the epithelial defects. In summary, our findings support a role of Snap29 at key steps of membrane trafficking, and predict that signaling defects may contribute to the pathogenesis of cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma (CEDNIK), a human congenital syndrome due to loss of Snap29.

Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae.

The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression-maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.