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Piezo1 is the stretch activated Ca 2+ channel in red blood cells that mediates homeostatic volume control. Here, we study the organization of Piezo1 in red blood cells using a combination of super-resolution microscopy techniques and electron microscopy. Piezo1 adopts a non-uniform distribution on the red blood cell surface, with a bias toward the biconcave ‘dimple’. Trajectories of diffusing Piezo1 molecules, which exhibit confined Brownian diffusion on short timescales and hopping on long timescales, also reflect a bias toward the dimple. This bias can be explained by ‘curvature coupling’ between the intrinsic curvature of the Piezo dome and the curvature of the red blood cell membrane. Piezo1 does not form clusters with itself, nor does it colocalize with F-actin, Spectrin, or the Gardos channel. Thus, Piezo1 exhibits the properties of a force-through-membrane sensor of curvature and lateral tension in the red blood cell.

Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change. The way in which metastatic tumour cells control endocytic vesicular trafficking networks to establish a front-rear polarity axis that facilitates motility remains unclear. Here, the authors show that the EMT activator ZEB1 influences vesicular trafficking dynamics to execute cell polarity change.

Tan discusses the cancer-associated secretory phenotypes (CASPs). Somatic mutations activate CASPs by initiating cargo-specific secretory vesicle biogenesis in the Golgi and accelerating anterograde trafficking of secretory vesicles from Golgi to plasma membrane. Embedded within the CASP regulatory network are Golgi-resident protein complexes that govern cargo sorting, Golgi membrane bending, and vesicle scission. Cargo-sorting proteins recognize and load specific cargos into secretory vesicles. In line with these observations, CASPs govern the secretion of specific cargos.

Collagen lysyl hydroxylases (LH1-3) are Fe 2+ - and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus . Loop residues in the double-stranded β-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe 2+ . The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe 2+ -binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase K m and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer. Collagen lysyl hydroxylases promote cancer progression. Here the authors present the crystal structure of the lysyl hydroxylase domain of L230 from Acanthamoeba polyphaga mimivirus , which is of interest for LH inhibitor development, and show that ectopic expression of L230 in tumors promotes collagen cross-linking and metastasis.

Bruck Syndrome is a connective tissue disease associated with inactivating mutations in lysyl hydroxylase 2 (LH2/PLOD2) or FK506 binding protein 65 (FKBP65/FKBP10). However, the functional relationship between LH2 and FKBP65 remains unclear. Here, we postulated that peptidyl prolyl isomerase (PPIase) activity of FKBP65 positively modulates LH2 enzymatic activity and is critical for the formation of hydroxylysine-aldehyde derived intermolecular collagen cross-links (HLCCs). To test this hypothesis, we analyzed collagen cross-links in Fkbp10 -null and –wild-type murine embryonic fibroblasts. Although LH2 protein levels did not change, FKBP65 deficiency significantly diminished HLCCs and increased the non-hydroxylated lysine-aldehyde–derived collagen cross-links (LCCs), a pattern consistent with loss of LH2 enzymatic activity. The HLCC-to-LCC ratio was rescued in FKBP65-deficient murine embryonic fibroblasts by reconstitution with wild-type but not mutant FKBP65 that lacks intact PPIase domains. Findings from co-immunoprecipitation, protein-fragment complementation, and co-immunofluorescence assays showed that LH2 and FKBP65 are part of a common protein complex. We conclude that FKBP65 regulates LH2-mediated collagen cross-linking. Because LH2 promotes fibrosis and cancer metastasis, our findings suggest that pharmacologic strategies to target FKBP65 and LH2 may have complementary therapeutic activities.

Cancer-associated fibroblasts (CAFs) regulate diverse intratumoral biological programs and can promote or inhibit tumorigenesis, but those CAF populations that negatively impact the clinical outcome of lung cancer patients have not been fully elucidated. Because Thy-1 (CD90) marks CAFs that promote tumor cell invasion in a murine model of Kras G12D –driven lung adenocarcinoma (Kras LA1 ), here we postulated that human lung adenocarcinomas containing Thy-1 + CAFs have a worse prognosis. We first examined the location of Thy-1 + CAFs within human lung adenocarcinomas. Cells that co-express Thy-1 and α-smooth muscle actin (αSMA), a CAF marker, were located on the tumor periphery surrounding collectively invading tumor cells and in perivascular regions. To interrogate a human lung cancer database for the presence of Thy-1 + CAFs, we isolated Thy-1 + CAFs and normal lung fibroblasts (LFs) from the lungs of Kras LA1 mice and wild-type littermates, respectively, and performed global proteomic analysis on the murine CAFs and LFs, which identified 425 proteins that were differentially expressed. Used as a probe to identify Thy-1 + CAF-enriched tumors in a compendium of 1,586 lung adenocarcinomas, the presence of the 425-gene signature predicted a significantly shorter survival. Thus, Thy-1 marks a CAF population that adversely impacts clinical outcome in human lung cancer.

Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen’s amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.Guo et al. determine the molecular basis of collagen lysyl hydroxylase 2 (LH2) substrate specificity. They further show that LH2 also functions as a collagen glucosyltransferase to promote lung cancer progression.

A fibrotic stroma accumulates in advanced cancers, and invasive cancer cells migrate along collagen fibers that facilitate dissemination from the primary tumor. However, the ways in which tumor cells govern these processes remain unclear. Here, we report that the epithelial-mesenchymal transition-activating transcription factor ZEB1 increased type I collagen (Col1) secretion and enhanced tumor cell adherence to Col1. Mechanistically, ZEB1 increased the levels of α1β1 integrin (encoded by Itga1 and Itgb1) by inhibiting PP2A activity, which reduced nuclear accumulation of HDAC4 and, thereby, derepressed Itga1 gene transcription. In parallel, ZEB1 relieved the miRNA-148a-mediated silencing of Itga1. High levels of Itga1 enhanced tumor cell adherence to Col1 and were essential for Col1-induced tumor growth and metastasis. Furthermore, ZEB1 enhanced Col1 secretion by increasing the expression of a kinesin protein that facilitated transport and secretion of Col1-containing vesicles. Our findings elucidate a transcriptional mechanism by which lung adenocarcinoma cells coordinate a collagen deposition and adhesion process that facilitates tumor progression.

In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to also include support from the National Institutes of Health grant T32GM008280 to Sarah Alvarado.

Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis.