Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.

DLC3 suppresses MT1-MMP-dependent matrix degradation by controlling RhoB and actin remodeling at endosomal membranes.

Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-ß-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-ß-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.

m6A RNA Degradation Products Are Catabolized by an Evolutionarily Conserved N6-Methyl-AMP Deaminase in Plant and Mammalian Cells.

N6-methylated adenine (m6A) is the most frequent posttranscriptional modification in eukaryotic mRNA. Turnover of RNA generates N6-methylated AMP (N6-mAMP), which has an unclear metabolic fate. We show that Arabidopsis thaliana and human cells require an N6-mAMP deaminase (ADAL, renamed MAPDA) to catabolize N6-mAMP to inosine monophosphate in vivo by hydrolytically removing the aminomethyl group. A phylogenetic, structural, and biochemical analysis revealed that many fungi partially or fully lack MAPDA, which coincides with a minor role of N6A-RNA methylation in these organisms. MAPDA likely protects RNA from m6A misincorporation. This is required because eukaryotic RNA polymerase can use N6-mATP as a substrate. Upon abrogation of MAPDA, root growth is slightly reduced, and the N6-methyladenosine, N6-mAMP, and N6-mATP concentrations are increased in Arabidopsis. Although this will potentially lead to m6A misincorporation into RNA, we show that the frequency is too low to be reliably detected in vivo. Since N6-mAMP was severalfold more abundant than N6-mATP in MAPDA mutants, we speculate that additional molecular filters suppress the generation of N6-mATP. Enzyme kinetic data indicate that adenylate kinases represent such filters being highly selective for AMP versus N6-mAMP phosphorylation. We conclude that a multilayer molecular protection system is in place preventing N6-mAMP accumulation and salvage.

The GEF-H1/PKD3 signaling pathway promotes the maintenance of triple-negative breast cancer stem cells.

Protein kinase D3 (PKD3) is upregulated in triple-negative breast cancer (TNBC) and associated with cell proliferation and metastasis development but its precise pro-oncogenic function is unknown. Here we show that PKD3 is required for the maintenance of the TNBC stem cell population. The depletion of PKD3 in MDA-MB-231 cells reduced the cancer stem cell frequency in vitro and tumor initiation potential in vivo. We further provide evidence that the RhoGEF GEF-H1 is upstream of PKD3 activation in TNBC stem cells. Most importantly, pharmacological PKD inhibition in combination with paclitaxel synergistically decreased oncosphere and colony formation efficiency in vitro and tumor recurrence in vivo. Based on our results we propose that targeting the GEF-H1/PKD3 signaling pathway in combination with chemotherapy might provide an effective therapeutic option for TNBC.

The tumor suppressor protein DLC1 maintains protein kinase D activity and Golgi secretory function.

Many newly synthesized cellular proteins pass through the Golgi complex from where secretory transport carriers sort them to the plasma membrane and the extracellular environment. The formation of these secretory carriers at the trans-Golgi network is promoted by the protein kinase D (PKD) family of serine/threonine kinases. Here, using mathematical modeling and experimental validation of the PKD activation and substrate phosphorylation kinetics, we reveal that the expression level of the PKD substrate deleted in liver cancer 1 (DLC1), a Rho GTPase-activating protein that is inhibited by PKD-mediated phosphorylation, determines PKD activity at the Golgi membranes. RNAi-mediated depletion of DLC1 reduced PKD activity in a Rho-Rho-associated protein kinase (ROCK)-dependent manner, impaired the exocytosis of the cargo protein horseradish peroxidase, and was associated with the accumulation of the small GTPase RAB6 on Golgi membranes, indicating a protein-trafficking defect. In summary, our findings reveal that DLC1 maintains basal activation of PKD at the Golgi and Golgi secretory activity, in part by down-regulating Rho-ROCK signaling. We propose that PKD senses cytoskeletal changes downstream of DLC1 to coordinate Rho signaling with Golgi secretory function.

The polarity protein Scribble positions DLC3 at adherens junctions to regulate Rho signaling.

The spatial regulation of cellular Rho signaling by GAP proteins is still poorly understood. By performing mass spectrometry, we here identify the polarity protein Scribble as a scaffold for the RhoGAP protein DLC3 (also known as StarD8) at cell-cell adhesions. This mutually dependent interaction is mediated by the PDZ domains of Scribble and a PDZ ligand (PDZL) motif in DLC3. Both Scribble depletion and PDZL deletion abrogated DLC3 junctional localization. Using a RhoA biosensor and a targeted GAP domain, we demonstrate that DLC3 activity locally regulates RhoA-ROCK signaling at and Scribble localization to adherens junctions, and is required for their functional integrity. In a 3D model of cyst development, we furthermore show that DLC3 depletion impairs polarized morphogenesis, phenocopying the effects observed upon Scribble knockdown. We thus propose a new function for Scribble in Rho regulation that entails positioning of DLC3 GAP activity at cell junctions in polarized epithelial cells.

miR-181 elevates Akt signaling by co-targeting PHLPP2 and INPP4B phosphatases in luminal breast cancer.

The PI3K-Akt pathway is one of the most commonly dysregulated cancer-associated signaling pathways. Here we report an oncogenic function for the miR-181 family in luminal breast cancer cells that involves Akt hyperactivation. We show that miR-181a and miR-181d posttranscriptionally suppress the expression of PHLPP2 and INPP4B phosphatases, resulting in elevated growth factor-induced Akt phosphorylation. Ectopic expression of miR-181a and miR-181d promoted S-phase entry and cell proliferation, which was reversed by pharmacological Akt inhibition. Importantly, the expression of miR-181 family members and PHLPP2/INPP2B are inversely correlated in primary human estrogen receptor-positive breast cancers, supporting the clinical relevance of our findings.

The Rho-specific GAP protein DLC3 coordinates endocytic membrane trafficking.

Membrane trafficking is known to be coordinated by small GTPases, but the identity of their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that ensure balanced GTPase activation at different subcellular sites is largely elusive. Here, we show in living cells that deleted in liver cancer 3 (DLC3, also known as STARD8) is a functional Rho-specific GAP protein, the loss of which enhances perinuclear RhoA activity. DLC3 is recruited to Rab8-positive membrane tubules and is required for the integrity of the Rab8 and Golgi compartments. Depletion of DLC3 impairs the transport of internalized transferrin to the endocytic recycling compartment (ERC), which is restored by the simultaneous downregulation of RhoA and RhoB. We further demonstrate that DLC3 loss interferes with epidermal growth factor receptor (EGFR) degradation associated with prolonged receptor signaling. Taken together, these findings identify DLC3 as a novel component of the endocytic trafficking machinery, wherein it maintains organelle integrity and regulates membrane transport through the control of Rho activity.

Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and Tbc1D20.

Chinese Hamster Ovary (CHO) cells are the most commonly used host for the production of biopharmaceuticals. Although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. In this study we show that ectopic expression of a human mitochondrial genome-encoded small RNA (mitosRNA-1978) in an IgG expressing CHO cell line strongly improved specific productivity by functioning in a microRNA-like fashion. By next generation sequencing we identified two endoplasmic reticulum (ER)-localized proteins, Ceramide Synthase 2 (CerS2) and the Rab1 GAP Tbc domain family member 20 (Tbc1D20), as target genes of mitosRNA-1978. Combined transient siRNA-mediated knockdown of CerS2 and Tbc1D20 resulted in increased specific productivity of CHO-IgG cells, thus recapitulating the mitosRNA-1978 phenotype. In support of a function in vesicular trafficking at the level of the ER, we provide evidence for altered cellular ceramide composition upon CerS2 knockdown and increased activity of Rab1 in CHO-IgG cells depleted of Tbc1D20. Importantly, in a fed-batch process, the combined stable knockdown of CerS2 and Tbc1D20 in CHO-IgG cells resulted in dramatically increased antibody production which was accompanied by enhanced cell growth. Thus, by identifying mitosRNA-1978 target genes in combination with an informed shRNA-mediated co-engineering approach we successfully optimized the secretory capacity of CHO producer cells used for the manufacturing of therapeutic proteins.

ATF6ß-based fine-tuning of the unfolded protein response enhances therapeutic antibody productivity of Chinese hamster ovary cells.

The dynamics of protein folding and secretion are key issues in improving the productivity and robustness of Chinese hamster ovary (CHO) producer cells. High recombinant protein secretion in CHO producer clones triggers the activation of the unfolded protein response (UPR), an intracellular response to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). We previously reported that the human microRNA (miRNA) miR-1287 enhances productivity in IgG-expressing CHO cells (CHO-IgG). Here, through next-generation sequencing (NGS), we identified the activating transcription factor 6 beta (ATF6ß), a repressor of the pro-survival and UPR promoting factor ATF6α, as a direct target gene of miR-1287 in CHO-IgG cells. We show that the transient depletion of ATF6ß resulted in enhanced specific productivity comparable to that of miR-1287-expressing CHO-IgG cells. Strikingly, stable ATF6ß knockdown in CHO-IgG cells significantly improved antibody titer and viable cell density under fed-batch conditions. This was associated with the elevated expression of the UPR genes glucose-regulated protein 78 (GRP78), homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1) and CCAAT/enhancer-binding protein homologous protein (CHOP). We hence demonstrate that ATF6ß-based cell line engineering is a promising strategy to improve the productivity of CHO producer cells by activating an optimally balanced UPR program. Biotechnol. Bioeng. 2017;114: 1310-1318. © 2017 Wiley Periodicals, Inc.

Elevated protein kinase D3 (PKD3) expression supports proliferation of triple-negative breast cancer cells and contributes to mTORC1-S6K1 pathway activation.

Here, we show that the expression of the Golgi-localized serine-threonine kinase protein kinase D3 (PKD3) is elevated in triple-negative breast cancer (TNBC). Using an antibody array, we identified PKD3 to trigger the activation of S6 kinase 1 (S6K1), a main downstream target of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Accordingly, PKD3 knockdown in TNBC cells led to reduced S6K1 phosphorylation, which was associated with impaired activation of mTORC1 at endolysosomal membranes, the accumulation of the mannose 6-phosphate receptor in and the recruitment of the autophagy marker light chain 3 to enlarged acidic vesicles. We further show that PKD3 depletion strongly inhibited cell spreading and proliferation of TNBC cells, identifying this kinase as a potential novel molecular therapeutic target in TNBC. Together, our data suggest that PKD3 in TNBC cells provides a molecular connection between the Golgi and endolysosomal compartments to enhance proliferative mTORC1-S6K1 signaling.

A WXW motif is required for the anticancer activity of the TAT-RasGAP317-326 peptide.

TAT-RasGAP317-326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317-326 sequence for the anticancer activities of TAT-RasGAP317-326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317-326.

Functional cross-talk between ras and rho pathways: a Ras-specific GTPase-activating protein (p120RasGAP) competitively inhibits the RhoGAP activity of deleted in liver cancer (DLC) tumor suppressor by masking the catalytic arginine finger.

The three deleted in liver cancer genes (DLC1-3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.

A global microRNA screen identifies regulators of the ErbB receptor signaling network.

BACKGROUND: The growth factor heregulin (HRG) potently stimulates epithelial cell survival and proliferation through the binding of its cognate receptor ErbB3 (also known as HER3). ErbB3-dependent signal transmission relies on the dimerization partner ErbB2, a receptor tyrosine kinase that is frequently overexpressed and/or amplified in breast cancer cells. Substantial evidence suggests that deregulated ErbB3 expression also contributes to the transformed phenotype of breast cancer cells. RESULTS: By genome-wide screening, we identify 43 microRNAs (miRNAs) that specifically impact HRG-induced activation of the PI3K-Akt pathway. Bioinformatic analysis combined with experimental validation reveals a highly connected molecular miRNA-gene interaction network particularly for the negative screen hits. For selected miRNAs, namely miR-149, miR-148b, miR-326, and miR-520a-3p, we demonstrate the simultaneous downregulation of the ErbB3 receptor and multiple downstream signaling molecules, explaining their efficient dampening of HRG responses and ascribing to these miRNAs potential context-dependent tumor suppressive functions. CONCLUSIONS: Given the contribution of HRG signaling and the PI3K-Akt pathway in particular to tumorigenesis, this study not only provides mechanistic insight into the function of miRNAs but also has implications for future clinical applications.

Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells.

Dynamic changes in the epigenome at defined genomic loci play crucial roles during cellular differentiation and disease development. Here, we developed dual-color bimolecular anchor detector (BiAD) sensors for high-sensitivity readout of locus-specific epigenome modifications by fluorescence microscopy. Our BiAD sensors comprise an sgRNA/dCas9 complex as anchor and double chromatin reader domains as detector modules, both fused to complementary parts of a split IFP2.0 fluorophore, enabling its reconstitution upon binding of both parts in close proximity. In addition, a YPet fluorophore is recruited to the sgRNA to mark the genomic locus of interest. With these dual-color BiAD sensors, we detected H3K9me2/3 and DNA methylation and their dynamic changes upon RNAi or inhibitor treatment with high sensitivity at endogenous genomic regions. Furthermore, we showcased locus-specific H3K36me2/3 readout as well as H3K27me3 and H3K9me2/3 enrichment on the inactive X chromosome, highlighting the broad applicability of our dual-color BiAD sensors for single-cell epigenome studies.

Repeat DNA methylation is modulated by adherens junction signaling.

Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.

Learning Dynamics from Multicellular Graphs with Deep Neural Networks.

The inference of multicellular self-assembly is the central quest of understanding morphogenesis, including embryos, organoids, tumors, and many others. However, it has been tremendously difficult to identify structural features that can indicate multicellular dynamics. Here we propose to harness the predictive power of graph-based deep neural networks (GNN) to discover important graph features that can predict dynamics. To demonstrate, we apply a physically informed GNN (piGNN) to predict the motility of multi-cellular collectives from a snapshot of their positions both in experiments and simulations. We demonstrate that piGNN is capable of navigating through complex graph features of multicellular living systems, which otherwise can not be achieved by classical mechanistic models. With increasing amounts of multicellular data, we propose that collaborative efforts can be made to create a multicellular data bank (MDB) from which it is possible to construct a large multicellular graph model (LMGM) for general-purposed predictions of multicellular organization.

GIT1 phosphorylation on serine 46 by PKD3 regulates paxillin trafficking and cellular protrusive activity.

The continuous assembly and disassembly of focal adhesions is required for efficient cell spreading and migration. The G-protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein whose dynamic localization to sites of cytoskeletal remodeling is critically involved in the regulation of these processes. Here we provide evidence that the subcellular localization of GIT1 is regulated by protein kinase D3 (PKD3) through direct phosphorylation on serine 46. GIT1 phosphorylation on serine 46 was abrograted by PKD3 depletion, thereby identifying GIT1 as the first specific substrate for this kinase. A GIT1 S46D phosphomimetic mutant localized to motile, paxillin-positive cytoplasmic complexes, whereas the phosphorylation-deficient GIT1 S46A was enriched in focal adhesions. We propose that phosphorylation of GIT1 on serine 46 by PKD3 represents a molecular switch by which GIT1 localization, paxillin trafficking, and cellular protrusive activity are regulated.

Integration of non-vesicular and vesicular transport processes at the Golgi complex by the PKD-CERT network.

Non-vesicular transport of ceramide from endoplasmic reticulum to Golgi membranes is essential for cellular lipid homeostasis. Protein kinase D (PKD) is a serine-threonine kinase that controls vesicle fission at Golgi membranes. Here we highlight the intimate connections between non-vesicular and vesicular transport at the level of the Golgi complex, and suggest that PKD and its substrate CERT, the ceramide transfer protein, play central roles in coordinating these processes by fine-tuning the local membrane lipid composition to maintain Golgi secretory function. This article is part of a Special Issue entitled Lipids and Vesicular Transport.

Stable microRNA expression enhances therapeutic antibody productivity of Chinese hamster ovary cells.

MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate the expression of different target genes and, thus, enable engineered gene networks to achieve complex phenotypic changes in mammalian cells. We hypothesized that exploiting this feature of miRNAs could improve therapeutic protein production processes by increasing viable cell densities and/or productivity of the mammalian cells used for manufacturing. To identify miRNAs that increase the productivity of producer cells, we performed a genome wide functional miRNA screen by transient transfection of Chinese hamster ovary (CHO) cells stably expressing an IgG1 antibody (CHO-IgG1). Using this approach, we identified nine human miRNAs that improved the productivities not only of the CHO-IgG1 cells but also of CHO cells expressing recombinant human serum albumin (HSA), demonstrating that the miRNAs act in a product-independent manner. We selected two miRNAs (miR-557 and miR-1287) positively impacting the viable cell density and the specific productivity, respectively, and then stably co-expressed them in IgG1 expressing CHO cells. In these cells, higher IgG1 titers were observed in fed-batch cultures whilst product quality was conserved, demonstrating that miRNA-based cell line engineering provides an attractive approach toward the genetic optimization of CHO producer cells for industrial applications.