Use of Single-Point Genome Signature Tags as a Universal Tagging Method for Microbial Genome Surveys

We developed single-point genome signature tags (SP-GSTs), a generally applicable, high-throughput sequencing-based method that targets specific genes to generate identifier tags from well-defined points in a genome. The technique yields identifier tags that can distinguish between closely related bacterial strains and allow for the identification of microbial community members. SP-GSTs are determined by three parameters: (i) the primer designed to recognize a conserved gene sequence, (ii) the anchoring enzyme recognition sequence, and (iii) the type IIS restriction enzyme which defines the tag length. We evaluated the SP-GST method in silico for bacterial identification using the genes rpoC, uvrB, and recA and the 16S rRNA gene. The best distinguishing tags were obtained with the restriction enzyme Csp6I upstream of the 16S rRNA gene, which discriminated all organisms in our data set to at least the genus level and most organisms to the species level. The method was successfully used to generate Csp6I-based tags upstream of the 16S rRNA gene and allowed us to discriminate between closely related strains of Bacillus cereus and Bacillus anthracis. This concept was further used successfully to identify the individual members of a defined microbial community.     

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell''s leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells.     

Hyperactivation of the G12-mediated signaling pathway in Caenorhabditis elegans induces a developmental growth arrest via protein kinase C

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.     

Wnt5a is strongly expressed at the leading edge in non-melanoma skin cancer, forming active gradients, while canonical Wnt signalling is repressed

Wnt5a is one of the so-called non-canonical Wnt ligands which do not act through β-catenin. In normal development, Wnt5a is secreted and directs the migration of target cells along concentration gradients. The effect of Wnt5a on target cells is regulated by many factors, including the expression level of inhibitors and receptors. Dysregulated Wnt5a signalling facilitates invasion of multiple tumor types into adjacent tissue. However, the expression and distribution of Wnt5a in cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), as well as the effect of Wnt5a on keratinocyte migration has not been studied in detail to date. We here report that Wnt5a is upregulated in SCC and BCC and localised to the leading edge of tumors, as well as tumor-associated fibroblasts. The Wnt5a-triggered bundling of its receptor Fzd3 provides evidence of Wnt5a concentration gradients projecting into the tumor. In vitro migration assays show that Wnt5a concentration gradients determine its effect on keratinoctye migration: While chemotactic migration is inhibited by Wnt5a present in homogenous concentrations, it is enhanced in the presence of a Wnt5a gradient. Expression profiling of the Wnt pathway shows that the upregulation of Wnt5a in SCC is coupled to repression of canonical Wnt signalling. This is confirmed by immunohistochemistry showing lack of nuclear β-catenin, as well as absent accumulation of Axin2. Since both types of Wnt signalling act mutually antogonistically at multiple levels, the concurrent repression of canonical Wnt signalling suggests hyper-active Wnt5a signal transduction. Significantly, this combination of gene dysregulation is not observed in the benign hyperproliferative inflammatory skin disease psoriasis. Collectively, our data strongly suggest that Wnt5a signalling contributes to tissue invasion by non-melanoma skin cancer.     

Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.     

The tumor suppressor Apc controls planar cell polarities central to gut homeostasis

The stem cells (SCs) at the bottom of intestinal crypts tightly contact niche-supporting cells and fuel the extraordinary tissue renewal of intestinal epithelia. Their fate is regulated stochastically by populational asymmetry, yet whether asymmetrical fate as a mode of SC division is relevant and whether the SC niche contains committed progenitors of the specialized cell types are under debate. We demonstrate spindle alignments and planar cell polarities, which form a novel functional unit that, in SCs, can yield daughter cell anisotropic movement away from niche-supporting cells. We propose that this contributes to SC homeostasis. Importantly, we demonstrate that some SC divisions are asymmetric with respect to cell fate and provide data suggesting that, in some SCs, mNumb displays asymmetric segregation. Some of these processes were altered in apparently normal crypts and microadenomas of mice carrying germline Apc mutations, shedding new light on the first stages of progression toward colorectal cancer.