Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse

Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years1,2. Detection of circulating tumor cells (CTCs) at the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of 100 patients enrolled into the TRACERx study3, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor (79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC

Alanine Zipper-Like Coiled-Coil Domains Are Necessary for Homotypic Dimerization of Plant GAGA-Factors in the Nucleus and Nucleolus

GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms

Plant aquaporin selectivity: where transport assays, computer simulations and physiology meet

Plants contain a large number of aquaporins with different selectivity. These channels generally conduct water, but some additionally conduct NH(3), CO(2) and/or H(2)O(2). The experimental evidence and molecular basis for the transport of a given solute, the validation with molecular dynamics simulations and the physiological impact of the selectivity are reviewed here. The aromatic/arginine (ar/R) constriction is most important for solute selection, but the exact pore requirements for efficient conduction of small solutes remain difficult to predict. Yeast growth assays are valuable for screening substrate selectivity and are explicitly shown for hydrogen peroxide and methylamine, a transport analog of ammonia. Independent assays need to address the relevance of different substrates for each channel in its physiological context. This is emphasized by the fact that several plant NIP channels, which conduct several solutes, are specifically involved in the transport of metalloids, such as silicic acid, arsenite, or boric acid in planta

Channel-like NH3 flux by ammonium transporter AtAMT2.

Prokaryotes, plants and animals control ammonium fluxes by the regulated expression of ammonium transporters (AMTs) and/or the related Rhesus (Rh) proteins. Plant AMTs were previously reported to mediate electrogenic transport. Functional analysis of AtAMT2 from Arabidopsis in yeast and oocytes suggests that NH(4)(+) is the recruited substrate, but the uncharged form NH(3) is conducted. AtAMT2 partially co-localized with electrogenic AMTs and conducted methylamine with low affinity. This transport mechanism may apply to other plant ammonium transporters and explains the different capacities of AMTs to accumulate ammonium in the plant cell

Molecular mechanisms of ammonium transport and accumulation in plants

The integral membrane proteins of the ammonium transporter (AMT/Rh) family provide the major route for shuttling ammonium (NH(4)(+)/NH(3)) across bacterial, archaeal, fungal and plant membranes. These proteins are distantly related to the Rh (rhesus) glycoproteins, which are absent in higher plants, but are present in many species, including bacteria and mammals. It appears that the large nitrogen requirement of plants resulted in unique strategies to acquire, capture and/or release ammonium. The biological function of plant ammonium transporters will be discussed and compared to other AMT/Rh proteins

Molecular determinants of ammonia and urea conductance in plant aquaporin homologs.

Aquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP-like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP-like mutants conducted urea but not NH(3), and a residue without direct contact to the pore lumen was critical for conduction in the mutants

Molecular determinants of ammonia and urea conductance in plant aquaporin homologs

AbstractAquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP-like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP-like mutants conducted urea but not NH3, and a residue without direct contact to the pore lumen was critical for conduction in the mutants

Regulation of NH(4)(+) Transport by Essential Cross Talk between AMT Monomers through the Carboxyl Tails

Ammonium transport across plant plasma membranes is facilitated by AMT/Rh-type ammonium transporters (AMTs), which also have homologs in most organisms. In the roots of the plant Arabidopsis (Arabidopsis thaliana), AMTs have been identified that function directly in the high-affinity NH(4)(+) acquisition from soil. Here, we show that AtAMT1;2 has a distinct role, as it is located in the plasma membrane of the root endodermis. AtAMT1;2 functions as a comparatively low-affinity NH(4)(+) transporter. Mutations at the highly conserved carboxyl terminus (C terminus) of AMTs, including one that mimics phosphorylation at a putative phosphorylation site, impair NH(4)(+) transport activity. Coexpressing these mutants along with wild-type AtAMT1;2 substantially reduced the activity of the wild-type transporter. A molecular model of AtAMT1;2 provides a plausible explanation for the dominant inhibition, as the C terminus of one monomer directly contacts the neighboring subunit. It is suggested that part of the cytoplasmic C terminus of a single monomer can gate the AMT trimer. This regulatory mechanism for rapid and efficient inactivation of NH(4)(+) transporters may apply to several AMT members to prevent excess influx of cytotoxic ammonium